Design and Synthesis of Cajanine Analogues against Hepatitis C

Article pubs.acs.org/jmc

Design and Synthesis of Cajanine Analogues against Hepatitis C Virus through Down-Regulating Host Chondroitin Sulfate N‑Acetylgalactosaminyltransferase 1 Xing-Yue Ji,† Jin-Hua Chen,† Guang-Hui Zheng, Meng-Hao Huang, Lei Zhang, Hong Yi, Jie Jin, Jian-Dong Jiang, Zong-Gen Peng,* and Zhuo-Rong Li* Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1, Tiantan Xili, Beijing 100050, China S Supporting Information *

ABSTRACT: There still remains a need to develop new anti-HCV agents with distinct mechanism of action (MOA) due to the occurrence of resistance to direct-acting antiviral agents (DAAs). Cajanine, a stilbenic component isolated from Cajanus cajan L., was identified as a potent HCV inhibitor by phenotypic screening in this work (EC50 = 3.17 ± 0.75 μM). The intensive structure optimization provided significant insights into the structure−activity relationships. Furthermore, the MOA study revealed that cajanine inhibited HCV replications via down-regulating a cellular protein chondroitin sulfate N-acetylgalactosaminyltransferase 1. In consistency with this host-targeting mechanism, cajanine showed the similar magnitude of inhibitory activity against both drug-resistant and wild-type HCV and synergistically inhibited HCV replication with approved DAAs. Taken together, our study not only presented cajanine derivatives as a novel class of anti-HCV agents but also discovered a promising anti-HCV target to combat drug resistance.

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INTRODUCTION Hepatitis C virus (HCV) infection is a major health problem, with as many as 200 million individuals infected worldwide.1 Approximately 20% of infected individuals would progress to cirrhosis, and 2−4% of the patient with cirrhosis are at risk of irreversible hepatic failure. In recent years, the therapy of HCV has been revolutionized with the approval of direct-acting antiviral agents (DAAs),2 especially with sofosbuvir, a nucleotide analogue inhibitor of HCV NS5B polymerase.3 Recently, sofosbuvir combined with ledipasvir (NS5A inhibitor) was approved by FDA under the brand name of Harvoni to treat patients with genotype 1 chronic hepatitis C. This is also the first approved regimen that is all oral and IFN- or RBVfree. Undoubtedly, sofosbuvir will be the new backbone of HCV treatment, and the overall therapeutic effect in HCVinfected patients will be continuously improved. However, challenges and issues still remain, including drug resistance,4 high cost,5 narrow genotype specificity,6 and the treatment of genotype 3 infections.7 It is still imperative for the development of new anti-HCV agents, especially the ones with novel mechanism of action (MOA). © 2016 American Chemical Society

Compared to DAAs, host-targeting antivirals (HTAs) have an advantage of no or decreased chance to induce drugresistant mutations.8 There are a broad range of HTAs being developed in preclinical or clinical trials, including entry inhibitors, translation inhibitors, replication inhibitors, and assembly inhibitors,9 and some promising results have been observed among these HTAs, especially in terms of higher barrier to resistance and synergistic effect with DAAs to reduce viral load. For example, alisporivir (ALV),10 a cyclophilin inhibitor, is effective against all DAAs-resistant HCV genotype 1b variants, with the half-maximal effective concentration (EC50) values in the range of 15.8−46.7 nM. In addition, the combination of ALV with daclatasvir (NS5A inhibitor) constantly showed synergistic effects on GT-2a and -3a HCV. In summary, it is recognized that an optimal HCV therapy regimen might be the combined treatment of several anti-HCV agents targeting different steps of HCV lifecycle.11 Because HCV infection requires the synergic action of viral and host Received: August 29, 2016 Published: October 26, 2016 10268

DOI: 10.1021/acs.jmedchem.6b01301 J. Med. Chem. 2016, 59, 10268−10284

Journal of Medicinal Chemistry

Article

Figure 1. 1 inhibited HCV replication in a dose-dependent manner in Huh7.5 cells. (A) Structure of 1. (B,C) 1 inhibited HCV replication at HCV RNA (B) and protein (C) level with no cytotoxicity (B, MTT assay). Intracellular HCV RNA and core protein were detected after 72 h treatment with 1. Data were normalized to the solvent control, which was regarded as 100%. *P < 0.05 and **P < 0.01 vs solvent control.

factors, it is anticipated that the combination of DAAs and HTAs would improve the overall therapeutic effect and reduce the occurrence of DAAs resistance in clinical practice. Natural products are always a good source for the discovery of novel lead compounds. Of the 877 small-molecule new chemical entities introduced between 1981 and 2002, nearly half (49%) of them were derived from natural products.12 Cajanine (also known as cajaninstilbene acid), a stilbene derivative isolated from the herb Cajanus cajan L., has recently drawn much attention due to its broad range of bioactivities, including antitumor,13 anti-inflammatory,14 antiosteoporotic,15 antioxidant,16 antibacterial,17 cytoprotective,18 and neuroprotective effects.19 Previously, we have reported the total synthesis of cajanine (1, Figure 1A).13 Given that there is a huge unmet need for effective and affordable therapies for HCV infections, 1 was subjected to a cell based phenotype anti-HCV screening in our lab and it was identified as a good inhibitor against HCV replication with an EC50 value of 3.17 ± 0.75 μM and no decrease in cell viability was observed throughout the experiments (Figure 1B,C). However, as shown in Figure 1A, 1 is extremely lipophilic with a calculated AlogP value of 5.0 (Discovery Studio 3.0),20 which is likely to raise poor physicochemical properties concern. Consequently, structural simplification was made to probe the importance of the two hydrophobic moieties (prenyl and phenyl ring B) for anti-HCV activity. In addition, intensive structure modifications were also conducted, and some significant structure−activity relationships were concluded herein. Furthermore, the MOA of 1 was also studied, and the results revealed that 1 inhibited HCV replication via a distinct mechanism, which involved a cellular protein chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). To our knowledge, the role of CSGalNAcT-1 in HCV replication has not yet been reported.

Figure 2. Indicated structural modifications to compound 1.

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RESULTS AND DISCUSSION Chemistry. Class I: Compound 13 was synthesized according to our previous reported method.13 As shown in Scheme 1, compound 13 was condensed with various ketones or aldehydes in the presence of NaH to yield compounds 14a− k. Selective demethylation with BCl3 of compounds 14a−k afforded 15a−k in high yield. The sodium salt of compounds 15a−k were condensed with prenyl bromide in anhydrous toluene to afford the desired C-alkylation products 2a−2k, and the O-alkylation products were also unavoidably formed at this step, which contributed to the moderate yield of the Calkylation products (50%−60%). Compound 2k was further deprotected in the presence of TBAF to give compound 2l with a free hydroxyl group. Compounds 2a−j and 2l were then hydrolyzed to form the target compounds 1 and 2m−v. Longistyline A (3) was also obtained when microwave irradiation was employed in the hydrolysis of 1. Class II: As shown in Scheme 2, compound 15a was hydrogenated to afford compound 16 in high yield. The sodium salt of compound 16 was reacted with prenyl bromide to yield the C-alkylation product 4 along with its O-alkylation counterpart. The hydrolysis of 4 afforded the target compound 5 (also known as amorfrutin A21) with the stilbenic double bond reduced. Class III: Given that 1, 14a, 15a, and 2a possessed an olefinic double bond in their structures, where the addition reaction could probably take place on the double bond along with the chlorination of ring A,22 compound 1713 was then chosen as the starting material to react with SO2Cl2 in diethyl ether to afford the chlorination product 18 (Scheme 3). Compound 20 was obtained after the bromination and Arbuzov reactions. The synthesis of compound 6 was straightforward by a similar method used to synthesize compounds of class I (Scheme 1). Longistyline C (7), a natural occurring analogue of 1, was also synthesized for the first time in this work. As shown in Scheme 4, compound 14a was brominated to afford compound 24, which was then reacted with tributylprenylstannane under

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DESIGN As shown in Figure 2, the structural modifications to 1 were primarily made to the indicated regions I, II, III, IV, and V, aiming to uncover the SARs at these positions. To address the hydrophobic issue of 1, compounds without ring B (2t, ALogP = 4.5) or prenyl (15a, ALogP = 3.4) were designed and synthesized. Meanwhile, the other two major stilbene compounds isolated from Cajanus cajan L., namely longstyline A and C, were also synthesized and assayed for their inhibitory activity against HCV herein. 10269



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Scheme 1. Synthesis of Class I Analoguesa

a

Reagents and conditions: (a) NaH, ketones or aldehydes, dry Dimethoxyethane (DME), N2, reflux, 1.5 h, 70−85%. R1: phenyl, 2-chlorophenyl, 2methylphenyl, 3-methylphenyl, 4-methylphenyl, 4-fluorophenyl, styryl, thienyl, ethyl, 4-OTBDMS-phenyl, or 4-OH-phenyl. R2: H (14a−i,k), or methyl (14j). (b) BCl3, −78 °C, CH2Cl2, 2 h, 85−95%. R1: phenyl, 2-chlorophenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 4fluorophenyl, styryl, thienyl, ethyl, 4-OTBDMS-phenyl, or 4-OH-phenyl. R2: H (15a−i,k), or methyl (15j). (c) NaH, dry toluene, prenyl bromide, 40 °C, 2 h, 50−60%. (d) TBAF, THF, rt, 2 h, 90%. (e) KOH, EtOH/H2O, reflux, 2 h, 90% (or KOH/EtOH, McW, 30 W, 100 °C, 40 psi, 0.5 h, 80%, for compound 3).

Scheme 2. Synthesis of Derivative 5 with the Stilbenic Double Bond Reduceda

Reagents and conditions: (a) H2, Pd/C (10%), EtOH, 50 psi, 4 h, 95%; (b) NaH, toluene, prenyl bromide, 40 °C, 4 h, 63%; (c) KOH, EtOH/H2O, reflux, 2 h, 92%.

a

Scheme 3. Synthesis of Class III Analoguea

Reagents and conditions: (a) SO2Cl2, ether, reflux, 4 h, 85%; (b) Br2, CCl4, hν, reflux, 3 h, 85%; (c) P(OEt)3, 150 °C, 2 h, 90%; (d) PhCHO, NaH, dry DME, reflux, 2 h, 70%; (e) BCl3, CH2Cl2, −78 °C, 2 h, 80%; (f) NaH, toluene, prenyl bromide, 40 °C, 4 h, 50%; (g) KOH, EtOH/H2O, reflux, 2 h, 90%. a

the conditions of Stille coupling reaction to give compound 25 in moderate yield. Selective demethylation of compound 25 in

the presence of BCl3 afforded compound 26 in 85% yield. The hydrolysis and decarboxylation of compound 26 were 10270



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Scheme 4. Synthesis of Longistyline Ca

Scheme 6. Synthesis of the Analogue with a Reduced Olefinic Double Bonda

a

Reagents and conditions: (a) H2, Pd/C, EtOH, 70 psi, 4 h, 95%.

Biology. Anti-HCV Activity in Vitro. The infectious virus J6/ JFH/JC-1 (a recombinant HCV genotype 2a) was employed to screen the HCV inhibition activity of the synthesized cajanine analogues in Huh7.5 cells. In the HCVcc system, HCV can propagate with complete life cycle including an early stage of the HCV life cycle. The Huh7.5 cells were infected with HCV viral stock (45 IU/cell) and treated simultaneously with test compounds or the positive control telaprevir. The intracellular HCV RNA was extracted and quantified with one-step qRTPCR. The cytotoxicity was determined using MTT assay. The EC50 and CC50 values were calculated with Reed & Muench methods. Class I derivatives were designed to probe the importance of ring B for anti-HCV activity. As shown in Table 1, most of the compounds exhibited definitive anti-HCV activity with selectivity index (SI) values higher than 10 in the HCVcc system and several derivatives exhibited more potent activity as compared to 1, especially compound 2v, which showed the most potent inhibitory activity among this series with an EC50 value of 0.33 ± 0.10 μM. In terms of SAR, the carboxylic derivatives were generally more potent than their esterified counterparts, and hence, a hydrogen bond donor or ionic interaction at this position may increase the inhibitory activity. On the other hand, the esterified compounds were less cytotoxic compared to their carboxylic counterparts. Replacing the proton at the para position of ring B with a hydroxyl group (2v) significantly increased the inhibitory activity. Substituting a chloro (2m) or fluoro (2q) group for the proton (1) at the

Reagents and conditions: (a) MeOH, Br2, 0 °C, 2 h, 90%; (b) Pd(Ph3P)4, DMF, tributyl(3-methylbut-2-enyl)stannane, 110 °C, 12 h, 50%; (c) BCl3, CH2Cl2, −78 °C, 2 h, 85%; (d) KOH/EtOH, Mcw, 30 W, 100 °C, 30 psi, 2 h, 70%.

a

accomplished in one step using microwave irradiation, and compound 7 was obtained with an overall yield of 27%. Class IV: As shown in Scheme 5, dimethylation of compound 2723 followed by bromination and condensation with triethyl phosphite yielded compound 30. Under the conditions of Stille coupling reaction, compound 31 was obtained in moderate yield. The target compound 9 was then obtained through a series of reactions including hydrogenation, condensation with benzaldehyde, selective demethylation, and hydrolysis. Meanwhile, compound 10 with all olefinic double bond reduced was also synthesized by hydrogenation of 1, as shown in Scheme 6. Additionally, as shown in Scheme 7, compound 11 with (E)3,7-dimethylocta-2,6-dienyl (geranyl) substituent at 3-position of ring A was also synthesized by a similar method used for the synthesis of 1 (Scheme 1). Class V: The carboxyl group in 1 was esterified or amidated by using N,N′-diisopropylcarbodiimide (DIC) as the coupling reagent. In the case of esterification, the alcohols were used both as reactant and solvent. Eventually, the target compounds 12a−d were obtained in 65−75% yield (Scheme 8).

