Synthesis of Either C2- or C4′-Alkylated Derivatives of Honokiol and Their Biological Evaluation for Anti-inflammatory Activity

Abstract

Honokiol, a biphenolic neolignan isolated from Magnolia officinalis, was reported to have a promising anti-inflammatory activity for the treatment of various diseases. There are many efforts on the synthesis and structure–activity relationship of honokiol derivatives. However, regioselective O-alkylation of honokiol remains a challenge and serves as a tool to provide not only some derivatives but also chemical probes for target identification and mode of action. In this study, we examined the reaction condition for regioselective O-alkylation, in which C2 and C4′-alkylated analogs of honokiol were synthesized and evaluated for inhibitory activity on nitric oxide production and cyclooxygenase-2 expression. Furthermore, we successfully synthesized a potential photoaffinity probe consisting of biotin and benzophenone based on a C4′-alkylated derivative.

Introduction

Macrophages and lymphocytes play an important role in the innate and adaptive immunity of hosts. These functions are mediated by various factors, such as cytokines, that have various defensive roles against pathogens.¹⁾ However, overproduction of inflammatory molecules can give the host severe immunopathological symptoms, such as acute and chronic inflammatory diseases.²⁾ Production of inflammatory molecules is triggered by mitogenic stimulation with various bacterial products, such as lipopolysaccharide (LPS). Nitric oxide (NO) has been implicated in physiological and pathological processes, such as nonspecific host defense, chronic inflammation, and vasodilation. Inducible nitric oxide synthase (iNOS) is involved in the pathological processes related to NO overproduction and is expressed in response to proinflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), in various cell types, including macrophages, endothelial cells, and smooth muscle cells.³⁾

Thus, effective modulation of the overproduction state has been considered as a therapeutic target in various types of inflammation.⁴⁾ Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely prescribed drugs because they exhibit anti-inflammatory effects by inhibiting cyclooxygenase (COX) in arachidonic acid metabolism.⁵⁾ There are two human enzymes that catalyze the same biochemical reactions, namely COX-1 and COX-2, but their amino acid sequence, tissue distribution, and physiology differ greatly. Long-term use of NSAIDs for treating pain and inflammation is often accompanied by side effects, such as gastrointestinal (GI) toxicity, hemorrhage, and inhibition of renal function.⁶⁾ Development of COX-2 selective inhibitors is a reasonable strategy for designing NSAIDS to eliminate side effects because the common side effects of these NSAIDs are due to COX-1 inhibition. Some COX-2 selective inhibitors, such as celecoxib, have an excellent effect with minimal adverse effects on humans. Concerns regarding cardiovascular events in thrombosis have been raised despite the initial success following introduction of selective COX-2 inhibitors. There has been continued progress in the development of new anti-inflammatory drugs with reduced risk of GI and cardiovascular diseases.⁷⁾

Biphenolic natural neolignans isolated from the bark of Magnolia officinalis, such as honokiol, 4′-O-methylhonokiol, magnolol, and obovatol, have been used as traditional medicines for treating gastrointestinal disorders, anxiety, and allergy. Recent studies revealed that honokiol and its analogs have various biological activities, such as antiangiogenesis,^8,9) anticancer,¹⁰⁾ anti-inflammatory,¹¹⁾ antibacterial,¹²⁾ osteoclast genesis-suppressing,¹³⁾ hepatoprotective,¹⁴⁾ and neuroprotective activities.¹⁵⁾ Honokiol and its derivatives were reported to target multiple signaling pathways, including the nuclear factor kappa light chain enhancer of activated B cells (NF-κB),¹⁶⁾ p53,¹⁷⁾ epidermal growth factor receptor (EGFR),¹⁸⁾ GPR55,¹⁹⁾ and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways.²⁰⁾ Previously, we have synthesized a series of derivatives of honokiol and 4′-O-methylhonokiol for improved inhibitory activities against COX-2 and PGF₁ production.¹¹⁾ Some analogs were found to prevent NO production in LPS-activated macrophages.²⁰⁾

Recently, the anti-neuroinflammatory effects of honokiol on heterocyclic bioisosteres in LPS-activated BV-2 cells have been reported.²¹⁾ However, the target of honokiol is still unknown, and to date, only one X-ray co-crystal structure of honokiol derivative has been suggested (PDB ID: 4OC7) to be selective for the retinoid X receptor (RXR)/coactivator interaction.²²⁾ These promising activities have prompted us to synthesize honokiol analogs to develop anti-inflammatory agents and photoaffinity probes to elucidate its binding targets and mechanisms of action.

Results and Discussion

Honokiol and 4′-O-methylhonokiol consist of a 5,3′-diallyl-biphenyl skeleton bearing hydroxy group in the C2 of the A ring, or hydroxyl and methoxy groups in the C4′ of the B ring, respectively. The previous study¹¹⁾ showed that 4′-O-methylhonokiol exerts higher anti-inflammatory activity than those of honokiol and various honokiol analogs, as examined using COX-1 and COX-2 enzyme assay and LTB4 formation assay. Thus, we contemplated efficient synthesis of mono-alkylated analogs on either the 2- or 4′-position for elongating the regioselective chain and designing site-selective photoaffinity probes, which would be useful to elucidate directly binding proteins, as shown in Fig. 1.

Fig. 1. Structure of Natural Neolignans and Photoaffinity Probe

Our first synthesis commenced with a study on regioselective O-methylation of honokiol to determine which phenol group is more reactive under various alkylation conditions.

Studies on regioselective O-methylation of honokiol were performed under different base and solvent conditions, as shown in Table 1. Briefly, 1 eq of commercially available honokiol, 1.2 eq of iodomethane, and 3 eq of base were used to yield 4′-O-methylhonokiol (1a), 2-O-methylhonkiol (1b), and 2,4′-dimethylhonokiol (1c). The methylation using LiOH and NaOH in aqueous dimethyl sulfoxide (DMSO) yielded three alkylated compounds (1a–c), whereas that using KOH and K₂CO₃ afforded 1b as a major product. Interestingly, C4′-alkylation occurred twice as much as C2-alkylation when Cs₂CO₃ was used as a base. The result was likely to be different depending on the metal cation. The yield and selectivity with strong bases, such as t-BuOK, potassium bis(trimethylsilyl)amide (KHMDS), and NaH, were not favorable, and the alkylation reaction did not work with amine bases, such as triethylamine and N,N-diisopropylethylamine. Because the chemical environment of the phenol group on the A ring and B ring was similar, regioselective alkylation was not completely achieved. However, with the automatic flash column chromatography system, both 4′-O- and 2-O-alkylated products were easily separated with more than 95% purity and fully analyzed by NMR, mass spectrometry before their biological evaluation.^8,23,24) Interestingly, the comparison of ¹³C-NMR spectra of honokiol, 4′-O-methylhonokiol (1a), 2-O-methylhonkiol (1b), and 2,4′-dimethylhonokiol (1c) enable us to confirm the aspect of their NMR spectra when other groups were alkylated at the C2 and C4′-position of honokiol, respectively, as shown in Fig. 2.

Table 1. Regioselective O-Methylation of Honokiol^a)

Entry	Base	Solvent	Isolated yield (%)
			1a	1b	1c
1	LiOH	DMSO/H2O	33	27	32
2	NaOH	DMSO/H2O	20	46	30
3	KOH	DMSO/H2O	21	50	10
4	K2CO3	DMSO/H2O	30	50	15
5	Cs2CO3	DMSO/H2O	35	21	15
6	Cs2CO3	Acetone	30	15	15
7	t-BuOK	THF	20	18	15
8	KHMDS	DMSO	7	15	7
9	NaH	THF	—	—	38
10	Et3N	THF	N/R
11	iPr2NEt	THF	N/R

a) Reaction conditions: a) for entries 1–6, 60°C, overnight; b) for entries 7–9, 0°C, 1 h; c) for entries 10–11, reflux, overnight.

