Preparation of Synthetic and Natural Derivatives of Flavonoids Using Suzuki–Miyaura Cross-Coupling Reaction

Herein, we report the use of the Suzuki–Miyaura cross-coupling reaction for the preparation of a library of synthetic derivatives of flavonoids for biological activity assays. We have investigated the reactivity of halogenated flavonoids with aryl boronates and with boronyl flavonoids. This reaction was used to prepare new synthetic derivatives of flavonoids substituted at C-8 with aryl, heteroaryl, alkyl, and boronate substituents. The formation of flavonoid boronate enabled a cross-coupling reaction with halogenated flavones yielding biflavonoids connected at C-8. This method was used for the preparation of natural compounds including C-8 prenylated compounds, such as sinoflavonoid NB. Flavonoid boronates were used for the preparation of rare C-8 hydroxyflavonoids (natural flavonoids gossypetin and hypolaetin). A series of previously unknown derivatives of quercetin and luteolin were prepared and fully characterized.


Introduction
Flavonoids are a large class of natural polyphenolic secondary metabolites that are a common component of the human diet. Flavonoids generally have low toxicity and possess numerous beneficial biological activities such as antioxidant, anti-inflammatory, hepatoprotective, and anticarcinogenic properties [1]. Quercetin (1; 3,3 ,4 ,5,7-pentahydroxyflavone) is a bioactive flavonol found as a plant pigment mainly in onions, apples, and citrus fruits. It is used as a dietary supplement because it reduces oxidative stress, inhibits low-density lipoprotein oxidation and platelet aggregation, and acts as a vasodilator in blood vessels [2]. Luteolin (2; 3 ,4 ,5,7-tetrahydroxyflavone) is a common flavone found in fruits, vegetables, and medicinal plants. Luteolin (2) is known for its anti-allergic and anti-cancer activity [3]. Another flavone, chrysin (3; 5,7-dihydroxyflavone), occurs naturally in many plants, honey, and propolis and exhibits antioxidant, anti-inflammatory, anticancer, and antiviral properties (Figure 1) [4]. In this study, we focused on the preparation of new synthetic derivatives of the flavonoids quercetin (1), luteolin (2), and chrysin (3) substituted on the A-ring with alkyl or aryl substituents using palladium-catalyzed cross-coupling reactions.
Cross-coupling reactions are metal-promoted hetero couplings used to make new C-C or C-N bonds. These reactions typically use halide and a coupling partner (organoboronate, organosilicate, or amine) in the presence of a transition metal catalyst (Pd, Ni, or Zn) and a base [5]. Widely used palladium-catalyzed cross-coupling reactions include Heck coupling, Negishi, Stille, and Suzuki-Miyaura cross-coupling [6]. Buchwald-Haartwig amination is a Pd-catalyzed coupling reaction of aryl (pseudo)halides with amines used to form C(sp 2 )-N bonds [7,8].
Prenylated flavonoids are secondary plant metabolites that are considered to act as phytoalexins and play an important role in defense against pathogenic organisms [18]. Prenylation at C-8 increases the lipophilicity of flavonoids and their affinity for biological membranes [18]. Prenylated derivatives of flavonoids have many beneficial biological effects, such as the ability to inhibit P-glycoprotein (Pgp), which is related to the modulation of multidrug resistance [19]; they were also reported to have strong antioxidant activity [20,21]. Prenylated derivatives of flavonoids are generally prepared by Claisen rearrangement of O-prenylated compounds or by direct C-alkylation in alkaline solution [22]. These synthetic methods have many drawbacks, mainly low yields and complicated separation of the products. Suzuki-Miyaura cross-coupling is an interesting alternative for the preparation of prenylated and alkylated derivatives of flavonoids. This reaction has not been previously described for the prenylation of flavonoids. Prenylated benzene was prepared by a Suzuki cross-coupling reaction using benzylboronic acid and prenyl bromide [23]. The prenyl group can also cyclize to form 2,2-dimethyldihydropyranone group, resulting in sinoflavonoids [24].
Biflavonoids are a small subclass of flavonoids, and their occurrence in nature is rare. Biflavonoids are flavonoid dimers consisting of two identical or non-identical flavonoid/flavone units linked by a C-C bond [25]. Biflavonoids have been reported to have anti-amyloidogenic effects by inhibiting aggregation of Aβ plaques and promoting the disaggregation of Aβ fibrils; this effect makes biflavonoids lead compounds in the treatment of Alzheimer's disease [26]. The preparation of biflavonoids using metal-catalyzed cross-coupling reactions has been previously reported in the literature. Palladium-catalyzed coupling of aryl triflates with organostannanes yielded biflavonoids linked via C-7 [27]. Suzuki cross-coupling reaction of boronate and iodo-flavonoid yielded biflavonoid con-nected via C-8 (ring A) and C-3'(ring B) [28]. Kohari et al. reported the preparation of biflavones by Suzuki cross-coupling of boronylflavone and bromoflavone (flavones without HO-groups) [11]. Ulmann condensation of halogenated flavonoids was reported for the preparation of ginkgetin, a natural biflavone first isolated from Ginkgo biloba [29]. To our knowledge, the preparation of biflavonoids linked via C-8 has not yet been described in the literature.