Scheme 5. Synthesis of the Analogue with a Reduced Prenylic Double Bonda

Reagents and conditions: (a) MeI, K2CO3, DMF, 60 °C, 2 h, 85%; (b) Br2, hν, CCl4, reflux, 2 h, 85%; (c) P(OEt)3, 150 °C, reflux, 6 h, 90%; (d) Pd(Ph3P)4, tributyl(3-methylbut-2-enyl)stannane, DMF, 110 °C, 12 h, 70%; (e) H2, Pd/C (10%), EtOH, 50 psi, 4 h, 95%; (f) NaH, PhCHO, dry DME, reflux, 2 h, 84%; (g) BCl3, CH2Cl2, −78 °C, 2 h, 85%; (h) KOH, EtOH/H2O, reflux, 2 h, 90%. a

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Scheme 7. Synthesis of the Analogue with Geranyl Substituent at 3-Position of Ring Aa

a

Reagents and conditions: (a) NaH, toluene, geranyl bromide, 40 °C, 2 h, 50%; (b) KOH, EtOH/H2O, reflux, 2 h, 90%.

Scheme 8. Synthesis of the Class V Analoguesa

compared to 1. Replacement of the ring B with a styryl (2r) or thienyl (2s) decreased the inhibitory activity. Unexpectedly, substituting an ethyl group (2t) for ring B did not decrease the activity but slightly increased the activity instead. This effect suggested that ring B was not indispensable for anti-HCV activity. Compound 2t also showed the highest SI value (71) among this series of analogues. Classes II (4 and 5) and III (6 and 7) were designed to probe the importance of the stilbenic double bond and the R3 group for the anti-HCV activity, respectively. As shown in Table 2, compound 5 was slightly more potent than compound 1 (Table 1), which meant the stilbenic double bond was not necessary for the anti-HCV activity. The introduction of a chloro group at 4-position of ring A (6) decreased the inhibitory activity. Compound 7, another stilbene component isolated from Cajanus cajan L., also showed a moderate level of inhibitory activity against HCV, but its inhibitory potency was inferior to that of 1.

a

Reagents and conditions: (a) X = O: DIC, RXH (reactant and solvent), rt, 4 h, 70−75%; or X = NH: DIC, CH2Cl2, RXH, rt, 4 h, 65%.

ortho or para positions of phenyl ring B, respectively, increased the inhibitory activity, while replacing the proton at ortho or para position with a methyl group (2n or 2p) decreased the activity. The introduction of a methyl group at the meta position of ring B (2o) had little effect on the activity as

Table 1. Structures and Inhibitory Activity of Class I Compounds against HCV in Huh7.5 Cells

HCV (J6/JFH gt2a full replication) compd

R1

R2

R3

CC50 (μM)a

EC50 (μM)b

SIc

2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 1 2m 2n 2o 2p 2q 2r 2s 2t 2u 2v telaprevir

Me Me Me Me Me Me Me Me Me Me H H H H H H H H H H H

phenyl 2-Cl-phenyl 2-Me-phenyl 3-Me-phenyl 4-Me-phenyl 4-F-phenyl styryl thienyl Et phenyl phenyl 2-Cl-phenyl 2-Me-phenyl 3-Me-phenyl 4-Me-phenyl 4-F-phenyl styryl thienyl Et phenyl 4-OH-phenyl

H H H H H H H H H Me H H H H H H H H H Me H

>200 >200 171.06 ± 10.16 137.79 ± 14.65 127.60 ± 9.93 180.80 ± 14.90 193.08 ± 6.03 >200 89.27 ± 10.47 102.05 ± 14.71 90.99 ± 8.18 39.52 ± 0.94 55.29 ± 3.67 82.45 ± 9.24 82.86 ± 9.12 69.22 ± 3.76 96.90 ± 5.99 83.01 ± 7.88 72.85 ± 2.43 76.84 ± 2.98 16.15 ± 0.10 44.85 ± 2.05

4.87 ± 2.81 10.93 ± 3.43 25.57 ± 7.49 18.31 ± 8.06 32.83 ± 3.71 3.18 ± 0.21 >66.67 12.32 ± 0.15 24.76 ± 1.85 2.89 ± 0.95 3.17 ± 0.75 1.03 ± 0.04 5.92 ± 0.13 1.95 ± 1.03 6.54 ± 2.01 1.49 ± 0.08 13.40 ± 3.65 3.70 ± 0.53 1.02 ± 0.46 1.70 ± 0.51 0.33 ± 0.10 0.008 ± 0.0003

>41 18 7 8 4 57 16 4 35 40 39 9 42 13 46 7 22 71 45 49 5704

CC50: 50% cytotoxicity concentration. bEC50: 50% effective concentration. cSI: selective index. The EC50 and CC50 values were indicated as mean ± SD values and were calculated from three independent experiments.

a

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Table 2. Structures and Inhibitory Activity of Classes II and III Analogues against HCV in Huh7.5 Cellsa

HCV (J6/JFH gt2a full replication)

a

compd

R1

X

R2

R3

4 5 6 7 telaprevir

COOMe COOH COOH H

CH2-CH2 CH2-CH2 CHCH CHCH

H H Cl prenyl

prenyl prenyl prenyl H

CC50 (μM) 121.86 59.35 108.94 18.54 44.85

± ± ± ± ±

6.69 9.04 0.90 2.14 2.05

EC50 (μM)

SI

± ± ± ± ±

8 35 14 2.2 5704

16.09 1.68 7.61 8.37 0.008

8.15 0.03 0.91 2.44 0.0003

The EC50 and CC50 values were indicated as mean ± SD values, and were calculated from three independent experiments.

Table 3. Structures and Inhibitory Activity of Class IV Analogues against HCV in Huh7.5 Cellsa


a

compd

R1

X

R2

CC50 (μM)

15a 8 9 10 11 telaprevir

COOMe COOMe COOH COOH COOH

CHCH CHCH CHCH CH2-CH2 CHCH

H iso-pentyl iso-pentyl iso-pentyl geranyl

160.26 ± 27.89 >200 97.76 ± 3.21 72.16 ± 6.90 46.74 ± 5.17 44.85 ± 2.05

EC50 (μM)

SI

± ± ± ± ± ±

113 >6 27 92 10 5704

1.42 32.31 3.58 0.78 4.66 0.008

0.31 4.32 0.77 0.42 0.10 0.0003


Table 4. Structures and Inhibitory Activity of Class V Analogues against HCV in Huh 7.5 Cellsa


a

compd

X

R

CC50 (μM)

12a 12b 12c 12d 3 telaprevir

O O O NH

Et iso-Pro t-Bu cyclopropyl

195.36 ± 7.79 >200 101.31 ± 7.77 33.06 ± 1.86 21.39 ± 0.41 44.85 ± 2.05

EC50 (μM)

SI

± ± ± ± ± ±

47 >15 8 8 1.8 5704

4.13 12.93 13.50 4.31 12.04 0.008

0.27 4.87 0.99 1.25 0.51 0.0003


effect indicated that the prenyl group was also not necessary for the anti-HCV activity. Actually, this effect is what we would like to observe. As shown in the chemical structure of 1, its core structure primarily consists of two hydrophobic groups (prenyl and styryl), which make this molecule extremely lipophilic (AlogP = 5.0). It is conceivable that compound 1 may suffer from potentially poor PK profiles including poor water solubility, poor bioavailability, and vulnerability to metabolism, among others. Compound 15a (AlogP = 3.4), a less lipophilic molecule compared to 1, possessed less hydrophobic burden and hence may present better PK profiles compared to 1. Additionally, compound 15a not only showed more potent inhibitory activity but also displayed the highest SI value (113)

Class IV derivatives were designed and synthesized to investigate the importance of prenyl substituent for the antiHCV activity. As shown in Table 3, replacement of the prenyl (1) with iso-pentyl (9) or a bulkier geranyl group (11) retained the inhibitory activity, with a slight decrease in the activity when compared to 1. On the basis of those results, it can be concluded that a saturated or unsaturated alkyl substituent was well tolerated at this position. Compounds 10 with both olefinic double bond reduced showed even more potent activity as compared to 1. Unexpectedly, removal of the prenyl substituent at R3 position (15a) did not lead to the loss in activity but slightly increased the inhibitory potency instead as compared to 1. This 10273



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among all the synthesized compounds. Consequently, compound 15a is a more promising alternative for further optimization. Class V derivatives were designed to investigate the importance of carboxyl group for the anti-HCV activity. As shown in Table 4, esterification (12a−c), amidation (12d), or removal of the carboxyl group (3) resulted in a decrease in activity, and these results further highlighted the importance of the carboxyl group for the anti-HCV activity. Additionally, with the increase in bulk of the R group, the inhibitory activity decreased. However, the EC50 value of compound 12d was lower than that of compound 12b and 12c, and this effect may be attributed to the amide proton, which could function as a hydrogen bond donor, as does the carboxyl group in 1. Anti-HCV Mechanism of 1. Having confirmed that 1 and its derivatives showed strong inhibitory activity against HCV in Huh7.5 cells, we next studied the MOA underlying 1’s antiHCV effect. 1 was initially screened against several known viral proteins, including NS5B, NS5A, and NS3/4A, and the results showed that 1 exhibited no inhibitory effect against these proteins (data not shown). Consequently, we speculated that 1 and its analogues might inhibit HCV replication by targeting host proteins. Along with this clue, 1 was employed as a representative compound to study its MOA. The early evidence from geneexpression chip assay showed that treatment of Huh7.5 cells with 1 decreased mRNA level of CSGalNAcT-1, which is an essential enzyme involved in the initiation of chondroitin sulfate (CS) synthesis.24 This result was further validated by a dose-dependent decrease in the protein level of CSGalNAcT-1 after 72 h treatment of 1 either in native Huh7.5 cells (Figure 3A, left) or in HCV-infected Huh7.5 cells (Figure 3B, left). It seems that 1 showed more effective results in terms of decreasing CSGalNAcT-1 protein levels in native Huh7.5 cells than HCV-infected ones (Figure 3A left vs Figure 3B left). Such discrepancy presumably resulted from the higher expression level of CSGalNAcT-1 protein in HCV infected cells as compared to the native ones (Figure 3C). Unexpectedly, 1 had no effect on the mRNA level of CSGalNAcT-1 after 72 h treatment of 1 either in native Huh7.5 cells (Figure 3A, right) or HCV-infected Huh7.5 cells (Figure 3B, right), which contradicted with the results obtained from gene chip assay. The reasons for such contradiction are possibly 2-fold: The short sequence of probe is not specific enough, or the target gene obtained from gene chip assay is homologous to CSGalNAcT-1, and hence other genes are possibly also involved in the anti-HCV mechanism of compound 1. Nonetheless, our results suggested that 1 may mainly downregulate CSGalNAcT-1 at the protein level. Next, we tested whether 1 had an effect on destabilizing CSGalNAcT-1 protein in native Huh7.5 cells. The results showed that the protease inhibitor MG132 markedly reversed 1-induced reduction of the CSGalNAcT-1 protein level (Figure 3D), suggesting that 1 inhibited HCV infection, presumably by destabilizing CSGalNAcT-1 protein. We next probed whether compound 1 specifically acts on CSGalNAcT-1 protein. To this end, we tested the effect of 1 on the expression level of other cellular proteins such as membrane protein cluster of differentiation 36 (CD36) and cytoplasmic protein heat-stress cognate 70 (Hsc70), both of which are related to HCV replication and are potential anti-HCV targets.25,26 The results showed that 1 possessed no effect on the expression level of CD36 and Hsc70 (Figure 3E), suggesting that 1 indeed shows some specificity to

Figure 3. 1 inhibited HCV replication via destabilizing CSGalNAcT-1 protein. (A,B) 1 down-regulated cellular CSGalNAcT-1 protein (left) with no role at the mRNA level of CSGalNAcT-1 (right) in native Huh7.5 cells (A) or HCV-infected Huh7.5 cells (B). (C) HCV infection up-regulated cellular CSGalNAcT-1 protein. Huh7.5 cells were infected with HCV (45 IU/cell) for 48 h. (D) 1 destabilized the CSGalNAcT-1 protein. MG132 (1 μM) reversed the 1 (25 μM) induced degradation of CSGalNAcT-1, **P < 0.01 vs. 1 alone. (E) Compound 1 had no effect on CD36 or Hsc70. (F) Compound 15a decreased the protein level of cellular CSGalNAcT-1 in naive Huh7.5 cells. (G) Compound 15a had no effect on CD36 or Hsc70. *P < 0.05 10274



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together, it can be concluded that CSGalNAcT-1 is essential for HCV replication. To explore the possible action step(s) of 1 in the viral lifecycle, we first evaluated its anti-HCV activity in GS4.3 replicon cell line carrying an HCV subgenomic replicon I3773′del.S,27 where HCV can replicate without early steps such as attachment, absorption, and fusion. As depicted in Figure 5A,

Figure 3. continued and **P < 0.01 vs control. Error bars represented the standard deviation of three independent experiments.