Fig. 2. ¹³C-NMR Comparison of Natural Honokiol and Its Methylated Analogs (1a–1c)

We turned to the alkylation using various alkylating agents and Cs₂CO₃ in acetone, as shown in Chart 1. Similar to that of methylation, the reactivity of aliphatic alkylation was sluggish and the yield was not satisfactory. However, a slightly C4′-selectively alkylated compound was found as a major product. Ethyl, n-butyl, cyclopropylmethyl, and cyclobutylmethyl were not well incorporated on the phenol groups of honokiol. Allyl and prenyl were introduced on the C4′-position slightly selectively, similar to methyl. The O-alkylation with phenacyl bromides, such as 2-bromo-4′-fluoroacetophenone, 2-bromo-2′,4′-dichloroacetophenone, and 2-bromo-4′-nitroacetophenone, as alkylating agents afforded C4′-alkylated analogs (8a–10a) of honokiol in moderate yield. The reaction of tert-butyl bromoacetate afforded a C4′-alkylated analog (11a) in moderate yield.

Chart 1. Synthesis of O-Alkylation Analogs of Honokiol

Reagents and conditions: a) for 1–7, iodomethane, iodoethane, 1-bromobutane, allyl bromide, 3,3-dimethylallyl bromide, cyclopropylmethyl bromide, cyclobutylmethyl bromide, Cs₂CO₃, acetone, reflux, 24 h; b) for 8–11, 2-bromo-4′-fluoroacetophenone, 2-bromo-2′,4′-dichloroacetophenone, 2-bromo-4′-nitroacetophenone, tert-butyl bromoacetate, Cs₂CO₃, THF, 1 h. Yields based on recovered starting material in parentheses.

In a previous study, we found that modifications of 4′-O-methylhonokiol through introduction of methyl, isopropyl, and prenyl groups at phenol slightly decrease the inhibitory activity of COX-2 enzyme.¹¹⁾ Compared with other synthesized analogs, 11a showed the most promising anti-inflammatory activity; thus, it was exploited as an intermediate to obtain C4′-labeled photoaffinity probe of honokiol.²⁵⁾ By the treatment with trifluoroacetic acid in dichloromethane, the tert-butyl ester group of 11a was removed to afford carboxylic acid 12, as shown in Chart 2. Acid 12 was coupled with the bifunctional molecule 13, which consists of benzophenone²⁶⁾ and biotin, under PyAOP/HOAt-mediated reaction condition to finally afford the desired photoaffinity probe 14 in good yield.

Chart 2. Synthesis of C4′-Labeled Photoaffinity Probe of Honokiol

The cell viability of RAW264.7 cells following treatment with honokiol and its analogs was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were treated in the presence or absence of honokiol analogs at different concentrations (0–20 µM). As shown in Table 2, it did not show cell cytotoxicity at 5 µM concentration of all new synthetic compounds similar to honokiol, suggesting that the derivatives are little toxic. The inhibitory activities of honokiol analogs on NO production and COX-2 expression were evaluated in RAW264.7 cells treated with 10 ng/mL or 100 ng/mL LPS in the presence or absence of compounds at different concentrations (0–10 µM). The inhibitory effects of the synthesized honokiol analogs on NO production are shown in Fig. 3 and Table 2. The carbon elongation in the 2- or 4′-hydroxy position with methoxy, ethoxy, allyl, prenyl, cyclobutylmethyl, and cyclopropylmethyl groups does not improve the inhibitory activity on NO production regardless of the C2 or C4′ position, although ethyl, prenyl and cyclobutyl derivatives exhibited the similar activity with honokiol (2a, 2b, 5a, 5b, 7a, and 7b; 5.1 < IC₅₀ < 8.3 µM). Meanwhile, the introduction of two phenacyl groups on the hydroxyl of the C4′ position increased the NO inhibitory activity (8a and 9a; IC₅₀ = 4.8 and 4.5 µM). Next, we examined the expression level of COX-2 enzyme by Western blotting. All compounds showed direct inhibition of COX-2 enzyme, with a range of 65–93% inhibition in the presence of LPS 100 nM. Contrary to the results of NO production, the analog 3a, whose n-butoxy group was replaced with hydroxyl group, significantly suppressed the expression of COX-2. Some analogs, such as 2a, 2b, 6a, 6b, and 7a, moderately inhibited the expression of COX-2. Fortunately, tert-butoxyacyl derivative (11a) and a photoaffinity probe (15) of honokiol maintained a moderate inhibition of NO production and COX-2 expression compared to those of honokiol without drastic loss of activity. Based on the prediction of log P value (S log P), most alkylated compounds were regarded to have slightly more lipophilic properties than honokiol.

Table 2. Anti-inflammatory Effect of Compounds on NO Production and COX-2 Expression, log P Value and, Cell Viability

Compound	IC50 (µM) of NO productiona)		COX-2 expression (%)b)	S log Pc)	Cell viability (%)c)
	LPS 10 ng/mL	LPS 100 ng/mL
Honokiol	6.2	6.7	75 ± 0.85	3.9	104.7 ± 5.8
1a	24.9	24.2	94 ± 9.7	4.2	99.9 ± 1.7
1b	23.7	25.9	86 ± 4.7	4.2	104.3 ± 2.2
2a	6.1	6.3	84 ± 8.1	4.6	107.6 ± 1.8
2b	5.3	5.4	89 ± 4.7	4.6	99.3 ± 4.3
3a	>100	>100	70 ± 7.1	5.4	97.4 ± 4.6
3b	>100	>100	93 ± 11	5.4	103.3 ± 0.9
4a	18.5	25.7	89 ± 7.0	4.6	97.1 ± 5.8
4b	18.8	19.6	100 ± 1.3	4.6	106.2 ± 1.2
5a	7.8	7.4	93 ± 8.0	5.5	104.0 ± 3.8
5b	5.1	5.0	NDd)	5.5	89.2 ± 3.6
6a	39.9	41.0	83 ± 3.8	5.0	96.7 ± 2.5
6b	>100	>100	88 ± 6. 9	5.0	104.5 ± 1.8
7a	8.3	8.7	86 ± 8.2	5.4	100.5 ± 7.9
7b	5.9	6.0	ND	5.4	101.9 ± 4.4
8a	4.8	5.0	91 ± 13	5.9	91.8 ± 3.8
9a	4.5	4.6	87 ± 4.0	6.8	87.4 ± 1.6
10a	16.9	18.4	93 ± 12	5.3	109.9 ± 3.9
11a	13.3	13.3	91 ± 10	4.9	91.1 ± 4.6
11b	25.7	24.2	99 ± 4.5	4.9	102.0 ± 2.3
12	103.8	86.4	ND	3.7	104.2 ± 0.8
14	11.2	10.7	86 ± 4.0	6.5	95.6 ± 3.1

a) Each compound was defined as the concentration (µM) that caused 50% inhibition of NO production in LPS-activated RAW264.7 cells. b) The expression ratio was calculated as a band density ratio of COX-2 to β-actin in the treatment with 5 µM of each compound. Representative data from three independent experiments. c) S log P was derived by applying the rules of Wildman and Crippen and represents a good estimate of the actual log P of the molecule. d) The effect of treatment with 5 µM of each compound was determined by MTT assay, and the results are expressed in percentage (%).

Fig. 3. Inhibitory Activity of Honokiol Analogs on NO Production in LPS-Activated RAW264.7 Cells

Conclusion

The regioselective O-alkylation of honokiol was aimed to synthesize either C2- or C4′-alkylated derivatives for developing anti-inflammatory agents. To investigate regioselectivity, we conducted O-methylation under various reaction conditions, such as iodomethane and dimethyl sulfate as an alkylating agent, as well as potassium carbonate, cesium carbonate, Et₃N, and NaH as a base. The yield of O-methylation was moderate, and all compounds, including recovered honokiol, could be separated successfully. By treatment with Cs₂CO₃ and DMSO/H₂O, 4′-O-methylhonokiol, which is also a natural product, was obtained as a major product. Several analogs, namely C4′-alkyl-, C2-alkyl-, and C2,C4′-dialkyl-honokiol were synthesized through O-alkylation using Cs₂CO₃ as a base.