Results and Discussion
8-Bromo-3,3 ,4 ,5,7-penta-O-isopropylquercetin (4) was prepared using α,β-dibromohy drocinnamic acid [30]. The Suzuki cross-coupling reaction was carried out using 8-bromo derivative (4) and 3-thienylboronic acid under various conditions ( Table 1). The outcome of this reaction was the desired reaction product 6 (isolated yield ca 8-10%), a product of debromination (3,3 ,4 ,5,7-penta-O-isopropylquercetin, 80%) and unreacted starting material (10%). Partial dehalogenation of the starting material is one of the drawbacks of the Suzuki cross-coupling reaction carried out in aqueous conditions. The outcome of this reaction indicates that the reaction stops after the oxidative addition step of the cross-coupling reaction. Varying the amount of base, boronic acid, or palladium catalyst did not affect the result of this reaction.  (12), and 8-iodo-5,7-di-O-isopropyl chrysin were prepared with N-iodosuccinimide in excellent yields (90-100%) [13]. 8-Iodo-3,3 ,4 ,5,7-penta-O-isopropylquercetin (5) was subsequently used for the Suzuki cross-coupling reaction. The outcome of this reaction was product 6 of the cross-coupling reaction in ca 24% yield (Entry 5, Table 1). Under the same reaction conditions, 50% of the desired product was obtained upon microwave (MW) irradiation (120 • C, 2 h, Entry 6, Table 1). The products of the cross-coupling reaction were isolated in excellent yields after optimization of the reaction conditions (Pd(PPh 3 ) 4 , NaOH, DMF, 10% H 2 O, 120 • C, 2 h, MW). The optimization of the reaction conditions with the isolated yields is summarized in Table 1. The optimized reaction conditions were used for the reactions with various boronic acids. In general, the reactions with aromatic boronic acids substituted with electron-donating groups or without substitution proceeded in good yields. In the case of aromatic boronic acids substituted with electron-withdrawing groups, the isolated yields were lower (30%), which was probably due to the low reactivity of the boronic acids, as the decomposition of the starting material was faster than the cross-coupling reaction. Exceptions were the reactions with tert-butylphenylboronic acid with a lower isolated yield (25%) and 4-trifluoromethylphenylboronic acid with a good isolated yield (71%). Low yields were also obtained after the cross-coupling reaction with 4-pyridinylboronic acid and pyrimidine-5-boronic acid due to their low reactivity towards cross-coupling reactions. The reaction with 4-mercaptophenylboronic acid and 2-fluoropyridine-4-boronic acid was unsuccessful.
The preparation of biflavonoids by cross-coupling reaction between 8-iodo-3 ,4 ,5,7tetra-O-isopropylluteolin (13) and 8-boronyl-3 ,4 ,3,5,7-penta-O-isopropylquercetin (7) failed under various reaction conditions. The products of these reactions were products of dehalogenation and deborylation of the starting materials. This reaction was also unsuccessful under other reaction conditions. The reactivity of boronate was studied to determine the effect of o-substitution. The reaction of 8-boronyl-3,3 ,4 ,5,7-penta-O-isopropylquercetin (7), and 6-bromoflavone (17) gave the product in 70% yield. This reaction proved that the low reactivity of iodo-and boronyl-flavonoids in the formation of biflavonoids is related to steric hindrance of the protecting group in the o-position and that the reaction proceeds when no substituent is present on the A-ring of the coupling partner. Pan et al. reported the synthesis of 8-(6 -umbelliferyl)-quercetin cross-coupling reaction of 8-iodo-3 ,4 ,3,5,7penta-O-isopropylquercetin and 7-methoxy-6-boronylcoumarin in 73% yield, suggesting that the cross-coupling should work for substrates protected in the o-position [13]. To determine whether the reaction would also work with a sterically less demanding protecting group, cross-coupling of 8-boronyl-3,3 ,4 ,5,7-penta-O-isopropylquercetin (7) and 8-iodo-3 ,4 ,5,7-tetra-O-methylluteolin (14) was performed but was also unsuccessful. This reaction confirmed that the low reactivity of flavonoids in our experiments to form C-8linked biflavonoids was related to the steric hindrance of protecting groups on the A-rings of coupling partners (Scheme 2). The main goal of our work was to create a library of compounds for biological activity testing. According to our preliminary experiments, the ether derivatives of flavonoids are highly cytotoxic to the tested cell lines. For further biological assays, all prepared products were deprotected with 1 M BCl 3 in dichloromethane and fully characterized by