CSGalNAcT-1. The inferior anti-HCV activity of 1 and its analogues to that of the positive control telaprevir is presumably ascribed to this distinct host-targeting instead of direct acting on viral protein mechanism. Having confirmed that 1 inhibited HCV infection by targeting cellular CSGalNAcT-1 protein, we next probed whether its analogues also possessed the similar MOA. Compound 15a was chosen for such purpose because it showed promising in vitro anti-HCV activity with the highest SI value (113) and also possessed the most different chemical structure among the other analogues as compared to 1. As shown in Figure 3F, compound 15a also decreased the level of CSGalNAcT-1 in a concentration dependent manner and showed no effect on CD36 and Hsc70 (Figure 3G), suggesting that removal of the prenyl group from 1 did not make a difference in terms of anti-HCV MOA. To better understand the MOA of 1, we investigated the role of CSGalNAcT-1 in HCV lifecycle. Huh7.5 cells were transfected with expression plasmid pcDNA3.1(+)-CSGalNAcT-1 followed by HCV infection, and the incubation was continued for another 72 h. The results showed that intracellular HCV RNA and core protein were significantly higher in the Huh7.5 cells transfected with pcDNA3.1(+)CSGalNAcT-1, as compared to the control plasmid pcDNA3.1(+) (Figure 4A, left for RNA level, right for protein level). Meanwhile, after the silence of endogenous CSGalNAcT-1 with specific siRNA, the intracellular HCV RNA load was largely reduced, and the HCV core protein was declined as well (Figure 4B, left for RNA level, right for protein level). Taken

Figure 5. CSGalNAcT-1 facilitated HCV replication at early steps in the viral life cycle. (A) 1 did not inhibit HCV replication in GS4.3 cells (HCV subgenome replicon cell). (B) CSGalNAcT-1 facilitated HCV replication at early steps in the viral life cycle at RNA level (left) and protein level (right). (C) 1 did not directly inhibited HCV replication at early steps in the viral life cycle at RNA level (left) and protein level (right). Data were normalized to the control, which was regarded as 100%. *P < 0.05 and **P < 0.01, vs control. Error bars represented the standard deviation of three independent experiments.

no inhibitory effect was observed in 1-treated GS4.3 cells, while the positive control telaprevir exhibited strong anti-HCV activity, suggesting that 1 did not inhibit HCV replication at the replicative steps of HCV lifecycle. Then, we conducted further experiment to probe whether 1 inhibited HCV replication at early steps. Huh7.5 cells were transfected with plasmid pcDNA3.1(+)-CSGalNAcT-1. After 48 h, the cells were infected with HCV (150 IU/cell) for 2 h. After washing with PBS, the cells were cultured with fresh medium. At 24 h postinfection, intracellular proteins and RNAs were extracted to detect CSGalNAcT-1 expression or the cells were passaged and cultured with fresh medium for 72 h. The results showed that the intracellular HCV RNA and HCV core protein was remarkably increased in Huh7.5 cells transfected with pcDNA3.1(+)-CSGalNAcT-1 when compared with that in cells transfected with control plasmid pcDNA3.1(+) (Figure

Figure 4. CSGalNAcT-1 was a cofactor for HCV replication. (A) High-expression of CSGalNAcT-1 with expression vector increased HCV RNA level (left) and HCV core protein (right). (B) Silence of CSGalNAcT-1 with siRNA decreased HCV RNA level (left) and HCV core protein (right). *P < 0.05 and **P < 0.01 vs control. Error bars represented the standard deviation of three independent experiments. 10275



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Table 6. Synergism of 1 plus DAAs against HCVa

5B, left for RNA level, right for protein level), suggesting that CSGalNAcT-1 is important to support HCV replication at the early stage. We then studied whether 1 directly inhibited HCV replication at early steps in the viral life cycle. Huh7.5 cells were infected with HCV and simultaneously treated with 1 for 2 h, and then the intracellular proteins were detected. Results showed that 1 did not directly inhibit HCV replication at the early steps of HCV life cycle (Figure 5C, left for RNA level, right for protein level). Collectively, 1 might inhibit HCV replication at early steps of the viral life cycle via downregulating of cellular CSGalNAcT-1. Anti-HCV Activity of 1 against Variants Resistant to DAAs. Because HTAs show advantages over DAAs in terms of combating DAAs drug resistance, we next probed the antiviral activities of 1 against HCV variants resistant to DAAs. As reported, D168V and A156T mutants showed the most frequent resistance to NS3/4A protease inhibitors, such as simeprevir and telaprevir,28 and S282T mutant was resistant to sofosbuvir.29 Therefore, 1 was assayed for its inhibitory activity against these mutant viruses. As expected, telaprevir, simeprevir, and sofosbuvir showed decreased inhibitory activity against these mutant variants, however, 1 exhibited equivalent inhibitory activities against both wild-type and mutant HCV virus (Table 5). Collectively, 1 showed promising potential for combating drug resistance in HCV treatment.

DRIc

A156T

D168V

S282T

FORa

1 telaprevir simeprevir sofosbuvir

3.03 0.02 0.04 0.064

6.44 0.62

3.80

8.59

1.3−2.8 31.0 23.3 4.5

0.93 0.29

1 simeprevir sofosbuvir daclatasvir Combination 1 + simeprevir 1 + sofosbuvir 1 + daclatasvir

12.02 0.04 0.11 17.67d 5.89 + 0.02 2.18 + 0.06 3.87 + 7.74d

CI value

1

DAAs

0.953 0.693 0.760

2.0 5.5 3.1

2.2 1.9 2.3

The anti-HCV activity was determined with qRT-PCR in Huh7.5 cells infected with HCV (J6/JFH/JC-1). bCombination index (CI) value at EC50 was calculated by the Chou−Talalay method using CompuSyn version 1.0. cFolds of dose reduction allowed for each drug caused by synergism at a given effect level (e.g., the dose reduction index (DRI) values at EC50 effect level); dThe concentration unit of daclatasvir was pM.

that both the hydrophobic moieties (ring B and prenyl) were not required for the inhibitory activity. Such SARs provided valuable implications for further lead optimizations. Compound 15a, which is analogous to 1 without prenyl substitution, showed comparable inhibitory activity to 1 and it possessed less hydrophobic burden with molecular weight and ALogP values being 284.3 and 3.4, respectively. Moreover, it shared the same MOA to 1 by targeting CSGalNAcT-1 despite that it possessed simplified structure as compared to 1. Therefore, compound 15a represented a more promising scaffold for further optimization. The MOA study showed 1 inhibited HCV replication by down-regulating cellular CSGalNAcT-1, which is demonstrated to be essential for viral replication in the early life cycles. In line with this distinct MOA, 1 showed the similar magnitude of inhibitory activity against both wild-type and DAAs-resistant variants and synergistically inhibited HCV replication with approved DAAs, including simeprevir, sofosbuvir, or daclatasvir. All these results highlighted cellular CSGalNAcT-1 as a potentially promising target to combat drug resistance. However, the detailed mechanism of how 1 destabilized CSGalNAcT-1 remains unclear at this stage and other mechanism might also be involved. The detailed studies are ongoing in our lab. In summary, our study not only presented 1 and its derivatives as a novel class of anti-HCV agents but also discovered a novel anti-HCV target to combat drug resistance.

EC50 (μM) wild-type

EC50 (μM)

a

Table 5. Inhibitory Activity of 1 against Wild-Type or DrugResistant HCV compd

compd

b

a

FOR: fold of resistance. The anti-HCV activity was determined with qRT-PCR in Huh7.5 cells infected with HCV (J6/JFH/JC-1) or mutant variant. A156T, D168V, and S282T were mutant variants from wild-type J6/JFH/JC-1.

Synergism of 1 plus DAAs against HCV. Because 1 inhibited HCV replication via targeting host factor CSGalNAcT-1, which is distinct from the MOA of DAAs, we then tested whether 1 possessed any synergistic effects against HCV infection with approved DAAs, including simeprevir (NS3/4A inhibitor), sofosbuvir (NS5B inhibitor), and daclatasvir (NS5A inhibitor). As shown in Table 6, 1 synergistically inhibited HCV replication with simeprevir, sofosbuvir, or daclatasvir at the RNA level with combination index (CI) < 1.0 and dose reduction index (DRI) > 1. The similar trend was also observed at the protein level (Figure 6). Altogether, 1 showed synergistic effects with DAAs to reduce HCV viral load.

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EXPERIMENTAL SECTION

General. All reagents and solvent were purchased from commercial sources. THF, toluene, and DME were distilled from sodium and benzophenone before use. CCl4, CH2Cl2, and CH3CN were distilled from P2O5. All reactions were carried out in flame-dried glassware and monitored by thin layer chromatography using aluminum TLC plate 60F254D (Merck Millipore). 1H NMR and 13C NMR spectra were recorded in CDCl3 or DMSO-d6 using a Varian Inova 400, 500, or 600 MHz spectrometer (Varian, San Francisco, CA, USA). Chemical shifts are reported in parts per million relative to tetramethylsilane as internal standard. The mass spectra (MS) were recorded on a Thermo Scientific LTQ ORBITRAP instrument with an ESI mass selective detector. Melting points were determined using an X6 microscope melting point apparatus and are uncorrected. The microwave experiment was conducted using a Discover microwave oven (CEM, Matthews, NC, USA). The purities of tested compounds were assessed as being at least 95% with analytical HPLC, which was performed

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CONCLUSIONS 1 was identified as a potent HCV inhibitor for the first time in this work. A series of novel cajanine derivatives along with its naturally occurring counterpart 7 were synthesized and assayed for anti-HCV activity in vitro. The intensive structure modifications led to several novel compounds with more potent inhibitory activity compared to 1, especially compounds 2v and 10, with EC50 values in the submicromolar range. Additionally, some significant SARs were uncovered, especially 10276



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Figure 6. 1 enhanced the inhibitory activities against HCV of DAAs. Huh7.5 cells were infected with HCV (45 IU/cell) and simultaneously treated with simeprevir (A, 0.025 μM), sofosbuvir (B, 0.1 μM), or daclatasvir (C, 16 pM) alone or with 1 (6.25 μM). *P < 0.05 vs 1 alone; #P < 0.05 and ##P < 0.01 vs simeprevir, sofosbuvir, or daclatasvir alone. All of the experiments were at least triplicated. The protein bands showed the results of a representative experiment. The Data presented are mean ± standard deviation and Student’s t test was used. H]− 365.17474, found 365.17496. HPLC: tR = 8.21 min, normalization method purity 95.04%. (E)-Methyl 6-(4-Methylstyryl)-2-hydroxy-4-methoxy-3-prenylbenzoate (2e). Yield: 50%; white solid; mp 95−97 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.68 (s, 3H), 1.79 (s, 3H), 2.37 (s, 3H), 3.37 (d, J = 7.2 Hz, 2H), 3.92 (s, 6H), 5.22 (t, J = 7.2 Hz, 1H), 6.60 (s, 1H), 6.76 (d, J = 16.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 16.0 Hz, 1H), 11.67 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.7, 21.0, 21.6, 25.7, 51.8, 55.6, 99.9, 102.6, 104.3, 116.4, 121.7, 126.4, 129.4, 129.6, 130.0, 131.7, 134.5, 137.4, 140.6, 161.2, 171.9. ESI-HRMS calcd for C23H25O4 [M − H]− 365.17474, found 365.17462. HPLC: tR = 8.22 min, normalization method purity 96.95%. (E)-Methyl 6-(4-Fluorostyryl)-2-hydroxy-4-methoxy-3-prenylbenzoate (2f). Yield 50%; white solid; mp 109−111 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.68 (s, 3H), 1.79 (s, 3H), 3.37 (d, J = 6.8 Hz, 2H), 3.92 (s, 3H), 3.93 (s, 3H), 5.21 (t, J = 6.8 Hz, 1H), 6.58 (s, 1H), 6.73 (d, J = 16.0 Hz, 1H), 7.06 (t, J = 8.4 Hz, 2H), 7.44−7.48 (m, 2H), 7.63 (d, J = 16.0 Hz, 1H), 11.65 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.8, 22.1, 25.8, 52.2, 55.8, 102.8, 104.5, 115.6, 115.7, 116.8, 122.0, 128.0, 128.1, 128.8, 130.7, 131.8, 133.6, 140.1, 161.3, 161.4, 161.4, 163.3, 171.8. ESI-HRMS calcd for C22H22O4F [M − H]− 369.14966, found 369.14970. HPLC: tR = 7.31 min, normalization method purity 99.17%. Methyl 2-Hydroxy-4-methoxy-3-prenyl-6-((1E,3E)-4-phenylbuta1,3-dienyl)benzoate (2g). Yellow solid; yield 50%; mp 77−79 °C. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 1.60 (s, 3H), 1.70 (s, 3H), 3.24 (d, J = 6.8 Hz, 2H), 3.87 (s, 3H), 3.88 (s, 3H), 5.09 (t, J = 6.8 Hz, 1H), 6.76 (d, J = 15.2 Hz, 1H), 6.80 (d, J = 15.2 Hz, 1H), 6.93−7.15 (m, 3H), 7.24 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.6 Hz, 2H), 7.51 (d, J = 7.6 Hz, 2H), 10.44 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.5, 22.0, 25.5, 52.1, 55.4, 102.2, 116.9, 121.9, 126.4, 127.9, 128.8, 129.3, 130.6, 130.9, 133.08, 133.2, 134.2, 134.6, 137.1,139.9, 161.7, 171.9. ESI-HRMS calcd for C24H25O4 [M − H]− 377.17474, found 377.17531. HPLC: tR = 7.76 min, normalization method purity 97.94%. (E)-Methyl 2-Hydroxy-4-methoxy-3-prenyl-6-(2-(thiophen-2-yl)vinyl)benzoate (2h). Yield 50%; yellow solid; mp 118−120 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.68 (s, 3H), 1.78 (s, 3H), 3.36 (d, J = 7.2 Hz, 2H), 3.91 (s, 3H), 3.95 (s, 3H), 5.21 (t, J = 7.2 Hz, 1H), 6.58 (s, 1H), 6.92 (d, J = 16.0 Hz, 1H), 7.01 (t, J = 4.0 Hz, 1H), 7.06 (d, J = 4.0 Hz, 1H), 7.20 (d, J = 4.0 Hz, 1H), 7.57 (d, J = 16.0 Hz, 1H), 11.67 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.7, 22.28, 25.5, 52.3, 56.4, 102.1, 104.4, 116.7, 121.7, 122.8, 124.5, 125.8, 127.0, 130.4, 131.5, 139.3, 142.8, 161.0, 161.5, 171.5. ESI-HRMS calcd for C20H21O4S [M − H]− 357.11551, found 357.11496. HPLC: tR = 7.00 min, normalization method purity 97.49%.