We confirmed that almost all compounds showed low cytotoxicity in macrophages, and several analogs showed improved inhibitory activities than that of honokiol against NO production and/or COX-2 expression. Moreover, a photoaffinity probe in which biotin and benzophenone were linked to the C4′ position was synthesized, and it showed a moderate inhibitory activity without drastic loss of activity. This study provided a basis for further development of these compounds as novel therapeutics for inflammatory diseases and cancer. Our next step is to identify the putative binding targets essential for signaling pathways in inflammation by using the honokiol-based photoaffinity probe.

Experimental

Chemistry

All starting materials and reagents were obtained from commercial suppliers and used without further purification. Air- and moisture-sensitive reactions were performed under nitrogen atmosphere. Flash column chromatography was performed using silica gel 60 (230–400 mesh; Merck, Darmstadt, Germany) with the indicated solvents. Thin-layer chromatography was performed using 0.25 mm silica gel plates (Merck). ¹H-(600 MHz) and ¹³C-NMR (150 MHz) spectra were recorded on an AVANCE III System 600 MHz spectrometer (Bruker, Billerica, MA, U.S.A.) as solutions in CDCl₃ or DMSO-d₆. High-resolution (HR) MS were obtained using JMS-700 (JEOL) with electron ionization or Thermo Scientific, Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Germering, Germany) using electrospray ionization (ESI). Low-resolution (LR) MS were obtained using a Waters Auto Purification instrument. Spectral data are available in Supplementary Materials.

Synthesis of Honokiol Analogs

3′,5-Diallyl-4′-methoxy-[1,1′-biphenyl]-2-ol (1a) and 3,5′-Diallyl-2′-methoxy-[1,1′-biphenyl]-4-ol (1b)

To an acetone solution (10 mL) of honokiol (1.13 g, 4.24 mmol) was added Cs₂CO₃ (1.38 g, 4.24 mmol), by 1 M iodomethane in tetrahydrofuran (THF) (0.5 mL, 5.08 mmol). After stirring at reflux overnight, the reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 1a (330 mg, 30%, BORSM (Based on recovered starting material) 36%), 1b (210 mg, 15%, BORSM 23%), 1c (182 mg, 15%, BORSM 19%), and recovered honokiol (280 mg). For 1a, ¹H-NMR (600 MHz, CDCl₃) δ: 7.28 (d, J = 8.3 Hz, 1H), 7.23 (s, 1H), 7.05 (d, J = 8.2 Hz, 1H), 7.03 (s, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.05–5.93 (m, 2H), 5.11–5.06 (m, 4H), 5.04 (bs, 1H), 3.88 (s, 3H), 3.43 (d, J = 6.6 Hz, 2H), 3.35 (d, J = 6.6 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 157.2, 151, 138, 136.6, 132.3, 130.6, 130.3, 129.9, 129.1, 128.9, 128.0, 128, 116.0, 115.7, 115.7, 111.1, 55.7, 39.6, 34.4; HRMS (electron ionization (EI)) m/z: [M] Calcd for C₁₉H₂₀O₂ 280.1463. Found 280.1466. For 1b, ¹H-NMR (600 MHz, CDCl₃) δ: 7.31 (dd, J = 8.2, 2.2 Hz, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.11–.09 (m, 2H), 6.90 (d, J = 8.1 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.10–5.95 (m, 2H), 5.24–5.20 (m, 2H), 5.11–5.04 (m, 2H), 5.03 (bs, 1H) 3.79 (s, 3H), 3.46 (d, J = 6.4 Hz, 2H), 3.37 (d, J = 6.7 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 154.8, 153.3, 137.8, 136.5, 132.3, 131.5, 131.2, 131, 130.3, 129.1, 128, 124.7, 116.6, 115.6, 115.5, 111.3, 55.7, 39.4, 35.4; HRMS (EI) m/z: [M] Calcd for C₁₉H₂₀O₂ 280.1463. Found 280.1461. For 1c, ¹H-NMR (600 MHz, CDCl₃) δ: 7.41–7.39 (m, 1H), 7.34 (s, 1H), 7.15 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.92 (dd, J = 8.3, 0.9 Hz, 2H), 6.09–5.98 (m, 2H), 5.14–5.10 (m, 2H), 5.09–5.06 (m, 2H), 3.88 (d, J = 1.0 Hz, 3H), 3.81 (d, J = 1.2 Hz, 3H), 3.46 (d, J = 6.5 Hz, 2H), 3.40 (d, J = 6.4 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 156.5, 155.0, 137.9, 137.2, 132.3, 131.1, 131.1, 130.8, 130.6, 128.5, 128.2, 128.0, 115.6, 115.5, 111.4, 110.0, 55.8, 55.6, 39.6, 34.5; LRMS (ESI) m/z: Calcd for C₂₀H₂₂O₂ [M + Na]⁺ 317.15. Found 317.25.

3′,5-Diallyl-4′-ethoxy-[1,1′-biphenyl]-2-ol (2a) and 3,5′-Diallyl-2′-ethoxy-[1,1′-biphenyl]-4-ol (2b)

To an acetone solution (10 mL) of honokiol (30 mg, 0.11 mmol) was added Cs₂CO₃ (100 mg, 0.3 mmol), followed by iodoethane (21 µL, 0.11 mmol) diluted in 5 mL of acetone. After stirring at reflux overnight, the reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 2a (16 mg, 26%, BORSM 47%), 2b (8 mg, 15%, BORSM 25%), 2c (5 mg, 10%, BORSM 16%), and recovered honokiol (22 mg). For 2a, ¹H-NMR (600 MHz, CDCl₃) δ: 7.26–7.25 (m, 1H), 7.23 (d, J = 1.9 Hz, 1H), 7.06 (dd, J = 8.2, 2.1 Hz, 1H), 7.03 (d, J = 1.7 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.05–5.95 (m, 2H), 5.14 (d, J = 1.1 Hz, 1H), 5.13–5.08 (m, 2H), 5.06 (d, J = 10.0 Hz, 1H), 4.09 (q, J = 7.0 Hz, 2H), 3.44 (d, J = 6.7 Hz, 2H), 3.35 (d, J = 6.7 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 156.6, 151, 138, 136.7, 132.3, 130.6, 130.3, 130.1, 128.9, 128.9, 128.0, 128, 116, 115.7, 115.6, 112.0, 63.9, 39.6, 34.6, 15.0; HRMS (EI) m/z: [M] Calcd for C₂₀H₂₂O₂ 294.1620. Found 294.1626. For 2b, ¹H-NMR (600 MHz, CDCl₃) δ: 7.34–7.33 (m, 2H), 7.13 (d, J = 2.2 Hz, 1H) 7.07–7.06 (m, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.09–6.03 (m, 1H), 6.02–5.95 (m, 1H), 5.23–5.16 (m, 2H), 5.11–5.04 (m, 2H), 4.95 (s, 1H), 4.01–3.98 (q, J = 7.0 Hz, 2H), 3.45 (d, J = 6.4 Hz, 2H), 3.37 (d, J = 6.7 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H); ¹³C-NMR (150 MHz, CDCl₃) δ: 154.4, 153.2, 138, 136.7, 132.5, 131.8, 131.5, 131, 130.6, 129.1, 128.1, 124.7, 116.7, 115.7, 115.5, 113.1, 64.3, 39.6, 35.4, 15.0; HRMS (EI) m/z: [M] Calcd for C₂₀H₂₂O₂ 294.1620. Found 293.1638. For 2c, ¹H-NMR (600 MHz, CDCl₃) δ: 7.40–7.36 (m, 2H), 7.14 (d, J = 2.3 Hz, 1H), 7.06 (dd, J = 8.3, 2.3 Hz, 1H), 6.88 (t, J = 8.2 Hz, 2H), 6.07–5.96 (m, 2H), 5.13–5.08 (m, 2H), 5.07–5.03 (m, 2H), 4.08 (q, J = 7.0 Hz, 2H), 4.00 (q, J = 7.0 Hz, 2H), 3.44 (d, J = 6.7 Hz, 2H), 3.37 (d, J = 6.7 Hz, 2H), 1.44 (t, J = 7.0 Hz, 3H), 1.34 (t, J = 7.0 Hz, 3H); ¹³C-NMR (150 MHz, CDCl₃) δ: 155.8, 154.4, 138.0, 137.3, 132.4, 131.2, 131.0, 130.8, 130.7, 128.3, 128.1, 127.9, 115.6, 115.5, 113.0, 111, 64.3, 63.8, 39.6, 34.6, 15.1, 15.0; LRMS (ESI) m/z: Calcd for C₂₂H₂₆O₂ [M + H]⁺ 322.20. Found 323.29.