General Procedure A for Suzuki Cross-Coupling Reaction
8-Iodo flavonoid (1 eq) and the corresponding boronic acid (2 eq) were dissolved in a DMF/H 2 O mixture (9:1) in a microwave vial. The reaction mixture was degassed with nitrogen for 15 min. Pd(PPh 3 ) 4 (3 mol%) and NaOH (4 eq) were added. The reaction mixture was irradiated in a microwave reactor at 120 • C for 2 h. After the reaction mixture cooled to ambient temperature, it was filtered through a microfilter (PTFE, 0.45 µm). The reaction mixture was poured into water and extracted with dichloromethane (3 × 10 mL). The combined organic layers were washed with water (3 × 10 mL), brine (3 × 10 mL), dried over Na 2 SO 4, and evaporated to dryness in vacuo. The residue was dissolved in dry dichloromethane (5 mL) at −10 • C. A solution of boron trichloride (10 eq, 1 M solution in dichloromethane) was added to the reaction mixture, which was then stirred at −10 • C for 30 min. The reaction mixture was then heated to 40 • C and stirred for 2 h, then cooled to 0 • C, and an excess of methanol was added. The reaction mixture was evaporated in vacuo, and the residue was purified by preparative HPLC chromatography (ASAHIPAK, 5 mL/min, MeOH isocratic) yielding the corresponding product.

Conclusions
The reactivity of halogen derivatives of flavonoids and their boronic acid pinacol esters in the Suzuki-Miyaura cross-coupling reaction was investigated. The reactivity of these derivatives depends strongly on the substitution of HO-7 and the reactivity of the boronate used. In the case of biflavonoid preparation, the reactivity was affected by the substitution on the A-ring of flavonoid. Optimized reaction conditions were used for the preparation of new synthetic derivatives of quercetin (1), luteolin (2), and chrysin (3), as well as flavonoid boronates. This method was also used for the first reported synthesis of sinoflavonoids (11a, 11b). Unsymmetrical biflavonoids connecting flavonoid-flavone (18,19) via C-8 were prepared. Boronyl flavonoids are also useful intermediates for the introduction of a new hydroxyl group at C-8, resulting in gossypetin (30) and hypolaetin (34) (naturally occurring flavonoids). All derivatives prepared have been fully characterized and are currently being tested for their biological activity.
Supplementary Materials: The following supporting information can be downloaded online. Experimental procedures, spectral data, 1 H and 13 C NMR spectra for products.