using a C18 (5 μm, 250 mm × 4.6 mm) column, UV detected at 254 nm and with elution 15% (0.01 M KH2PO4 solution)/85% (MeOH) for the carboxylic compounds, or 2% (0.01 M KH2PO4 solution)/98% (MeOH) for the noncarboxylic compounds at 1 mL/min. For the synthesis and spectra data of compounds 13, 14a, 15a, 2a, 1, and 3, please refer to our previous report.13 General Procedure A for the Prenylation of Demethylation Compounds. Compound 15a−k (1 equiv) was dissolved in dry toluene (30 mL), and NaH (1.3 equiv) was added at room temperature. The reaction mixture was stirred at room temperature for 0.5 h, after which a portion of the toluene was evaporated away and prenyl bromide (1.5 equiv) was added at room temperature. The mixture was stirred for 2 h at 40 °C and quenched by the addition of ice−water. The organic layer was separated and dried over anhydrous MgSO4. It was then filtered and concentrated to give a residue, which was purified over silica gel to give a crude product, which was recrystallized from petroleum ether to afford the target compounds. (E)-Methyl 6-(2-Chlorostyryl)-2-hydroxy-4-methoxy-3-prenylbenzoate (2b). Yield 50%; white solid; mp 94−96 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.68 (s, 3H), 1.79 (s, 3H), 3.38 (d, J = 6.8 Hz, 2H), 3.93 (s, 3H), 5.22 (t, J = 6.8 Hz, 1H), 6.64 (s, 1H), 7.15 (d, J = 16.0 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H),7.40 (d, J = 7.6 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.69 (d, J = 16.0 Hz, 1H), 11.66 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ (ppm): 17.8, 22.1, 25.9, 52.1, 55.4, 103.3, 104.6, 117.2, 121.8, 126.1, 126.4, 126.7, 128.8, 129.8, 131.7, 133.0, 133.3, 135.4, 140.0, 161.6, 171.7. ESI-HRMS calcd for C22H22O4Cl [M − H]− 385.12011, found 385.12070. HPLC: tR = 9.99 min, normalization method purity 98.36%. (E)-Methyl 6-(2-Methylstyryl)-2-hydroxy-4-methoxy-3-prenylbenzoate (2c). Yield 50%; white solid; mp 89−91 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.68 (s, 3H), 1.79 (s, 3H), 2.42 (s, 3H), 3.38 (d, J = 7.2 Hz, 2H), 3.93 (s, 6H), 5.22 (t, J = 7.2 Hz, 1H), 6.60 (s, 1H), 6.96 (d, J = 16.0 Hz, 1H), 7.19−7.24 (m, 3H), 7.56 (d, J = 6.8 Hz, 1H), 7.60 (d, J = 16.0 Hz, 1H), 11.69 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.8, 19.9, 22.1, 25.8, 52.2, 55.6, 103.1, 104.6, 116.7, 122.1, 125.6, 126.2, 127.6, 128.0, 130.4, 131.8, 131.96 135.80 136.53 140.79 161.35 161.49 172.0. ESI-HRMS calcd for C23H25O4 [M − H]− 365.17474, found 365.17454. HPLC: tR = 8.43 min, normalization method purity 97.62%. (E)-Methyl 6-(3-Methylstyryl)-2-hydroxy-4-methoxy-3-prenylbenzoate (2d). Yield 50%; white solid; mp 64−66 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.68 (s, 3H), 1.79 (s, 3H), 2.38 (s, 3H), 3.37 (d, J = 7.2 Hz, 2H), 3.92 (s, 3H), 3.94 (s, 3H), 5.21 (t, J = 7.2 Hz, 1H), 6.60 (s, 1H), 6.75 (d, J = 16.0 Hz, 1H), 7.10 (d, J = 6.8 Hz, 1H), 7.30 (m, 3H), 7.70 (d, J = 16.0 Hz, 1H), 11.67 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.8, 21.2, 22.2, 25.8, 52.2, 55.6, 102.8, 104.5, 116.5, 122.1, 123.6, 127.5, 128.5, 128.6, 130.2, 130.5, 131.8, 137.5, 138.3, 140.4, 161.4, 161.5, 171.9. ESI-HRMS calcd for C23H25O4 [M − 10277



Article

(E)-Methyl 6-(But-1-enyl)-2-hydroxy-4-methoxy-3-prenylbenzoate (2i). Yield 60%; white solid; mp 50−52 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.11 (t, J = 7.2 Hz, 3H), 1.67 (s, 3H), 1.78 (s, 3H), 2.22 (m, 2H), 3.34 (d, J = 7.2 Hz, 2H), 3.88 (s, 3H), 3.91 (s, 3H), 5.19 (t, J = 7.2 Hz, 1H), 5.97 (m, 1H), 6.45 (s, 1H), 6.93 (d, J = 16.0 Hz, 1H), 11.60 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 13.4, 17.8, 21.9, 25.8, 25.9, 51.6, 55.6, 102.8, 104.3, 115.8, 122.1, 130.64, 131.6, 133.4, 141.0, 161.2, 161.2, 171.9. ESI-HRMS calcd for C18H23O4 [M − H]− 303.15909, found 303.15896. HPLC: tR = 6.74 min, normalization method purity 95.56%. (E)-Methyl 2-Hydroxy-4-methoxy-3-(3-methylbut-2-enyl)-6-(2phenylprop-1-enyl)benzoate (2j). Yield 55%; white solid; mp 93− 95 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.65 (s, 3H), 1.73 (s, 3H), 2.22 (s, 3H), 3.24 (d, J = 7.2 Hz, 2H), 3.30 (s, 3H), 3.94 (s, 3H), 5.12 (t, J = 7.2 Hz, 1H), 5.86 (s, 1H), 6.77 (s, 1H), 7.08−7.20 (m, 5H), 11.60 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.7, 21.9, 25.4, 25.8, 52.0, 55.1, 105.1, 107.5, 115.1, 122.3, 126.5, 127.9, 128.3, 128.6, 131.5, 136.4, 139.9, 141.8, 160.7, 161.0, 171.9. ESIHRMS calcd for C23H25O4 [M − H]− 365.17474, found 365.17471. HPLC: tR = 6.58 min, normalization method purity 96.16%. (E)-Methyl 6-(4-(tert-Butyldimethylsilyloxy)styryl)-2-hydroxy-4methoxy-3-prenylbenzoate (2k). White solid; mp 81−83 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.22 (s, 6H), 0.99 (s, 9H), 1.68 (s, 3H), 1.79 (s, 3H), 3.37 (d, J = 7.2 Hz, 2H), 3.91 (s, 3H), 3.92 (s, 3H), 5.22 (t, J = 7.2 Hz, 1H), 6.60 (s, 1H), 6.74 (d, J = 16.0 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 16.0 Hz, 1H), 11.66 (s, 1H). ESI-MS, [M + H] +, m/z: 483.73. (E)-Methyl 6-(4-Hydroxystyryl)-2-hydroxy-4-methoxy-3-prenylbenzoate (2l). To a solution of compound 2k (1 equiv) in THF was added TBAF (1.5 equiv) at room temperature, and the resulting mixture was stirred for another 2 h at room temperature, after which the mixture was poured into water and extracted with ethyl acetate for three times. The combined organic layer was washed with brine and dried over anhydrous MgSO4. After filtration and concentration, the obtained residue was recrystallized from petroleum ether to produce the target compound as a pale-yellow solid. Yield 90%; mp 153−155 °C. 1H NMR (500 MHz, CDCl3) δ (ppm): 1.54 (s, 3H), 1.68 (s, 3H), 3. 37 (d, J = 7.0 Hz, 2H), 3.88 (s, 3H), 3.92 (s, 3H), 4.78 (br, 1H), 5.22 (t, J = 7.0 Hz, 1H), 6.60 (s, 1H), 6.73 (d, J = 15.5 Hz, 1H), 6.84 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 16.0 Hz, 1H), 11.65 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.7, 22.0, 25.8, 52.5, 55.3, 102.6, 104.3, 115.4, 116.5, 122.1, 128.0, 128.6, 129.5, 130.4, 131.9, 140.3, 155.1, 161.7, 161.4, 171.1. ESI-HRMS calcd for C22H25O5 [M + H]+ 369.16965, found 369.16962. HPLC: tR = 5.00 min, normalization method purity 98.72%. General Procedure B for the Hydrolysis (1, 2m−v). Compounds 2a−2l (1 equiv) was dissolved in EtOH/H2O (20/5 mL), and KOH (1.5 equiv) was added. The reaction mixture was heated under reflux for 2 h, after which the mixture was poured into ice water and acidified with 15% HCl. The formed precipitate was filtered and dried under vacuum. Recrystallization from petroleum ether/ethyl acetate produced the target compounds. (E)-6-(2-Chlorostyryl)-2-hydroxy-4-methoxy-3-prenylbenzoic Acid (2m). White solid; mp 151−154 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.61 (s, 3H), 1.71 (s, 3H), 3.25 (d, J = 7.2 Hz, 2H), 3.90 (s, 3H), 5.11 (t, J = 7.2 Hz, 1H), 6.72 (s, 1H), 7.12 (d, J = 16.0 Hz, 1H), 7.30−7.39 (m, 2H), 7.48 (d, J = 7.6 Hz, 1H), 7.72 (d. J = 7.6 Hz, 1H), 7.85 (d, J = 16.0 Hz, 1H), 12.41 (br, 1H), 14.15 (br, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.5, 21.8, 25.5, 55.3, 102.8, 103.6, 116.7, 121.7, 126.9, 126.9, 127.0, 128.8, 129.8, 132.0, 132.8, 133.4, 135.3, 141.4, 162.3, 162.4, 174.4. ESI-HRMS calcd for C21H20O4Cl [M − H]− 371.10446, found 371.10497. HPLC: tR = 6.49 min, normalization method purity 98.00%. (E)-6-(2-Methylstyryl)-2-hydroxy-4-methoxy-3-prenylbenzoic Acid (2n). White solid; mp 138−140 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.68 (s, 3H), 1.79 (s, 3H), 2.42 (s, 3H), 3.38 (d, J = 7.2 Hz, 2H), 3.95 (s, 3H), 5.21 (t, J = 7.2 Hz, 1H), 6.64 (s, 1H), 7.01 (d, J = 15.6 Hz, 1H), 7.19−7.24 (m, 3H), 7.59 (d, J = 7.2 Hz, 1H), 7.70 (d, J = 15.6 Hz, 1H), 11.54 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.9, 20.1, 22.0, 25.5, 55.6, 103.0, 103.5, 116.7, 121.9, 125.8,