3′,5-Diallyl-4′-butoxy-[1,1′-biphenyl]-2-ol (3a) and 3,5′-Diallyl-2′-butoxy-[1,1′-biphenyl]-4-ol (3b)

To an acetone solution (2 mL) of honokiol (50 mg, 0.18 mmol) was added Cs₂CO₃ (183 mg, 0.5 mmol), followed by 1-bromobutane (20 µL, 0.18 mmol). After stirring at reflux overnight, the reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford pure 3a (8 mg, 15%, BORSM 28%), 3b (9 mg, 14%, BORSM 26%), 3c (5 mg, 7%, BORSM 13%), and recovered honokiol (23 mg). For 3a, ¹H-NMR (600 MHz, CDCl₃) δ: 7.26–7.23 (m, 1H), 7.22 (d, J = 2.2 Hz, 1H), 7.05 (dd, J = 8.2, 2.2 Hz, 1H), 7.02 (d, J = 2.1 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.04–5.94 (m, 2H), 5.14 (bs, 1H), 5.12–5.03 (m, 4H), 4.02 (t, J = 6.3 Hz, 2H), 3.43 (d, J = 6.8 Hz, 2H), 3.35 (d, J = 6.7 Hz, 2H), 1.84–1.79 (m, 2H), 1.57–1.50 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H); ¹³C-NMR (150 MHz, CDCl₃) δ: 156.9, 151.1, 138.1, 136.9, 132.4, 130.7, 130.5, 130.2, 129.0, 129.0, 128.2, 128.1, 116.1, 115.8, 115.8, 112.0, 68.1, 39.7, 34.8, 31.7, 19.7, 14.2; HRMS (EI) m/z: [M] Calcd for C₂₂H₂₆O₂ 322.1933. Found 322.1932. For 3b, ¹H-NMR (600 MHz, CDCl₃) δ: 7.34 (d, J = 2.1 Hz, 1H), 7.31 (dd, J = 8.2, 2.2 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 7.06 (dd, J = 8.3, 2.3 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.10–6.02 (m, 1H), 6.02–5.88 (m, 1H), 5.22–5.15 (m, 2H), 5.12–5.02 (m, 2H), 3.92 (t, J = 6.4 Hz, 2H), 3.45 (d, J = 6.4 Hz, 2H), 3.36 (d, J = 6.7 Hz, 2H), 1.72–1.64 (m, 2H), 1.47–1.38 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H); HRMS (EI) m/z: [M] Calcd for C₂₂H₂₆O₂ 322.1933. Found 321.1926. For 3c, ¹H-NMR (600 MHz, CDCl₃) δ: 7.37 (d, J = 2.2 Hz, 1H), 7.33 (dd, J = 8.4, 2.3 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.3, 2.3 Hz, 1H), 6.87 (t, J = 8.7 Hz, 2H), 6.00 (m, 2H), 5.11–5.00 (m, 4H), 4.00 (t, J = 6.4 Hz, 2H), 3.92 (t, J = 6.4 Hz, 2H), 3.42 (d, J = 6.7 Hz, 2H), 3.36 (d, J = 6.7 Hz, 2H), 1.83–1.77 (m, 2H), 1.72–1.66 (m, 2H), 1.53 (dd, J = 15.1, 7.5 Hz, 2H), 1.43 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H); ¹³C-NMR (150 MHz, CDCl₃) δ: 159.4, 154.4, 137.9, 137.2, 132.1, 131.1, 130.9, 130.6, 128.1, 127.9, 127.7, 115.4, 115.2, 112.5, 110.6, 68.2, 67.7, 39.5, 34.6, 31.5, 31.4, 29.7, 19.4, 19.4, 13.9, 13.8; LRMS (ESI) m/z: Calcd for C₂₆H₃₄O₂ [M + Na]⁺ 401.25. Found 401.36.

3,5′-Diallyl-4′-(allyloxy)-[1,1′-biphenyl]-2-ol (4a) and 3,5′-Diallyl-2′-(allyloxy)-[1,1′-biphenyl]-4-ol (4b)

To an acetone solution (2 mL) of honokiol (60 mg, 0.22 mmol) was added Cs₂CO₃ (220 mg, 0.67 mmol), followed by allyl bromide (20 µL, 0.22 mmol). After stirring at reflux overnight, the reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 4a (21 mg, 31%, BORSM 46%), 4b (11 mg, 16%, BORSM 24%), 4c (7.5 mg, 9%, BORSM 14%), and recovered honokiol (20 mg). For 4a, ¹H-NMR (600 MHz, CDCl₃) δ: 7.29–7.22 (m, 2H), 7.08–7.00 (m, 3H), 6.94 (d, J = 8.2 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.13–5.99 (m, 1H), 6.03–5.93 (m, 1H), 5.46 (dd, J = 17.3, 1.6 Hz, 1H), 5.31 (dd, J = 10.6, 1.5 Hz, 1H), 5.13–5.04 (m, 5H), 4.60 (d, J = 5.0 Hz, 1H), 3.47 (d, J = 6.7 Hz, 3H), 3.35 (d, J = 6.7 Hz, 3H); ¹³C-NMR (150 MHz, CDCl₃) δ: 156.1, 150.9, 137.9, 136.6, 133.4, 132.3, 130.7, 130.3, 130.2, 129.3, 128.9, 127.9, 127.9, 117.3, 116.0, 115.6, 115.6, 112.3, 69.0, 39.5, 34.6; HRMS (EI) m/z: [M] Calcd for C₂₁H₂₂O₂ 306.1620. Found 306.1614. For 4b, ¹H-NMR (600 MHz, CDCl₃) δ: 7.37 (dd, J = 6.9, 2.0 Hz, 2H), 7.13 (d, J = 2.2 Hz, 1H), 7.07 (dd, J = 8.3, 2.3 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.85 (d, J = 8.9 Hz, 1H), 6.09–5.95 (m, 3H), 5.33 (dd, J = 17.3, 1.7 Hz, 1H), 5.24–5.15 (m, 3H), 5.12–5.04 (m, 2H), 4.96 (bs, 1H), 4.50 (d, J = 4.8 Hz, 2H), 3.46 (d, J = 6.4 Hz, 2H), 3.37 (d, J = 6.7 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 153.9, 153.3, 137.9, 136.6, 133.6, 132.7, 131.8, 131.3, 131.0, 130.8, 129.2, 128.0, 124.7, 116.8, 116.7, 115.7, 115.5, 113.3, 69.4, 39.5, 35.4; HRMS (EI) m/z: [M] Calcd for C₂₁H₂₂O₂ 306.1620. Found 306.1614. For 4c, ¹H-NMR (600 MHz, CDCl₃) δ: 7.44–7.35 (m, 2H), 7.16 (d, J = 2.3 Hz, 1H), 7.09 (dd, J = 8.3, 2.3 Hz, 1H), 6.91 (dd, J = 8.3, 6.2 Hz, 2H), 6.14–5.97 (m, 4H), 5.49–5.46 (m, 1H), 5.38–5.37 (m, 1H), 5.32–5.29 (m, 1H), 5.23–5.21 (m, 1H), 5.14–5.10 (m, 2H), 5.08–5.05 (m, 2H), 4.61–4.60 (m, 2H), 4.53–4.52 (m, 2H), 3.48 (d, J = 6.6 Hz, 2H), 3.39 (d, J = 6.7 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 155.5, 154.0, 137.9, 137.2, 133.8, 133.6, 132.7, 131.3, 131.1, 131.0, 130.9, 128.4, 128, 117.0, 116.9, 115.7, 115.6, 113.3, 111.3, 69.5, 69, 39.6, 34.7; LRMS (ESI) m/z: Calcd for C₂₄H₂₆O₂ [M + Na]⁺ 401.24. Found 401.36.