126.4, 127.8, 128.7, 130.4, 131.7, 131.9, 135.7, 136.3, 142.2, 162.2, 162.4, 174.9. ESI-HRMS calcd for C22H23O4 [M − H]− 351.15909, found 351.15939. HPLC: tR = 5.92 min, normalization method purity 95.17%. (E)-6-(3-Methylstyryl)-2-hydroxy-4-methoxy-3-prenylbenzoic Acid (2o). White solid; mp 141−142 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.68 (s, 3H), 1.79 (s, 3H), 2.38 (s, 3H), 3.37 (d, J = 6.8 Hz, 2H), 3.94 (s, 3H), 5.20 (t, J = 6.8 Hz, 1H), 6.64 (s, 1H), 6.80 (d, J = 15.6 Hz, 1H), 7.10 (d, J = 7.2 Hz, 1H), 7.28 (m, 2H), 7.32 (s, 1H), 7.80 (d, J = 15.6 Hz, 1H), 11.55 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.7, 21.2, 22.1, 25.7, 55.8, 103.0, 103.3, 116.6, 121.9, 123.9, 127.5, 128.7, 128.4, 130.7, 131.4, 131.9, 137.2, 138.3, 141.9, 162.2, 162.4, 175.2. ESI-HRMS calcd for C22H23O4 [M − H]− 351.15909, found 351.15862. HPLC: tR = 5.94 min, normalization method purity 95.45%. (E)-6-(4-Methylstyryl)-2-hydroxy-4-methoxy-3-prenylbenzoic Acid (2p). White solid; mp 147−149 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.60 (s, 3H), 1.71 (s, 3H), 2.30 (s, 3H), 3.23 (d, J = 7.2 Hz, 2H), 3.90 (s, 3H), 5.11 (t, J = 7.2 Hz, 1H), 6.76 (s, 1H), 6.96 (d, J = 16.0 Hz, 1H), 7.18 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 16.0 Hz, 1H), 12.29 (br, 1H), 13.97 (br, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.8, 21.3, 22.1, 25.8, 55.8, 103.1, 103.2, 116.6, 121.9, 126.7, 129.3, 129.4, 130.8, 131.9, 134.5, 137.8, 142.0, 162.2, 162.4, 175.2. ESI-HRMS calcd for C22H23O4 [M − H]− 351.15909, found 351.15883. HPLC: tR = 5.95 min, normalization method purity 96.99%. (E)-6-(4-Fluorostyryl)-2-hydroxy-4-methoxy-3-prenylbenzoic Acid (2q). White solid; mp 169−172 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.68 (s, 3H), 1.79 (s, 3H), 3.37 (d, J = 7.2 Hz, 2H), 3.94 (s, 3H), 5.21 (t, J = 7.2 Hz, 1H), 6.62 (s, 1H), 6.78 (d, J = 16.0 Hz, 1H), 7.06 (t, J = 8.4 Hz, 2H), 7.46−7.50 (m, 2H), 7.73 (d, J = 16.0 Hz, 1H), 11.58 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.7, 21.9, 25.5, 55.6, 102.9, 103.3, 115.6, 115.8, 116.9, 121.8, 128.3, 128.3, 129.6, 130.1, 132.0, 133.4, 141.6, 161.4, 162.4, 162.5, 163.5, 175.2. ESIHRMS calcd for C21H20O4F [M − H]− 355.13401, found 355.13386. HPLC: tR = 6.86 min, normalization method purity 99.68%. 2-Hydroxy-4-methoxy-3-prenyl-6-((1E,3E)-4-phenylbuta-1,3dienyl)benzoic Acid (2r). Yellow solid; mp 176−178 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.60 (s, 3H), 1.70 (s, 3H), 3.23 (d, J = 7.2 Hz, 2H), 3.89 (s, 3H), 5.10 (t, J = 7.2 Hz, 1H), 6.74 (d, J = 15.6 Hz, 1H), 6.76 (s, 1H), 6.92 (dd, J = 15.2 Hz, 15.2 Hz, 1H), 7.10 (dd, J = 15.6 Hz, 15.2 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.34 (t, J = 7.6 Hz, 2H), 7.45 (d, J = 15.2 Hz, 1H), 7.51 (d, J = 7.6 Hz, 2H), 12.24 (br, 1H), 14.05 (br, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.6, 21.8, 25.7, 55.6, 99.9, 102.8, 116.8, 121.9, 126.5, 127.7, 128.6, 129.2, 131.5, 131.9, 133.6, 133.9, 137.2, 141.2, 162.1, 162.4, 174.5. ESIHRMS calcd for C23H23O4 [M − H]− 363.15909, found 363.15969. HPLC: tR = 6.26 min, normalization method purity 96.51%. (E)-2-Hydroxy-4-methoxy-3-prenyl-6-(2-(thiophen-2-yl)vinyl)benzoic Acid (2s). Yield 50%; yellow solid; mp 146−148 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.61 (s, 3H), 1.72 (s, 3H), 3.24 (d, J = 7.2 Hz, 2H), 3.91 (s, 3H), 5.11 (t, J = 7.2 Hz, 1H), 6.79 (s, 1H), 7.06 (dd, J = 3.2 Hz, 4.0 Hz, 1H), 7.18 (d, J = 3.2 Hz, 1H), 7.26 (d, J = 16.0 Hz, 1H), 7.46 (d, J = 4.0 Hz, 1H), 7.67 (d, J = 16.0 Hz, 1H), 12.30 (br, 1H), 14.10 (br, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.5, 22.0, 25.7, 55.6, 103.0, 116.9, 121.9, 123.8, 124.9, 126.3, 127.6, 129.8, 131.9, 141.1, 142.7, 162.2, 162.4, 174.9. ESI-HRMS calcd for C19H19O4S [M − H]− 343.09986, found 343.09952. HPLC: tR = 4.63 min, normalization method purity 95.90%. (E)-6-(But-1-enyl)-2-hydroxy-4-methoxy-3-prenylbenzoic Acid (2t). Yield 50%; white solid; mp 122−124 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.12 (t, J = 7.2 Hz, 3H), 1.67 (s, 3H), 1.78 (s, 3H), 2.26 (m, 2H), 3.34 (d, J = 6.8 Hz, 2H), 3.90 (s, 3H), 5.20 (t, J = 6.8 Hz, 1H), 6.01 (m, 1H), 6.48 (s, 1H), 7.05 (d, J = 15.6 Hz, 1H), 10.38 (br, 1H), 11.53 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 13.5, 17.8, 22.0, 25.8, 26.0, 55.6, 102.8, 103.6, 116.0, 122.1, 130.2, 131.5, 134.7, 142.3, 162.2, 175.5. ESI-HRMS calcd for C17H21O4 [M − H]− 289.14344, found 289.14396. HPLC: tR = 4.46 min, normalization method purity 97.58%. 10278



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(E)-2-Hydroxy-4-methoxy-3-(3-methylbut-2-enyl)-6-(2-phenylprop-1-enyl)benzoic Acid (2u). White solid; mp 165−167 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.65 (s, 3H), 1.73 (s, 3H), 2.27 (s, 3H), 3.25 (d, J = 7.2 Hz, 2H), 3.33 (s, 3H), 5.12 (t, J = 7.2 Hz, 1H), 5.90 (s, 1H), 6.86 (s, 1H), 7.11−7.22 (m, 5H), 11.57 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.7, 21.8, 25.6, 25.8, 55.2, 103.7, 108.0, 115.4, 122.2, 126.7, 128.1, 128.2, 128.5, 131.7, 138.0, 141.3, 141.6, 161.5, 162.1, 175.0. ESI-HRMS calcd for C22H23O4 [M − H]− 351.15909, found 351.15876. HPLC: tR = 5.29 min, normalization method purity 96.52%. (E)-6-(4-Hydroxystyryl)-2-hydroxy-4-methoxy-3-prenylbenzoic Acid (2v). Yield 90%; White solid; mp >220 °C. 1H NMR (500 MHz, DMSO-d6) δ (ppm) 1.54 (s, 3H), 1.69 (s, 3H), 3.32 (d, J = 7.0 Hz, 1H), 3.77 (s, 3H), 5.14 (t, J = 7.0 Hz, 1H), 6.45 (s, 1H), 6.73 (d, J = 16.5 Hz, 1H), 6.75 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 8.54 (d, J = 16.5 Hz, 1H),9.52 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ (ppm): 17.6, 22.0, 25.5, 55.0, 57.3, 97.0, 111.7, 113.8, 115.3, 123.9, 126.0, 127.4, 128.8, 129.1, 129.4, 138.4, 156.5, 157.6, 163.2, 171.9. ESIHRMS calcd for C21H23O5 [M + H]+ 355.15400, found 355.15407. HPLC: tR = 3.84 min, normalization method purity 97.95%. Methyl 2-Hydroxy-4-methoxy-3-prenyl-6-phenethylbenzoate (4). Compound 16 was transformed into 4 as a white solid according to general procedure A. Yield 56%; mp 76−78 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.67 (s, 3H), 1.78 (s, 3H), 2.84 (t, J = 8.0 Hz, 2H), 3.18 (t, J = 8.0 Hz, 2H), 3.33 (d, J = 7.2 Hz, 2H), 3.79 (s, 3H), 3.95 (s, 3H), 5.20 (t, J = 7.2 Hz, 1H), 6.21 (s, 1H), 7.20 (m, 3H), 7.30 (t, J = 7.2 Hz, 2H), 11.70 (s, 1H). ESI-HRMS calcd for C22H25O4 [M − H]− 353.17474, found 353.17503. HPLC: tR = 6.97 min, normalization method purity 96.09%. 2-Hydroxy-4-methoxy-3-prenyl-6-phenethylbenzoic Acid (5). Compound 4 was transformed into 5 as a white solid according to general procedure B. Yield 90%; mp 146−148 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.59 (s, 3H), 1.69 (s, 3H), 2.80 (t, J = 7.6 Hz, 2H), 3.12 (t, J = 7.6 Hz, 2H), 3.21 (d, J = 6.8 Hz, 2H), 3.78 (s, 3H), 5.10 (t, J = 6.8 Hz, 1H), 6.45 (s, 1H), 7.17 (t, J = 7.2 Hz, 1H), 7.22 (d, J = 7.2 Hz, 2H), 7.28 (t, J = 7.2 Hz, 2H), 12.47 (br, 1H), 13.95 (br, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.6, 22.1, 25.7, 38.2, 39.1, 55.6, 103.6, 106.3, 115.4, 122.2, 125.9, 128.4, 128.5, 131.7, 141.9, 145.6, 162.1, 162.9, 175.2. ESI-HRMS calcd for C21H23O4 [M − H]− 339.15909, found 339.15955. HPLC: tR = 5.35 min, normalization method purity 96.98%. (E)-3-Chloro-6-hydroxy-4-methoxy-5-prenyl-2-styrylbenzoic Acid (6). Compound 6 was obtained as a yellow solid according to general procedure B. Yield 90%; mp 122−124 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.70 (s, 3H), 1.80 (s, 3H), 3.43(d, J = 6.8 Hz, 2H), 3.88 (s, 3H), 5.21(t, J = 6.8 Hz, 1H), 6.59 (d, J = 16.4 Hz, 1H), 7.20 (d, J = 16.4 Hz, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.38 (t, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 11.32 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.9, 23.5, 25.7, 61.0, 107.8, 119.9, 121.5, 124.4, 125.3, 126.7, 128.3, 128.8, 132.6, 135.6, 136.7, 138.0, 159.5, 161.0, 172.6. ESIHRMS calcd for C21H20O4Cl [M − H]− 371.10446, found 371.10497. HPLC: tR = 5.46 min, normalization method purity 98.20%. (E)-3-Methoxy-4-(3-methylbut-2-enyl)-5-styrylphenol (7). To a solution of compound 26 (1 equiv) in EtOH (10 mL) was added KOH (1.5 equiv). The resulting mixture was subjected to microwave irradiation (60W, 110 °C, 50 psi) for 30 min, after which the mixture was cooled to room temperature and poured into ice water. The mixture was then acidified with 10% HCl and extracted with ethyl acetate (3 × 40 mL). The organic layer was washed with NaHCO3 solution and brine successively. The obtained organic layer was dried over anhydrous MgSO4. After filtration and concentration, the residue was purified over silica gel to afford longistyline C as a white solid. Yield 70%; mp 95−97 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.59 (s, 3H), 1.72 (s, 3H), 3.28 (d, J =7.2 Hz, 2H), 3.73 (s, 3H), 4.98 (t, J = 7.2 Hz, 1H), 6.34 (d, J = 2.0 Hz, 1H), 6.64 (d, J = 2.0 Hz, 1H), 6.94(d, J = 16.0 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 7.30 (d, J = 16.0 Hz, 1H), 7.36 (t, J = 8.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 9.25 (s, 1H). 13 C NMR (100 MHz, DMSO-d6) δ (ppm): 17.7, 24.6, 25.4, 55.4, 98.8, 103.8, 118.6, 123.9, 126.4, 127.5, 128.7, 129.5, 130.1, 136.7, 137.1, 151.8, 156.2. EI-HRMS calcd for C20H21O2 [M − H]− 293.15361,

found 293.15383. HPLC: tR = 4.90 min, normalization method purity 95.99%. (E)-Methyl 2-Hydroxy-3-isopentyl-4-methoxy-6-styrylbenzoate (8). Compound 8 was obtained as a white solid according to procedure A. Yield 85%; mp 116−119 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.90 (d, J = 6.8 Hz, 6H), 1.31 (m, 2H), 1.51 (m, 1H), 2.57(t, J = 8.0 Hz, 2H), 3.90 (s, 6H), 6.82 (s, 1H), 7.03 (d, J = 16.0 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 2H), 7.54 (d, J = 7.6 Hz, 2H), 7.62 (d, J = 16.0 Hz, 1H), 10.97(s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 14.5, 21.0, 22.5, 28.4, 37.7, 55.6, 61.4, 102.6, 104.5, 118.3, 126.5, 126.6, 127.6, 128.7, 128.7, 129.6, 130.8, 137.5, 140.1, 161.5, 161.6, 171.6. ESI-HRMS calcd for C22H25O4 [M − H]− 353.17474, found 353.17493. HPLC: tR = 9.02 min, normalization method purity 98.13%. (E)-2-Hydroxy-3-isopentyl-4-methoxy-6-styrylbenzoic Acid (9). Compound 9 was obtained as a white solid according to general procedure B. Yield 90%; mp 154−156 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 0.90(d, J = 6.4 Hz, 6H), 1.30 (m,2H), 1.51 (m, 1H), 2.56 (t, J = 7.2 Hz, 2H), 3.90 (s, 3H), 6.77 (s, 1H), 6.99 (d, J = 16.0 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 2H), 7.52 (d, J = 7.6 Hz, 2H), 7.86 (d, J = 16.0 Hz, 1H), 12.40 (br, 1H), 13.98 (br, 1H). ESI-HRMS calcd for C21H23O4 [M − H]− 339.15909, found 339.15962. HPLC: tR = 5.90 min, normalization method purity 96.59%. 2-Hydroxy-3-isopentyl-4-methoxy-6-phenethylbenzoic Acid (10). Compound 10 was obtained as a pale-yellow solid by using a similar method used to synthesize compound 16. Yield 95%; mp 122−124 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.95 (d, J = 6.4 Hz, 6H), 1.33−1.39 (m, 2H), 1.57−1.61 (m, 1H), 2.63 (t, J = 7.6 Hz, 2H), 2.92 (t, J = 7.6 Hz, 2H), 3.26 (t, J = 7.6 Hz, 2H), 3.78 (s, 3H), 6.19 (s, 1H), 7.20 (d, J = 7.2 Hz, 2H), 7.27−7.31 (m, 3H), 10.5 (br, 1H), 11.63 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 20.7, 22.6, 28.3, 37.8, 38.2, 39.2, 55.5, 103.4, 106.3, 116.8, 125.9, 128.4, 128.5, 141.9, 145.3, 162.3, 162.9, 175.3. ESI-HRMS calcd for C21H25O4 [M − H]− 341.17474, found 341.17518. HPLC: tR = 6.04 min, normalization method purity 97.67%. (E)-3-(3,7-Dimethylocta-2,6-dienyl)-2-hydroxy-4-methoxy-6-phenethylbenzoic Acid (11). Compound 11 was obtained as a white solid according to general procedure B. Yield 90%; mp 159−161 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.56 (s, 3H), 1.64 (s, 3H), 1.79 (s, 3H), 1.94−2.08 (m, 4H), 3.27 (d, J = 6.4 Hz, 2H), 3.88 (s, 3H), 5.07 (t, J = 6.4 Hz, 1H), 5.18 (t, J = 6.8 Hz, 1H), 6.42 (s, 1H), 6.66 (d, J = 16.0 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.29 (t, J = 7.6 Hz, 2H), 7.41 (d, J = 7.6 Hz, 2H), 7.77 (d, J = 16.0 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 16.1, 17.6, 22.1, 25.6, 26.8, 39.8, 55.4, 102.3, 106.5, 116.3, 122.4, 124.5, 126.2, 127.8, 128.0, 129.1, 131.1, 132.4, 134.9, 137.1, 140.3, 160.6, 161.6, 175.4. ESI-HRMS calcd for C26H29O4 [M − H]− 405.20604, found 405.20626. HPLC: tR = 9.82 min, normalization method purity 99.34%. General Procedure C for the Synthesis of Compound 12a− d. To a solution of cajanine in corresponding alcohol (20 mL, in the case of 12d, 1 was dissolved in CH2Cl2 (30 mL), and amine (1.5 equiv) was added) was added DIC (1.5 equiv) at 0 °C. The resulting mixture was stirred at room temperature for a further 4 h, after which the mixture was poured into water and extracted with ethyl acetate (3 × 30 mL). The combined organic layer was washed with 10% HCl, 10% NaHCO3 solution, and brine successively. The obtained organic layer was dried over anhydrous MgSO4. After filtration and concentration, the residue was purified over silica gel to give the target compounds. (E)-Ethyl 2-Hydroxy-4-methoxy-3-(3-methylbut-2-enyl)-6-styrylbenzoate (12a). White solid; yield 70%; mp 84−86 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.38 (t, J = 7.2 Hz, 3H), 1.68 (s, 3H), 1.79 (s, 3H), 3.37 (d, J = 7.2 Hz, 2H), 3.92 (s, 3H), 4.39 (q, J = 7.2 Hz, 2H), 5.21 (t, J = 7.2 Hz, 1H), 6.60 (s, 1H), 6.75 (d, J = 16.0 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 2H), 7.50 (d, J = 7.6 Hz, 2H), 7.77 (d, J = 16.0 Hz, 1H), 11.76 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 14.4, 17.8, 22.2, 25.8, 55.7, 61.4, 102.8, 104.7, 116.6, 122.2, 126.5, 127.6, 128.7, 129.8, 130.8, 131.8, 137.4, 140.4, 161.3, 161.5, 171.6. ESI-HRMS calcd for C23H25O4 [M − H]− 10279



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(E)-Methyl 2,4-Dimethoxy-6-(4-methylstyryl)benzoate (14e). Yield 80%; white solid; mp 142−145 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.36 (s, 3H), 3.82 (s, 3H), 3.88 (s, 3H), 3.92 (s, 3H), 6.40 (s, 1H), 6.76 (s, 1H), 7.01 (d, J = 16.0 Hz, 1H), 7.05 (d, J = 16.0 Hz, 1H), 7.15 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H). MS (ESI) calcd for C19H21O4 [M + H]+ 313, found 313. (E)-Methyl 2-(4-Fluorostyryl)-4,6-dimethoxybenzoate (14f). Yield 80%; white solid; mp 99−101 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.83 (s, 3H), 3.87 (s, 3H), 3.92 (s, 3H), 6.42 (s, 1H), 6.73 (s, 1H), 7.00−7.06 (m, 4H), 7.42−7.45 (m, 2H). MS (ESI) calcd for C18H18FO4 [M + H]+ 317, found 317. Methyl 2,4-Dimethoxy-6-((1E,3E)-4-phenylbuta-1,3-dienyl)benzoate (14g). Yield 80%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.76 (s, 3H), 3.80 (s, 3H), 3.88 (s, 3H), 6.53 (d, J = 2.0 Hz, 1H), 6.54 (d, J = 15.2 Hz, 1H), 6.78 (d, J = 15.2 Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 7.09−7.15 (m, 2H), 7.26 (t, J = 7.6 Hz, 1H), 7.34 (t, J = 7.6 Hz, 2H), 7.52 (d, J = 7.6 Hz, 2H). MS(ESI) calcd for C20H21O4 [M + H]+ 325, found 325. (E)-Methyl 2,4-Dimethoxy-6-(2-(thiophen-2-yl)vinyl)benzoate (14h). Yield 80%; yellow solid; mp 112−114 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.82 (s, 3H), 3.86 (s, 3H), 3.92 (s, 3H), 6.40 (s, 1H), 6.70 (s, 1H), 6.90 (d, J = 15.6 Hz, 1H), 6.99 (t, J = 4.0 Hz, 1H), 7.07 (t, J = 4.0 Hz, 1H), 7.14 (dd, J = 2.8 Hz, 15.6 Hz, 1H), 7.21 (d, J = 4.2 Hz, 1H). (E)-Methyl 2-(But-1-enyl)-4,6-dimethoxybenzoate (14i). Yield 80%; oil. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.06 (t, J = 7.2 Hz, 3H), 2.20 (m, 2H), 3.81 (s, 3H), 3.82 (s, 3H), 3.88 (s, 3H), 6.21 (m, 1H), 6.34 (d, J = 16.0 Hz, 1H), 6.35 (d, J = 2.0 Hz, 1H), 6.59 (d, J = 2.0 Hz, 1H). (E)-Methyl 2,4-Dimethoxy-6-(2-phenylprop-1-enyl)benzoate (14j). Yield 80%; white solid; mp 90−93 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.19 (s, 3H), 3.31 (s, 3H), 3.76 (s, 3H), 3.90 (s, 3H), 5.84 (d, J = 1.6 Hz, 1H), 6.20 (d, J = 1.6 Hz, 1H), 6.47 (s, 1H), 7.16−7.25 (m, 5H). MS(ESI) calcd for C19H21O4 [M + H]+ 313, found 313. (E)-Methyl 2-(4-(tert-Butyldimethylsilyloxy)styryl)-4,6-dimethoxybenzoate (14k). Yield 75%; white solid; mp 105−106 °C. 1H NMR (400 MHz, CDCl3) δ (ppm) 0.21 (s, 6H), 0.99 (s, 9H), 3.82 (s, 3H), 3.87 (s, 3H), 3.92 (s, 3H), 6.39 (d, J = 2.0 Hz, 2H), 6.74 (d, J = 2.0 Hz, 2H), 6.82 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 16.0 Hz, 1H), 7.01 (d, J = 16.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H). General Procedure E for the Selective Demethylation. To a solution of compounds 14a−k (1 equiv) in anhydrous CH2Cl2 (20 mL) was added a solution of BCl3 in CH2Cl2 (1.3 equiv) at −78 °C and the mixture was slowly warmed to room temperature for another 2 h, after which the reaction was quenched by the addition of a solution of NaHCO3 (10 mL). The organic layer was separated and dried over anhydrous MgSO4. After filtration and concentration, the residue was purified over silica gel to afford the target compounds. (E)-Methyl 2-(2-Chlorostyryl)-6-hydroxy-4-methoxybenzoate (15b). Yield 83%; white solid; mp 118−120 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.86 (s, 3H), 3.95 (s, 3H), 6.46 (d, J = 2.4 Hz, 1H), 6.67 (d, J = 2.4 Hz, 1H), 7.18 (d, J = 16.0 Hz, 1H), 7.20−7.26 (m, 2H), 7.40 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 16.0 Hz, 1H), 11.66 (s, 1H). MS (ESI) calcd for C17H17ClO4 [M + H]+ 319, found 319. (E)-Methyl 2-(2-Methylstyryl)-6-hydroxy-4-methoxybenzoate (15c). Yield 83%; white solid; mp 93−95 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.42 (s, 3H), 3.86 (s, 3H), 3.95 (s, 3H), 6.45 (d, J = 2.4 Hz, 1H), 6.63 (d, J = 2.4 Hz, 1H), 7.01 (d, J = 16.0 Hz, 1H), 7.19− 7.23 (m, 3H), 7.56 (d, J = 6.4 Hz, 1H), 7.58 (d, J = 16.0 Hz, 1H), 11.71 (s, 1H). MS(ESI) calcd for C18H19O4 [M + H]+ 299, found 299. (E)-Methyl 2-(3-Methylstyryl)-6-hydroxy-4-methoxybenzoate (15d). Yield 83%; white solid; mp 96−100 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.38 (s, 3H), 3.85 (s, 3H), 3.93 (s, 3H), 6.43 (d, J = 2.0 Hz, 1H), 6.62 (d, J = 2.0 Hz, 1H), 6.77 (d, J = 16.0 Hz, 1H), 7.10 (d, J = 7.2 Hz, 1H), 7.27−7.32 (m, 3H), 7.67 (d, J = 16.0 Hz, 1H), 11.67 (s, 1H). MS (ESI) calcd for C18H19O4 [M + H]+ 299, found 299. (E)-Methyl 2-(4-Methylstyryl)-6-hydroxy-4-methoxybenzoate (15e). Yield 83%; white solid; mp 69−71 °C. 1H NMR (400 MHz,

365.17474, found 365.17512. HPLC: tR = 8.13 min, normalization method purity 99.77%. (E)-Isopropyl 2-Hydroxy-4-methoxy-3-(3-methylbut-2-enyl)-6styrylbenzoate (12b). White solid; yield 73%; mp 67−69 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.35 (d, J = 6.4 Hz, 6H), 1.68 (s, 3H), 1.79 (s, 3H), 3.37 (d, J = 6.8 Hz, 2H), 3.92 (s, 3H), 5.22 (t, J = 6.8 Hz, 1H), 5.29 (q, J = 6.4 Hz, 1H), 6.59 (s, 1H), 6.73 (d, J = 16.0 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 2H), 7.50 (d, J = 7.6 Hz, 2H), 7.76 (d, J = 16.0 Hz, 1H), 11.83 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.7, 22.1, 25.6, 55.3, 69.3, 102.6, 104.9, 116.7, 122.2, 126.4, 127.6, 128.7, 129.5, 130.9, 131.8, 137.5, 140.3, 161.2, 161.5, 171.1. ESI-HRMS calcd for C24H27O4 [M − H]− 379.19039, found 379.19068. HPLC: tR = 8.81 min, normalization method purity 97.26%. (E)-tert-Butyl 2-Hydroxy-4-methoxy-3-(3-methylbut-2-enyl)-6styrylbenzoate (12c). White solid; yield 73%; mp 130−132 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.55 (s, 9H), 1.68 (s, 3H), 1.79 (s, 3H), 3.37 (d, J = 7.2 Hz, 2H), 3.90 (s, 3H), 5.22 (t, J = 7.2 Hz, 1H), 6.55 (s, 1H), 6.71 (d, J = 16.0 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 2H), 7.49 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 16.0 Hz, 1H), 11.93 (s, 1H). 13C NMR (126 MHz, CDCl3) δ (ppm): 17.8, 22.1, 25.8, 28.5, 55.6, 82.9, 102.8, 106.1, 116.7, 122.2, 126.4, 127.5, 128.6, 129.4, 131.2, 131.7, 137.4, 140.2, 160.8, 161.5, 171.0. ESI-HRMS calcd for C25H29O4 [M − H]− 393.20604, found 393.20652. HPLC: tR = 9.55 min, normalization method purity 98.96%. (E)-N-Cyclopropyl-2-hydroxy-4-methoxy-3-(3-methylbut-2-enyl)6-styrylbenzamide (12d). White solid; yield 65%; mp 121−123 °C. 1 H NMR (400 MHz, CDCl3) δ (ppm): 0.48 (m, 2H), 0.83 (m, 2H), 1.68 (s, 3H), 1.79 (s, 3H), 2.88 (m, 1H), 3.37 (d, J = 7.2 Hz, 2H), 3.89 (s, 3H), 5.22 (t, J = 7.2 Hz, 1H), 6.23 (br, 1H), 6.46 (s, 1H), 6.89 (d, J = 16.0 Hz, 1H), 7.24 (d, J = 16.0 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.41 (t, J = 7.6 Hz, 2H), 7.47 (d, J = 7.6 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ (ppm): 7.1,17.8, 22.2, 22.9, 25.8, 55.4, 102.4, 108.4, 117.4, 122.1, 126.4, 128.0, 128.4, 129.1, 131.8, 132.5, 136.1, 136.3, 159.7, 160.1, 172.0. ESI-HRMS calcd for C24H26O3N [M − H]− 376.19072, found 376.19024. HPLC: tR = 4.31 min, normalization method purity 99.61%. General Procedure D for the Horner−Wadsworth−Emmons Reaction (14a−k). Compounds 13 (1 equiv) and aldehydes (1.3 equiv) were dissolved in anhydrous DME (20 mL), and NaH (1.4 equiv) was added to the solution carefully at room temperature. The resulting mixture was heated gently to 80 °C under N2 atmosphere and was kept for another 2 h. After that, most of the DME was evaporated away under vacuum and the residue was dissolved in ether (40 mL) and washed with water and brine successively. The obtained organic layer was dried over anhydrous MgSO4. After filtration and concentration, the residue was recrystallized from ethyl acetate and petroleum ether to afford the target compounds. The double bond formed was assigned to “E” configuration as verified by the coupling constant (16.0 Hz) between the two protons on the double bond. (E)-Methyl 2-(2-Chlorostyryl)-4, 6-dimethoxybenzoate (14b). Yield 80%; white solid; mp 85−87 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.83 (s, 3H), 3.89 (s, 3H), 3.92 (s, 3H), 6.44 (s, 1H), 6.80 (s, 1H), 7.08 (d, J = 16.0 Hz, 1H), 7.19−7.25 (m, 2H), 7.38 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 16.0 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H). MS (ESI) calcd for C18H18ClO4 [M + H]+ 333, found 333. (E)-Methyl 2,4-Dimethoxy-6-(2-methylstyryl)benzoate (14c). Yield 80%; white solid; mp 69−71 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.41 (s, 3H), 3.83 (s, 3H), 3.88 (s, 3H), 3.91 (s, 3H), 6.42 (d, J = 2.4 Hz, 1H), 6.76 (d, J = 2.4 Hz, 1H), 6.97 (d, J = 16.0 Hz, 1H), 7.20−7.25 (m, 3H), 7.24 (d, J = 16.0 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H). MS (ESI) calcd for C19H21O4 [M + H]+ 313, found 313. (E)-Methyl 2,4-Dimethoxy-6-(3-methylstyryl)benzoate (14d). Yield 80%; white solid; mp 72−74 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.36 (s, 3H), 3.82 (s, 3H), 3.87 (s, 3H), 3.92 (s, 3H), 6.40 (s, 1H), 6.76 (s, 1H), 7.06 (d, J = 16.0 Hz, 1H), 7.09 (d, J = 16.0 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 7.24−7.26 (m, 3H). MS (ESI) calcd for C19H21O4 [M + H]+ 313, found 313. 10280