3′,5-Diallyl-4′-((3-methylbut-2-en-1-yl)oxy)-[1,1′-biphenyl]-2-ol (5a) and 3,5′-Diallyl-2′-((3-methylbut-2-en-1-yl)oxy)-[1,1′-biphenyl]-4-ol (5b)

To an acetone solution (2 mL) of honokiol (40 mg, 0.15 mmol) was added Cs₂CO₃ (146 mg, 0.45 mmol), followed by 3,3-dimethylallyl bromide (22 mg, 0.15 mmol). After stirring at reflux overnight, the reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 5a (25 mg, 50%, BORSM 73%), 5b (1.6 mg, 3%, BORSM 5%), and recovered honokiol (13 mg). For 5a, ¹H-NMR (600 MHz, CDCl₃) δ: 7.26–7.21 (m, 2H), 7.05–7.00 (m, 2H), 6.94 (d, 1H, J = 8.3 Hz), 6.90 (d, 1H, J = 8.2 Hz), 6.04–5.93 (m, 2H), 5.52–5.48 (m, 1H), 5.19 (s, 1H), 5.11–5.03 (m, 4H), 4.57 (d, 2H, J = 6.5 Hz), 3.43 (d, 2H, J = 6.8 Hz), 3.34 (d, 2H, J = 6.7 Hz), 1.80 (s, 3H), 1.75 (s, 3H); ¹³C-NMR (150 MHz, CDCl₃) δ: 156.3, 150.8, 137.8, 137.5, 136.5, 132.1, 130.4, 130.1, 128.7, 127.8, 127.7, 119.9, 115.8, 115.5, 115.4, 112.2, 65.2, 39.4, 34.4, 25.7, 18.3; HRMS (EI) m/z: [M] Calcd for C₂₃H₂₆O₂ 334.1933. Found 334.1935. For 5b, ¹H-NMR (600 MHz, CDCl₃) δ: 7.25–7.20 (m, 2H), 6.92–6.91 (m, 1H), 6.90–6.88 (m, 1H), 6.09–5.91 (m, 3H), 5.37–5.33 (m, 1H), 5.25 (s, 1H), 5.23–5.17 (m, 2H), 5.11–5.06 (m, 2H), 5.05–5.02 (m, 1H), 3.46 (d, 2H, J = 6.4 Hz), 3.37 (d, 2H, J = 7.2 Hz), 3.32 (d, 2H, J = 6.8 Hz), 1.75 (s, 6H); ¹³C-NMR (150 MHz, CDCl₃) δ: 153.9, 149.0, 138.0, 136.2, 133.6, 131.8, 131.4, 130.2, 129.2, 128.9, 128.1, 127.9, 127.7, 126.1, 122.4, 117.0, 116.5, 115.5, 39.6, 35.4, 29.6, 25.9, 18.0; HRMS (EI) m/z: [M] Calcd for C₂₃H₂₆O₂ 334.1933. Found 334.1927.

3′,5-Diallyl-4′-(cyclopropylmethoxy)-[1,1′-biphenyl]-2-ol (6a) and 4-(2-(Cyclopropylmethoxy)-5-(prop-2-en-1-yl)phenyl)-2-(prop-2-en-1-yl)phenol (6b)

To an acetone solution (2 mL) of honokiol (50 mg, 0.18 mmol) was added Cs₂CO₃ (0.15 mg, 0.45 mmol), followed by cyclopropylmethyl bromide (25 mg, 0.18 mmol). After stirring at reflux overnight, the reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 6a (10 mg, 16%, BORSM 40%), 6b (2 mg, 3%, BORSM 8%), 6c (5 mg, 7%, BORSM 18%), and recovered honokiol (30 mg). For 6a, ¹H-NMR (600 MHz, CDCl₃) δ: 7.25 (d, J = 2.4 Hz, 2H), 7.04 (dd, J = 8.2, 2.2 Hz, 1H), 7.02 (d, J = 2.1 Hz, 1H) 6.92–6.89 (m, 2H), 6.06–5.93 (m, 2H), 5.13–5.0.4 (m, 5H), 3.88 (d, 2H, J = 6.7 Hz), 3.46 (d, 2H, J = 6.8 Hz), 3.34 (d, 2H, J = 6.8 Hz), 1.32–1.27 (m, 1H), 0.64–0.63 (m, 2H), 0.38–0.37 (m, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 156.4, 150.8, 137.8, 136.6, 132.1, 130.4, 130.1, 128.8, 128.7, 127.8, 127.8, 115.8, 115.5, 115.4, 112.1, 72.7, 39.4, 34.5, 10.3, 3.0; HRMS (EI) m/z: [M] Calcd for C₂₂H₂₄O₂ 320.1776. Found 320.1774. For 6b, ¹H-NMR (600 MHz, CDCl₃) δ: 7.46–7.32 (m, 2H), 7.13 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.3, 2.3 Hz, 1H), 6.86 (dd, J = 17.2, 8.3 Hz, 2H), 6.1–6.03 (m, 1H), 6.01–5.94 (m, 1H), 5.23–5.22 (m, 1H), 5.17–5.15 (m, 1H), 5.10–5.05 (m, 1H), 5.04 (m, 1H), 4.96 (bs, 1H), 3.77 (d, J = 6.7 Hz, 2H), 3.46 (d, J = 6.4 Hz, 2H), 3.36 (d, J = 6.7 Hz, 2H), 1.20–1.16 (m, 1H), 0.55–0.52 (m, 2H), 0.28–0.25 (m, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 154.4, 153.2, 137.9, 136.6, 132.6, 131.9, 131.4, 130.9, 130.8, 129.1, 128.0, 124.6, 116.6, 115.6, 115.5, 113.8, 73.5, 39.6, 35.4, 10.4, 3.2; HRMS (EI) m/z: [M] Calcd for C₂₂H₂₄O₂ 320.1776. Found 320.1775. For 6c, ¹H-NMR (600 MHz, CDCl₃) δ: 7.44 (s, 1H), 7.39–7.35 (m, 1H), 7.14 (s, 1H), 7.06 (dd, J = 8.2, 1.7 Hz, 1H), 6.90–6.85 (m, 2H), 6.11–6.03 (m, 1H), 6.03–5.95 (m, 1H), 5.16–5.08 (m, 2H), 5.08–5.02 (m, 2H), 3.88 (d, J = 6.6 Hz, 2H), 3.77 (d, J = 6.7 Hz, 2H), 3.48 (d, J = 5.8 Hz, 2H), 3.37 (d, J = 6.5 Hz, 2H), 1.33–1.28 (m, 1H), 1.23–1.16 (m, 1H), 0.63 (d, J = 7.0 Hz, 2H), 0.55 (d, J = 8.0 Hz, 2H), 0.38 (d, J = 4.8 Hz, 2H), 0.28 (d, J = 4.8 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 155.9, 154.5, 138, 137.4, 132.6, 131.3, 131.1, 131.0, 130.9, 128.3, 128.3, 127.9, 115.6, 115.5, 113.7, 111.3, 73.5, 72.7, 39.6, 34.8, 10.6, 10.5, 3.2, 3.2; LRMS (ESI) m/z: Calcd for C₂₆H₃₀O₂ [M + Na]⁺ 397.21. Found 397.38.

3′,5-Diallyl-4′-(cyclobutylmethoxy)-[1,1′-biphenyl]-2-ol (7a) and 3,5′-Diallyl-2′-(cyclobutylmethoxy)-[1,1′-biphenyl]-4-ol (7b)

To an acetone solution (4 mL) of honokiol (140 mg, 0.52 mmol) was added Cs₂CO₃ (0.51 g, 1.6 mmol), followed by cyclobutylmethyl bromide (72 µL, 0.63 mmol). After stirring at reflux overnight, the reaction mixture was diluted with ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1/19 : 1/1) to afford 7a (10 mg, 6%, BORSM 36%), 7b (4.5 mg, 3%, BORSM 16%), 7c (1 mg, 0.5%, BORSM 3%), and recovered honokiol (112 mg). For 7a, ¹H-NMR (600 MHz, CDCl₃) δ: 7.27–7.23 (m, 2H), 7.05 (dd, J = 8.2, 2.2 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.04–5.94 (m, 2H), 5.12–5.04 (m, 5H), 3.97 (d, J = 6.3 Hz, 2H), 3.44 (d, J = 6.8 Hz, 2H), 3.34 (d, J = 6.7 Hz, 2H), 2.86–2.79 (m, 2H), 2.17–2.13 (m, 2H), 2.01–1.92 (m, 4H); ¹³C-NMR (150 MHz, CDCl₃) δ: 156.1, 150.9, 137.9, 136.6, 133.4, 132.3, 130.7, 130.3, 130.2, 129.3, 128.9, 127.9, 127.9, 117.3, 116.0, 115.6, 115.6, 112.3, 69.0, 39.5, 34.6; HRMS (EI) m/z: [M] Calcd for C₂₃H₂₆O₂ 334.1933. Found 334.1935.. For 7b, ¹H-NMR (600 MHz, CDCl₃) δ: 7.35 (d, J = 2.1 Hz, 1H), 7.32 (dd, J = 8.2, 2.2 Hz, 1H), 7.13 (d, J = 2.2 Hz, 1H), 7.06 (dd, J = 8.3, 2.3 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.09–6.01 (m, 1H), 6.00–5.95 (m, 1H), 5.22–5.18 (m, 1H), 5.18–5.16 (m, 1H), 5.10–5.07 (m, 1H), 5.06–5.04 (m, 1H), 4.94 (bs, 1H), 3.88 (d, J = 6.3 Hz, 2H), 3.45 (d, J = 6.3 Hz, 2H), 3.36 (d, J = 6.7 Hz, 2H), 2.74–2.66 (m, 1H), 2.06–2.00 (m, 2H), 1.94–1.78 (m, 4H); ¹³C-NMR (150 MHz, CDCl₃) δ: 154.6, 153.3, 138.0, 136.7, 132.4, 132, 131.5, 130.9, 130.6, 129.2, 128.0, 124.5, 116.7, 115.6, 115.5, 113, 72.7, 39.6, 35.6, 34.9, 25, 18.6.; HRMS (EI) m/z: [M] Calcd for C₂₃H₂₆O₂ 334.1933. Found 334.1940.

2-((3,5′-Diallyl-2′-hydroxy-[1,1′-biphenyl]-4-yl)oxy)-1-(4-fluorophenyl)ethan-1-one (8a) and 2-((3′,5-Diallyl-4′-hydroxy-[1,1′-biphenyl]-2-yl)oxy)-1-(4-fluorophenyl)ethan-1-one (8b)

To a Tetrahydrofuran solution (4 mL) of honokiol (140 mg, 0.52 mmol) was added Cs₂CO₃ (0.51 g, 1.6 mmol), followed by 4-fluorophenacyl bromide (0.14 g, 0.62 mmol). After stirring 1 h at ambient temperature, the reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 8a (67 mg, 32%, BORSM 40%), 8b (21 mg, 10%, BORSM 12%), 8c (50 mg, 18%, BORSM 22%), and recovered honokiol (27 mg). For 8a, ¹H-NMR (600 MHz, CDCl₃) δ: 8.09–8.06 (m, 2H), 7.27 (d, J = 2.2 Hz, 1H), 7.24 (dd, J = 8.3, 2.3 Hz, 1H), 7.21–7.16 (m, 2H), 7.05 (dd, J = 8.2, 2.1 Hz, 1H), 7.00 (d, J = 2.1 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 6.04–5.93 (m, 2H), 5.27 (s, 2H), 5.10–5.04 (m, 5H), 3.50 (d, J = 6.6 Hz, 2H), 3.34 (d, J = 6.7 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 193.4, 155.5, 150.9, 137.9, 136.5, 132.4, 131.3, 131.2, 131.2, 130.3, 129.1, 128.1, 127.7, 116.3, 116.3, 116.2, 115.8, 115.7, 112.2, 71.3, 39.5, 34.5.; HRMS (EI) m/z: [M] Calcd for C₂₆H₂₃FO₃ 402.1631. Found 402.1635. For 8b, ¹H-NMR (600 MHz, CDCl₃) δ: 7.95–7.92 (m, 2H), 7.33–7.30 (m, 2H), 7.09–7.01 (m, 4H), 6.83 (dd, J = 8.3, 5.4 Hz, 2H), 6.04–5.97 (m, 2H), 5.10–5.06 (m, 5H), 3.41 (d, J = 6.4 Hz, 2H), 3.36 (d, J = 6.8 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 193.8, 166.9, 165.2, 153.4, 153.3, 137.5, 136.4, 131.7, 131.3, 131.2, 131.1, 129.1, 128.0, 116.6, 115.9, 115.8, 115.8, 115.5, 113.4, 71.9, 39.4, 35.3. For 8c, ¹H-NMR (600 MHz, CDCl₃) δ: 8.11–8.05 (m, 2H), 7.95–7.90 (m, 2H), 7.36–7.34 (m, 2H), 7.20–7.15 (m, 2H), 7.12 (d, J = 2.2 Hz, 1H), 7.10–7.03 (m, 3H), 6.83 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 9.0 Hz, 1H), 6.01–5.92 (m, 2H), 5.22 (s, 2H), 5.09 (s, 2H), 5.07–5.00 (m, 4H), 3.44 (d, J = 6.6 Hz, 2H), 3.35 (d, J = 6.7 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 193.8, 193.7, 155.0, 153.4, 137.6, 136.9, 133.9, 131.7, 131.4, 131.3, 131.2, 131.0, 128.7, 128.3, 116.2, 116.0, 115.9, 115.9, 115.8, 113.4, 111.2, 72.0, 71.5, 39.5, 34.5; LRMS (ESI) m/z: Calcd for C₃₄H₂₈F₂O₂ [M + Na]⁺ 561.18. Found 561.32.

2-((3,5′-Diallyl-2′-hydroxy-[1,1′-biphenyl]-4-yl)oxy)-1-(2,4-dichlorophenyl)ethan-1-one (9a) and 2-((3′,5-Diallyl-4′-hydroxy-[1,1′-biphenyl]-2-yl)oxy)-1-(2,4-dichlorophenyl)ethan-1-one (9b)

To an acetone solution (4 mL) of honokiol (140 mg, 0.52 mmol) was added Cs₂CO₃ (0.51 g, 1.6 mmol), followed by 4-nitrophenacyl bromide (77 µL, 0.62 mmol). After stirring for 1 h at ambient temperature, the reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 9a (14 mg, 6%, BORSM 17%), 9b (14 mg, 6%, BORSM 17%), 9c (23 mg, 7%, BORSM 19%), and recovered honokiol (64 mg). For 9a, ¹H-NMR (600 MHz, CDCl₃) δ: 7.56 (d, J = 8.3 Hz, 1H), 7.48 (s, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.24 (s, 1H), 7.05 (d, J = 7.9 Hz, 1H), 7.00 (s, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 5.96–5.85 (m, 3H), 5.18 (s, 2H), 5.09–4.99 (m, 4H), 3.35 (t, J = 7.3 Hz, 4H); ¹³C-NMR (150 MHz, CDCl₃) δ: 197.5, 155.2, 150.9, 138.6, 137.9, 136.3, 134.9. 132.9, 132.4, 131.2, 130.5, 130.4, 130.3, 130.2, 129.1, 128.1, 127.7, 127.6, 116.1, 115.8, 115.7, 112.0, 73.1, 39.5, 34.4; HRMS (EI) m/z: [M] Calcd for C₂₆H₂₂Cl₂O₃ 452.0946. Found 452.0947. For 9c, ¹H-NMR (600 MHz, CDCl₃) δ: 7.54 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 1.9 Hz, 1H), 7.38–7.33 (m, 2H), 7.29 (d, J = 1.9 Hz, 1H), 7.22–7.16 (m, 3H), 7.08–7.04 (m, 2H), 6.79 (d, J = 8.2 Hz, 1H), 6.64 (d, J = 8.8 Hz, 1H), 5.99–5.92 (m, 1H), 5.89–5.82 (m, 1H), 5.13 (s, 2H), 5.10–5.04 (m, 2H), 4.99 (s, 2H), 4.97–4.91 (m, 2H), 3.35 (d, J = 6.7 Hz, 2H), 3.27 (d, J = 6.6 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 198.25, 197.92, 154.56, 153.13, 138.38, 138.02, 137.60, 136.73, 135.10, 135.04, 133.83, 132.85, 132.74, 131.57, 131.46, 131.38, 131.12, 130.98, 130.90, 130.43, 130.16, 128.46, 128.22, 127.57, 127.32, 115.94, 115.60, 112.95, 110.57, 73.68, 73.00, 39.49, 34.48; LRMS (ESI) m/z: Calcd for C₃₄H₂₆Cl₄O₄ [M + Na]⁺ 661.04. Found 661.25.