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CDCl3) δ (ppm): 2.38 (s, 3H), 3.85 (s, 3H), 3.94 (s, 3H), 6.43 (s, 1H), 6.63 (s, 1H), 6.78 (d, J = 16.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 16.0 Hz, 1H), 11.68 (s, 1H). MS (ESI) calcd for C18H19O4 [M + H]+ 299, found 299. (E)-Methyl 2-(4-Fluorostyryl)-6-hydroxy-4-methoxybenzoate (15f). Yield 83%; white solid; mp 85−87 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.85 (s, 3H), 3.94 (s, 3H), 6.44 (s, 1H), 6.60 (s, 1H), 6.74 (d, J = 16.0 Hz, 1H), 7.06 (t, J = 8.4 Hz, 2H), 7.43−7.47 (m, 2H), 7.60 (d, J = 16.0 Hz, 1H), 11.66 (s, 1H). MS(ESI) calcd for C17H16FO4 [M + H]+ 303, found 303. (E)-Methyl 2-Hydroxy-4-methoxy-6-(2-(thiophen-2-yl)vinyl)benzoate (15h). Yield 83%; white solid; mp 105−107 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.84 (s, 3H), 3.96 (s, 3H), 6.43 (s, 1H), 6.60 (s, 1H), 6.95 (d, J = 16.0 Hz, 1H), 7.01 (d, J = 4.4 Hz, 1H), 7.06 (t, J = 4.4 Hz, 1H), 7.21 (d, J = 4.4 Hz, 1H), 7.55 (d, J = 16.0 Hz, 1H), 11.68 (s, 1H). MS(ESI) calcd for C15H15SO4 [M + H]+ 311, found 311. (E)-Methyl 2-(But-1-enyl)-6-hydroxy-4-methoxybenzoate (15i). Yield 83%. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.10 (t, J = 7.2 Hz, 3H), 2.22 (m, 2H), 3.83 (s, 3H), 3.91(s, 3H), 5.97 (m, 1H), 6.37 (d, J = 2.4 Hz, 1H), 6.46 (d, J = 2.4 Hz, 1H), 6.91 (d, J = 16.0 Hz, 1H), 11.62 (s, 1H). (E)-Methyl 2-Hydroxy-4-methoxy-6-(2-phenylprop-1-enyl)benzoate (15j). Yield 83%; white solid; mp 90−93 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.19 (s, 3H), 3.49 (s, 3H), 3.94 (s, 3H), 5.88 (d, J = 1.6 Hz, 1H), 6.23 (d, J = 1.6 Hz, 1H), 6.73 (s, 1H), 7.06− 7.20 (m, 5H), 11.60 (s, 1H). MS(ESI) calcd for C18H19O4 [M + H]+ 299, found 299. (E)-Methyl 2-(4-(tert-Butyldimethylsilyloxy)styryl)-4,6-dimethoxybenzoate (15k). Yield 80%; mp 80−81 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.22 (s, 6H), 1.00 (s, 9H), 3.85 (s, 3H), 3.95 (s, 3H), 6.42 (d, J = 2.4 Hz, 1H), 6.62 (d, J = 2.4 Hz, 1H), 6.76 (d, J = 16.0 Hz, 1H), 6.84 (d, J = 8.0, 2H), 7.37 (dd, J = 8.0 Hz, 2H), 7.57 (d, J = 16.0 Hz, 1H), 11.67 (s, 1H). Methyl 2-Hydroxy-4-methoxy-6-phenethylbenzoate (16). Compound 15a (1 equiv) was dissolved in EtOH (20 mL), and 5% Pd/C (10 wt %) was added. The resulting mixture was hydrogenated under 70 psi hydrogen atmosphere at room temperature for 4 h. After that, the mixture was filtered and the filtrate was condensed in vacuum to give the target compound 16 as a white solid. Yield 95%; mp 45−47 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.75−2.77 (m, 2H), 2.82−2.84 (m, 2H), 3.70 (s, 3H), 3.80 (s, 3H), 6.30 (d, J = 2.4 Hz, 1H), 6.33 (d, J = 2.4 Hz, 1H), 7.18 (m, 3H), 7.27 (t, J = 7.6 Hz, 2H), 10.35 (s, 1H). MS (ESI) [M + H]+ m/z: 287. Methyl 3-Chloro-4,6-dimethoxy-2-methylbenzoate (18). To a solution of compound 17 (1 equiv) in ether (20 mL) was added SO2Cl2 (1.5 equiv) at room temperature, and the resulting mixture was heated under reflux for 4 h. Then the mixture was washed with water, saturated NaHCO3 solution, and brine successively. The organic layer was dried over anhydrous MgSO4. After filtration and concentration, the residue was purified over silica gel to afford the target compound 18 as a white solid. Yield 85%; mp 106−108 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.31 (s, 3H), 3.84 (s, 3H), 3.89 (s, 3H), 3.92 (s, 3H), 6.40 (s, 1H). Methyl 2-(Bromomethyl)-3-chloro-4,6-dimethoxybenzoate (19). Compound 18 (1 equiv) was dissolved in anhydrous CCl4 (100 mL) and the solution was heated under reflux in an N2 atmosphere and was irradiated with daylight lamp (200 W). Then a solution of Br2 (1.05 equiv) in CCl4 was added dropwise. After the completion of addition, the reaction mixture was stirred for another 2 h under reflux. The mixture was then cooled to room temperature and washed with water, saturated NaHCO3 solution, and brine successively. The obtained organic layer was dried over anhydrous MgSO4. After filtration and concentration, the residue was purified over silica gel to afford the target compound 19 as a white solid. Yield 85%; mp 106−108 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.86 (s, 3H), 3.94 (s, 6H), 4.62 (s, 2H), 6.50 (s, 1H). MS (ESI) [M + H]+ m/z: 323. Methyl 3-Chloro-2-((diethoxyphosphoryl)methyl)-4,6-dimethoxybenzoate (20). A mixture of compound 19 (1 equiv) and triethyl phosphite (3 equiv) was heated under reflux for 6 h. Then, the excess

of triethyl phosphite was evaporated away under vacuum, and the obtained residue was purified over silica gel to afford the title compound as a white solid. Yield 90%; mp 91−93 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.26 (t, J = 7.2 Hz, 6H), 3.70 (d, J = 22.8 Hz, 2H), 3.85 (s, 3H), 3.90 (s, 3H), 3.92 (s, 3H), 4.05 (m, 4H), 6.46 (s, 1H). MS (ESI) [M + H]+ m/z: 381. (E)-Methyl 3-Chloro-4,6-dimethoxy-2-styrylbenzoate (21). Compound 21 was obtained as a white solid according to general procedure D. Yield 70%; mp 85−87 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.78 (s, 3H), 3.89 (s, 3H), 3.95 (s, 3H), 6.49 (s, 1H), 6.79 (d, J = 16.0 Hz, 1H), 7.22 (d, J = 16.0 Hz, 1H), 7.28 (t, J = 7.2 Hz, 1H), 7.35 (t, J = 7.2 Hz, 2H), 7.48 (d, J = 7.2 Hz, 2H). MS(ESI) [M + H]+ m/z: 333. (E)-Methyl 3-Chloro-6-hydroxy-4-methoxy-2-styrylbenzoate (22). Compound 22 was obtained as a white solid according to general procedure E. Yield 80%; mp 111−113 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.80 (s, 3H), 3.93 (s, 3H), 6.38 (d, J = 16.0 Hz, 1H), 6.50 (s, 1H), 7.20 (d, J = 16.0 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.38 (t, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 11.23 (s, 1H). MS (ESI) [M + H]+ m/z: 319. (E)-Methyl 3-Chloro-6-hydroxy-4-methoxy-5-prenyl-2-styrylbenzoate (23). Compound 23 was obtained as a yellow oil according to general procedure A. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.63 (s, 3H), 1.73 (s, 3H), 3.36 (d, J = 6.8 Hz, 2H), 3.73 (s, 3H), 3.79 (s, 3H), 5.15 (t, J = 6.8 Hz, 1H), 6.38 (d, J = 16.4 Hz, 1H), 7.14 (d, J = 16.4 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.30 (t, J = 8.0 Hz, 2H), 7.41(d, J = 8.0 Hz, 2H), 10.85 (s, 1H). ESI-HRMS calcd for C22H22O4Cl [M − H]− 385.12011, found 385.12080. (E)-Methyl 3-Bromo-4,6-dimethoxy-2-styrylbenzoate (24). To a solution compound 14a (1 equiv) in MeOH (25 mL) was added Br2 (1.05 equiv) slowly at 0 °C. After the completion of addition, the mixture was stirred for another 2 h at room temperature, after which the majority of MeOH was evaporated away under vacuum. The obtained residue was dissolved in ether (40 mL) and washed with water and saturated NaHCO3 solution successively. The organic layer was dried over anhydrous MgSO4. After filtration and concentration, the crude product was recrystallized from ethyl acetate and petroleum ether to afford the title compound as a white solid. Yield 90%; mp 156−158 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.80 (s, 3H), 3.86 (s, 3H), 3.93 (s, 3H), 6.39 (d, J = 15.6 Hz, 1H), 6.50 (s, 1H), 7.20 (d, J = 15.6 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 2H), 7.49 (d, J = 7.6 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ (ppm): 52.4, 56.3, 56.5, 95.1, 104.5, 116.7, 126.6, 126.8, 128.3, 128.6, 134.4, 136.6, 138.0, 156.9, 157.3, 167.8. (E)-Methyl 4,6-Dimethoxy-3-prenyl-2-styrylbenzoate (25). A solution of compound 24 (1 equiv), tributylprenyl stanane (1.4 equiv), and Pd(PPh3)4 (10% equiv) in anhydrous DMF (20 mL) was stirred at 110 °C for 12 h. After that, the mixture was poured into water and extracted with ether (3 × 40 mL). The combined organic layer was washed with brine and dried over anhydrous MgSO4. After filtration and concentration, the obtained residue was purified over silica gel to afford compound 25 as a colorless oil. Yield 50%. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.59 (s, 3H), 1.61 (s, 3H), 3.25 (d, J = 6.4 Hz, 2H), 3.68 (s, 3H), 3.80 (s, 6H), 5.01 (t, J = 6.4 Hz, 1H), 6.37 (s, 1H), 6.58 (d, J = 16.0 Hz, 1H), 7.06 (d, J = 16.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.27 (t, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H). (E)-Methyl 6-Hydroxy-4-methoxy-3-(3-methylbut-2-enyl)-2-styrylbenzoate (26). Compound 26 was obtained as a pale-brown solid according to general procedure A. Yield 85%; mp 98−100 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.59 (s, 6H), 3.23 (d, J = 6.4 Hz, 2H), 3.62 (s, 3H), 3.76 (s, 3H), 4.99 (t, J = 6.4 Hz, 1H), 6.42 (d, J = 16.0 Hz, 1H), 6.47 (s, 1H), 7.18 (d, J = 16.0 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 7.36 (t, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 9.94 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 17.7, 24.8, 25.4, 51.7, 22.6, 98.2, 112.4, 118.7, 123.1, 126.2, 127.8, 128.7, 130.0, 132.6, 136.7, 136.8, 154.8, 158.7, 168.8. ESI-HRMS calcd for C22H23O4 [M − H]− 351.15909, found 351.15963. Methyl 3-Bromo-2,4-dimethoxy-6-methylbenzoate (28). A mixture of compound 27 (1 equiv), anhydrous K2CO3 (3 equiv), and MeI (3 equiv) in DMF (20 mL) was stirred at 60 °C for 2 h, after which the mixture was filtered and the filtrate was concentrated. The 10281