2-((3,5′-Diallyl-2′-hydroxy-[1,1′-biphenyl]-4-yl)oxy)-1-(4-nitrophenyl)ethan-1-one (10a)

To a tetrahydrofuran solution (2 mL) of honokiol (50 mg, 0.18 mmol) was added Cs₂CO₃ (183 mg, 0.56 mmol), followed by 2-bromo-4′-nitroacetophenone (46 mg, 0.18). After stirring 1 h at room temperature, the reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 10a (35 mg, 43%, BORSM 54%), 10c (10 mg, 9%, BORSM 11%), and recovered honokiol (10 mg). ¹H-NMR (600 MHz, CDCl₃) δ: 8.37–8.34 (m, 2H), 8.21–8.18 (m, 2H), 7.28 (dd, 2H, J = 5.1, 2.1 Hz), 7.05 (dd, 1H, J = 8.2, 2.2 Hz), 7.01 (d, 1H, J = 2.1 Hz), 6.88 (dd, 2H, J = 14.2, 8.2 Hz), 6.00–5.94 (m, 2H), 5.30 (s, 2H), 5.10–5.02 (m, 5H), 3.47 (d, 2H, J = 6.6 Hz), 3.34 (d, 2H, J = 6.7 Hz); ¹³C-NMR (150 MHz, CDCl₃) δ: 194.0, 155.1, 150.8, 150.8, 139.1, 137.8, 136.3, 132.4, 131.4, 130.9, 130.3, 130.2, 129.7, 129.1, 128.1, 127.5, 124.1, 116.3, 115.8, 115.7, 112.0, 71.6, 39.5, 34.4; HRMS (EI) m/z: [M] Calcd for C₂₆H₂₃NO₅ 429.1576. Found 429.1574. For 10c, ¹H-NMR (600 MHz, CDCl₃) δ: 8.24 (dd, J = 8.9, 1.7 Hz, 2H), 7.98 (dd, J = 9.0, 1.0 Hz, 2H), 7.80 (dd, J = 9.0, 1.1 Hz, 2H), 7.42 (dd, J = 9.0, 2.1 Hz, 1H), 7.20 (d, J = 2.0 Hz, 1H), 7.16–7.11 (m, 1H), 7.08–7.00 (m, 1H), 6.86–6.76 (m, 1H), 5.99–5.88 (m, 1H), 5.09–4.98 (m, 2H), 4.82 (d, J = 15.0 Hz, 1H), 4.57 (d, J = 10.8 Hz, 1H), 4.23–4.18 (m, 1H), 3.90 (dd, J = 9.2, 6.9 Hz, 1H), 3.34 (dd, J = 19.1, 7.0 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 209.8, 155.2, 153.5, 151.1, 147.5, 147.1, 137.6, 136.9, 133.6, 131.9, 131.7, 131.2, 130.9, 128.8, 128.6, 126.6, 126.5, 123.7, 123.3, 116, 113.2, 111.0, 75.0, 74.6, 49.3, 39.5; LRMS (ESI) m/z: Calcd for C₃₄H₂₈O₂ [M + H]⁺ 593.19. Found 593.35.

tert-Butyl 2-((3,5′-diallyl-2′-hydroxy-[1,1′-biphenyl]-4-yl)oxy)acetate (11a) and tert-Butyl 2-((3′,5-diallyl-4′-hydroxy-[1,1′-biphenyl]-2-yl)oxy)acetate (11b)

To a tetrahydrofuran solution (2 mL) of honokiol (200 mg, 0.75 mmol) was added Cs₂CO₃ (245 mg, 0.75 mmol), followed by tert-butyl bromoacetate (220 mg, 1.13). After stirring 1 h at ambient temperature, the reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 11a (107 mg, 37%, BORSM 48%), 11b (36 mg, 13%, BORSM 16%), 11c (97 mg, 26%, BORSM 33%), and recovered honokiol (43 mg). For 11a, ¹H-NMR (600 MHz, CDCl₃) δ: 7.24 (d, 2H, J = 2.3 Hz), 7.06–7.04 (m, 1H), 7.02 (d, 1H, J = 2.2 Hz), 6.90 (d, 1H, J = 8.2 Hz), 6.80 (d, 1H, J = 8.0 Hz), 6.01 (dd, 2H, J = 44.3, 6.9 Hz), 5.14–5.04 (m, 5H), 4.58 (s, 2H), 3.51 (d, 2H, J = 6.7 Hz), 3.35 (d, 2H, J = 6.7 Hz), 1.51 (s, 9H); ¹³C-NMR (150 MHz, CDCl₃) δ: 167.9, 155.4, 150.8, 137.7, 136.3, 132.1, 130.8, 130.2, 130.1, 129.9, 128.8, 127.7, 127.6, 116.0, 115.5, 115.5, 111.8, 82.4, 65.9, 39.4, 34.3, 28.1; HRMS (EI) m/z: [M] Calcd for C₂₄H₂₈O₄ 380.1988. Found 380.1987. For 11b, ¹H-NMR (600 MHz, CDCl₃) δ: 7.40 (dd, J = 8.2, 2.2 Hz, 1H), 7.36 (d, J = 2.1 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H), 7.06 (dd, J = 8.3, 2.2 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.10–6.03 (m, 1H), 6.00–5.93 (m, 1H), 5.23–5.20 (m, 1H), 5.18–5.16 (m, 1H), 5.10–5.04 (m, 3H), 5.01 (bs, 1H), 4.46 (s, 2H), 3.46 (d, J = 6.4 Hz, 2H), 3.37 (d, J = 6.7 Hz, 2H), 1.48 (s, 9H); ¹³C-NMR (150 MHz, CDCl₃) δ: 168.5, 153.5, 153.3, 137.7, 136.7, 133.4, 131.6, 131.3, 130.8, 130.8, 129.2, 127.8, 125.0, 116.4, 115.7, 115.5, 112.8, 82.3, 66.5, 39.5, 35.3, 28.1; HRMS (EI) m/z: [M] Calcd for C₂₄H₂₈O₄ 380.1988. Found 380.1986. For 11c, ¹H-NMR (600 MHz, CDCl₃) δ: 7.44 (dd, J = 8.4, 2.3 Hz, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.13 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.3, 2.3 Hz, 1H), 6.77 (dd, J = 10.7, 8.4 Hz, 2H), 6.11–6.04 (m, 1H), 6.00–5.94 (m, 1H), 5.14–5.03 (m, 4H), 4.55 (s, 2H), 4.44 (s, 2H), 3.51 (d, J = 6.7 Hz, 2H), 3.36 (d, J = 6.7 Hz, 2H), 1.51 (s, 9H), 1.46 (s, 9H); ¹³C-NMR (150 MHz, CDCl₃) δ: 168.3, 168.3, 155.0, 153.4, 137.8, 137.1, 133.4, 131.5, 131.4, 131.4, 130.8, 128.8, 128.5, 128, 115.8, 115.6, 112.9, 111.1, 82.3, 82.2, 66.5, 66.4, 39.5, 34.6, 28.2, 28.2; LRMS (ESI) m/z: Calcd for C₃₀H₃₈O₆ [M + Na]⁺ 517.25. Found 517.37.

2-((3,5′-Diallyl-2′-hydroxy-[1,1′-biphenyl]-4-yl)oxy)acetic Acid (12)

To a CH₂Cl₂ solution (3.0 mL) of tert-butyl acetate (11a) (50 mg, 0.13 mmol) was added trifluoroacetic acid (1 mL). After stirring for 2 h at ambient temperature, the reaction mixture was concentrated under reduced pressure to afford crude the carboxylic acid (12) (45 mg, 95%). ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.25 (s, 1H), 7.30 (d, J = 8.5 Hz, 1H), 6.99 (s, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 6.05–5.97 (m, 1H), 5.97–5.90 (m, 1H), 5.12–4.98 (m, 6H), 4.69 (s, 2H), 3.39 (d, J = 6.6 Hz, 2H), 3.33 (s, 1H), 3.28 (d, J = 6.6 Hz, 2H). ¹³C-NMR (150 MHz, DMSO-d₆) δ: 170.4, 154.3, 152.4, 138.3, 137, 131.2, 130.4, 130.2, 130.1, 127.8, 127.8, 127.4, 127.3, 115.9, 115.6, 115.3, 111.1, 65.0, 38.8, 34.0; HRMS (EI) m/z: [M] Calcd for C₂₀H₂₀O₄ 324.1362. Found 324.1362.

N-(2-(2-(2-(2-(4-((4-(4-(3-(2-((3,5′-Diallyl-2′-hydroxy-[1,1′-biphenyl]-4-yl)oxy)acetamido)propoxy)benzoyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (14)

To a CH₂Cl₂ solution (3.0 mL) of tert-butyl acetate (12b) (23 mg, 0.05 mmol) was added trifluoroacetic acid (0.3 mL). After stirring for 2 h at ambient temperature, the reaction mixture was concentrated under reduced pressure to afford crude the carboxylic acid (13) (20 mg, 98%). To a dimethylformamide (DMF) solution (3 mL) of carboxylic acid (13) (50 mg, 0.15 mmol) were added HOAt (63 mg, 0.46 mmol), PyAOP (161 mg, 0.308 mmol), and N,N-diisopropylethylamine (DIPEA) (0.22 mL, 1.26 mmol). After stirring for 24 h, the reaction mixture was diluted in ethyl acetate, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (MeOH : CH₂Cl₂ = 1 : 10) to afford biotin probe (14) (25 mg, 20%, BORSM 31%) and recovered starting material 13 (18 mg). ¹H-NMR (600 MHz, CDCl₃) δ: 9.16 (s, 1H), 7.84 (s, 1H), 7.72–7.67 (m, 4H), 7.41 (dd, 1H, J = 8.4, 2.1 Hz), 7.37 (d, 1H, J = 1.7 Hz), 7.01 (dd, 2H, J = 10.5, 2.9 Hz), 6.98 (s, 1H), 6.91–6.85 (m, 4H), 6.80–6.77 (m, 1H), 6.72 (d, 1H, J = 4.6 Hz), 6.28 (s, 1H), 5.95–5.86 (m, 2H), 5.57 (s, 1H), 5.23 (s, 2H), 5.03–4.94 (m, 4H), 4.52 (t, 2H, J = 4.8 Hz), 4.49 (s, 1H), 4.42–4.38 (m, 1H), 4.23–4.19 (m, 1H), 4.00 (t, 2H, J = 5.6 Hz), 3.84 (t, 2H, J = 4.8 Hz), 3.56–3.54 (m, 2H), 3.52 (d, 8H, J = 1.2 Hz), 3.47 (t, 2H, J = 4.9 Hz), 3.37 (d, 2H, J = 5.9 Hz), 3.35–3.33 (m, 2H), 3.26 (d, 2H, J = 6.6 Hz), 3.07–2.97 (m, 2H), 2.82–2.78 (m, 1H), 2.66 (d, 1H, J = 12.7 Hz), 2.13 (dd, 2H, J = 9.7, 4.9 Hz), 2.05–2.00 (m, 2H), 1.69–1.53 (m, 6H); ¹³C-NMR (150 MHz, CDCl₃) δ: 194.3, 173.3, 168.8, 163.8, 162.0, 161.4, 153.9, 152.9, 143.2, 138.1, 137.5, 132.9, 132.2, 132.2, 131.8, 131.1, 130.6, 130.5, 130.2, 128.7, 128.2, 127.6, 127.3, 124.3, 116.2, 115.3, 115.2, 114.3, 114.0, 111.3, 70.5, 70.3, 70.3, 70.0, 69.8, 69.4, 67.5, 65.6, 62.0, 61.7, 60.2, 55.5, 50.3, 40.5, 39.4, 39.1, 36.2, 35.8, 35.1, 28.9, 28.2, 28.1, 25.5; HRMS (ESI/QTOF) m/z: [M + Na] Calcd for C₅₇H₆₉N₇NaO₁₁S 1082.4668. Found 1082.4673.

Biological Assays

Cell Viability Assay

The cell viability was measured using soluble MTT (Sigma, Cat. No. M5655) that converts into insoluble purple formazan by metabolic enzymes in live cells. 5 × 104 RAW264.7 cells were seeded in 96 well plates and treated with various concentration of honokiol derivatives. Following 24 h incubation, the MTT solution were added to each well and additionally incubated at 37°C for 2 h. Then, the supernatant was removed and the formazan was dissolved with DMSO (Sigma, D-2650). The quantitative absorbance was measured at 540 nm with enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, SpectraMax M2).

Nitric Oxide Production Assay

To observe the production of NO, 4 × 105 RAW264.7 cells were seeded and activated by LPS (InVivogen, Cat. No. tlrl-eklps) in 24 well plates. Consistent with LPS activation, the cells also treated with honokiol derivatives and incubated for 24 h at 37°C. After 24 h incubation, the supernatant was transferred to 96-well plate and Griess reagent A (1% Sulfanilamide, Sigma, Cat. No. S9251; 2% Phosphoric acid, Sigma, Cat. No. 345245) and Gries reagent B (N-(1-Naphthyl) ethylenediamine dihydrochloride (NED), TCI, Cat. No. N0063) were sequentially incubated for 20 min each at room temperature. The NO concentration was determined by absorbance at 540 nm using ELISA reader (Molecular Devices, SpectraMax M2).

Western Blotting

2.5 × 106 RAW264.7 cells were stimulated with LPS (10 ng/mL or 100 ng/mL) (InvivoGen, Cat. No. 13106MM) with or without honokiol derivatives (5 µM) respectively and lysed with a cold lysis buffer containing 50 mM Tris–HCl (Amresco, Cat No. 0826), 2 mM EDTA (Biosesang, Cat No. E2002), 1% NP-40 (Millipore, Cat. No. 492016), protease inhibitors (Roche, Cat No. 11836170001), and PhoSTOP (Roche, Cat. No. 04906837001). Lysates were clearly collected and electrophoresed by SDS-PAGE and transferred to PVDF blotting membrane (GE Healthcare, Cat. No. 10600023). The membrane was incubated with rabbit anti-COX-2 antibody (Cell Signaling Technology, Cat. No. 4842), mouse anti-iNOS antibody (BD Biosciences, Cat. No. 610329), mouse anti-β-actin antibody (Santa Cruz Biotechnology, Cat. No sc-47778) respectively, followed by detection using the corresponding horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Cat. No. 115-036-003), goat anti-rabbit IgG (Bio-Rad, Cat. No. 170-6515) respectively, and enhanced chemiluminescence (ECL) (Bio-Rad, Cat. No. 170-0561). The membranes were scanned with a Fusion Solo chemiluminescence imaging system (Vilber).

Acknowledgments

This work was supported by the Gachon University research fund of 2017 (GCU-2018-0666) and grant from the National Research Foundation of Korea grant funded by the Korean government (Grant number: NRF-2016R1D1A1A09917300).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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