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obtained residue was purified over silica gel to give the target compound as a white solid. Yield 85%; mp 56−57 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.30 (s, 3H), 3.86 (s, 3H), 3.89 (s, 3H), 3.91 (s, 3H), 6.52 (s, 1H). Methyl 3-Bromo-6-(bromomethyl)-2, 4-dimethoxybenzoate (29). Compound 29 was obtained as a white solid by using a similar method used to synthesize compound 19. Yield 85%; mp 142−145 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.90 (s, 3H), 3.94 (s, 3H), 3.96 (s, 3H), 4.53 (s, 2H), 6.73 (s, 1H). Methyl 3-Bromo-6-((diethoxyphosphoryl)methyl)-2,4-dimethoxybenzoate (30). Compound 30 was obtained as colorless oil by using a similar method used to synthesize compound 20. Yield 90%. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.26 (t, J = 7.2 Hz, 6H), 3.28 (d, J = 22.4 Hz, 2H), 3.87 (s, 3H), 3.92 (s, 6H), 4.02 (q, J = 7.2 Hz, 4H), 6.78 (s, 1H). Methyl 6-((Diethoxyphosphoryl)methyl)-2,4-dimethoxy-3-(3methylbut-2-enyl)benzoate (31). Compound 31 was obtained as colorless oil by using a similar method used to synthesize compound 25. Yield 70%. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.26 (t, J = 7.2 Hz, 6H), 1.62 (s, 3H), 1.75 (s, 3H), 3.30 (m, 4H), 3.73 (s, 3H), 3.84 (s, 3H), 3.90 (s, 3H), 4.02 (q, J = 7.2 Hz, 4H), 5.13 (t, J = 6.8 Hz, 1H), 6.73 (s, 1H). Methyl 6-((Diethoxyphosphoryl)methyl)-3-isopentyl-2,4-dimethoxybenzoate (32). Compound 32 was obtained as a color less oil by using a similar method used to synthesize compound 16. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.93 (d, J = 6.4 Hz, 6H), 1.26 (t, J = 7.2 Hz, 6H), 1.37 (m, 2H), 1.63 (m, 1H), 2.58 (t, J = 8.0 Hz, 2H), 3.32 (d, J = 21.6 Hz, 2H), 3.76 (s, 3H), 3.83 (s, 3H), 3.91 (s,3H), 4.02 (q, J = 7.2 Hz, 4H), 6.71 (s, 1H). (E)-Methyl 3-Isopentyl-2,4-dimethoxy-6-styrylbenzoate (33). Compound 33 was obtained as a colorless oil according to general procedure D. Yield: 84%. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.95 (d, J = 6.8 Hz, 6H), 1.39 (m, 2H), 1.62 (m, 1H), 2.62 (t, J = 8.0 Hz, 2H), 3.87 (s, 3H), 3.90 (s, 3H), 3.94 (s, 3H), 6.90 (s, 1H), 7.04 (d, J = 16.0 Hz, 1H), 7.14 (d, J = 16.0 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.6 Hz, 2H), 7.48 (d, J = 7.6 Hz, 2H). (E)-Methyl 3-(3,7-Dimethylocta-2,6-dienyl)-2-hydroxy-4-methoxy-6-phenethyl-benzoate (34). Compound 34 was obtained as a yellow oil according to general procedure A. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.57 (s, 3H), 1.64 (s, 3H), 1.78 (s, 3H), 1.95−2.08 (m, 4H), 3.78 (d, J = 6.8 Hz, 2H), 3.91 (s, 3H), 3.92 (s, 3H), 5.07 (t, J = 6.8 Hz, 1H), 5.22 (t, J = 6.8 Hz, 1H), 6.61 (s, 1H), 6.78 (d, J = 16.0 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 2H), 7.50 (d, J = 7.6 Hz, 2H), 7.72 (d, J = 16.0 Hz, 1H), 11.66 (s, 1H). The Calculation of AlogP Values. The chemical structure of the compound was constructed in ChemDraw, and was saved as “.mol” file, which was imported into Discovery Studio 3.0. The “Caculate Molecular Properties” model was employed for the caculation of AlogP values. Biology. Cells and Virus. Human hepatoma Huh7.5 cells and the plasmid pFL-J6/JFH/JC1 containing the full-length chimeric HCV complementary DNA (cDNA) were kindly provided by the Vertex Pharmaceuticals Inc. (Boston, USA). Huh7.5 cells and GS4.3 replicon cells, a human hepatoma Huh7 cell line carrying an HCV subgenomic replicon I377-3′del.S, were cultured as described previously.24 The S282T, D168V, and A156T mutant HCV variants were prepared with plasmids pHCV-S282T, pHCV-D168V, and pHCV-A156T derived from the plasmid pFL-J6/JFH/JC1, respectively. Wild-type and mutant HCV virus stock were prepared as described.25 Agents. Telaprevir, sofosbuvir, and simeprevir were purchased from the MedChemExpress (Princeton, NJ). MG132 (M7449, 200 μL) was purchased from Sigma. The siRNA for CSGalNAcT-1 (sc-77837) and negative control siRNA (siRNA-A, sc-37007), the mAb to CSGalNAcT-1(sc-87539), and mouse antigoat secondary antibody (sc-2354) were from the Santa Cruz Biotechnology. The mAbs to HCV Core (ab2740) and to HCV NS3 (ab13830) were from Abcam, Co. Ltd. The mAb to β-actin (3700S) and antimouse secondary antibody (7076S) were from the Cell Signaling Technology, Inc. Plasmid. Total RNAs were extracted from Huh7.5 cells and reversetranscripted into cDNA. The DNA of CSGalNAcT-1 was amplified

with primers 5′-CGCGGATCCATGATGATGGTTCGCCGGGG-3′ (sense) and 5′-CCGCTCGAGTCATGTTTTTTTGCTACTTGTCTTCTGT-3′ (antisense) with BamHI and XhoI restriction sites using the cDNA template. This product was then cloned into a pcDNA3.1(+) vector as an expression plasmid (pcDNA3.1(+)CSGalNAcT-1). Anti-HCV Activity Assay in Vitro. Huh7.5 cells or GS4.3 cells were seeded at a density of 3 × 104/cm2. After 24 h incubation, the Huh7.5 cells were incubated with wild-type or mutant HCV virus stock and simultaneously treated with compound or solvent control, or the GS4.3 cells were treated with the compound or solvent control. After 72 h, intracellular RNA was extracted with RNeasy Mini Kit, and total intracellular proteins were extracted with CytoBuster protein extraction reagent (Novagen) with 1 mM protease inhibitor cocktail. RNAs and proteins were analyzed by real-time quantitative reversetranscript PCR (qRT-PCR) and Western blot, respectively. Compound cytotoxicity in Huh7.5 cells or GS4.3 replicon cells was evaluated at 72 h with the tetrazolium (MTT, Amresco) assay. Influence of CSGalNAcT-1 of HCV Infection. Huh7.5 cells were infected with HCV (45 IU/cell) for 48 h. Then total intracellular proteins were extracted with PER and analyzed by Western blot. Destabilization of CSGalNAcT-1 Protein by 1. Huh7.5 cells were cotreated with 1 and a proteasome inhibitor MG132. Cells treated with 1 or MG132 only were used as controls. After 24 h, total intracellular proteins were extracted with PER and analyzed by Western blot.30 Overexpression and Silence of CSGalNAcT-1. After being seeded 24 h, the Huh7.5 cells were transfected with 0.2 μg of pcDNA3.1(+)CSGalNAcT-1 plasmid or control plasmid pcDNA3.1(+) using HD transfection reagent (Promega) or transfected with 25 or 100 pmol of siRNA for CSGalNAcT-1 or negative control siRNA-A using RNAiMAX transfection reagent. At 6 h, the cells were washed and then infected with HCV (45 IU/cell), and intracellular RNAs and proteins were extracted in 72 h and detected with qRT-PCR and WB, respectively. Influence on HCV Entry of CSGalNAcT-1. After being seeded 24 h, the Huh7.5 cells were transfected with 0.2 μg of pcDNA3.1(+)CSGalNAcT-1 plasmid or control plasmid pcDNA3.1(+) using HD transfection reagent (Promega). After 48 h, the cells were infected with HCV (150 IU/cell) for 2 h, followed by washing with PBS and continuously culturing with fresh medium, intracellular proteins and RNAs were extracted for detecting CSGalNAcT-1 and actin in 24 h and for detecting HCV core protein and HCV RNA in 72 h. Influence on HCV Entry of 1. After being seeded 24 h, the Huh7.5 cells were infected with HCV (150 IU/cell) and simultaneously treated with compound or solvent control for 2 h. The cells were then washed with PBS and cultured with fresh medium. Intracellular proteins and RNAs were extracted in 72 h and detected with Western blot and qRT-PCR, respectively. The Synergistic Effects against HCV Infections of 1 with DAAs. Huh7.5 cells were treated with different concentrations of 1 or DAA (simeprevir, sofosbuvir, or daclatasvir) only or cotreated with 1 and DAA. The cells were then simultaneously infected with HCV (45 IU/ cell). After 72 h of incubation, the intracellular RNAs and proteins were extracted and analyzed with qRT-PCR and Western blot, respectively. The combination index (CI) was calculated by the Chou−Talalay method using CompuSyn version 1.0, and CI > 1 indicates antagonism, CI = 1 indicates addition, while CI < 1 suggests synergy between the two drugs used.31,32.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01301. Molecular formula strings (CSV) 10282



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a group of real-world Japanese patients chronically infected with HCV genotype 1b. Hepatol. Int. 2015, 9, 424−430. (b) Sarrazin, C. The importance of resistance to direct antiviral drugs in HCV infection in clinical practice. J. Hepatol. 2016, 64, 486−504. (12) Koehn, F. E.; Carter, G. T. The evolving role of natural products in drug discovery. Nat. Rev. Drug Discovery 2005, 4, 206−220. (13) Ji, X.-y.; Xue, S.-t.; Zheng, G.-h.; Han, Y.-x.; Liu, Z.-y.; Jiang, J.d.; Li, Z.-r. Total synthesis of cajanine and its antiproliferative activity against human hepatoma cells. Acta Pharm. Sin. B 2011, 1, 93−99. (14) Huang, M.-Y.; Lin, J.; Lu, K.; Xu, H.-G.; Geng, Z.-Z.; Sun, P.-H.; Chen, W.-M. Anti-inflammatory effects of cajaninstilbene acid and its derivatives. J. Agric. Food Chem. 2016, 64, 2893−2900. (15) (a) Zheng, Y. Y.; Yang, J.; Chen, D. H.; Sun, L. Effects of the stilbene extracts from Cajanus cajan L. on ovariectomy-induced bone loss in rats. Yao Xue Xue Bao 2007, 42, 562−565. (b) Zheng, Y. Y.; Yang, J.; Chen, D. H.; Sun, L. Effects of the extracts of Cajanus cajan L. on cell functions in human osteoblast-like TE85 cells and the derivation of osteoclast-like cells. Yao Xue Xue Bao 2007, 42, 386−391. (16) (a) Wu, N.; Kong, Y.; Fu, Y.; Zu, Y.; Yang, Z.; Yang, M.; Peng, X.; Efferth, T. In vitro antioxidant properties, DNA damage protective activity, and xanthine oxidase inhibitory effect of cajaninstilbene acid, a stilbene compound derived from pigeon pea [Cajanus cajan (L.) Millsp.] leaves. J. Agric. Food Chem. 2011, 59, 437−443. (b) Kong, Y.; Fu, Y.-J.; Zu, Y.-G.; Liu, W.; Wang, W.; Hua, X.; Yang, M. Ethanol modified supercritical fluid extraction and antioxidant activity of cajaninstilbene acid and pinostrobin from pigeonpea [Cajanus cajan (L.) Millsp.] leaves. Food Chem. 2009, 117, 152−159. (17) (a) Kong, Y.; Fu, Y.-J.; Zu, Y.-G.; Chang, F.-R.; Chen, Y.-H.; Liu, X.-L.; Stelten, J.; Schiebel, H.-M. Cajanuslactone, a new coumarin with anti-bacterial activity from pigeon pea [Cajanus cajan (L.) Millsp.] leaves. Food Chem. 2010, 121, 1150−1155. (b) Geng, Z.-Z.; Zhang, J.J.; Lin, J.; Huang, M.-Y.; An, L.-K.; Zhang, H.-B.; Sun, P.-H.; Ye, W.C.; Chen, W.-M. Novel cajaninstilbene acid derivatives as antibacterial agents. Eur. J. Med. Chem. 2015, 100, 235−245. (18) Liang, L.; Luo, M.; Fu, Y.; Zu, Y.; Wang, W.; Gu, C.; Zhao, C.; Li, C.; Efferth, T. Cajaninstilbene acid (CSA) exerts cytoprotective effects against oxidative stress through the Nrf2-dependent antioxidant pathway. Toxicol. Lett. 2013, 219, 254−261. (19) Ruan, C. J.; Si, J. Y.; Zhang, L.; Chen, D. H.; Du, G. H.; Sun, L. Protective effect of stilbenes containing extract-fraction from Cajanus cajan L. on Abeta(25−35)-induced cognitive deficits in mice. Neurosci. Lett. 2009, 467, 159−163. (20) Wang, S.; Li, Y.; Wang, J.; Chen, L.; Zhang, L.; Yu, H.; Hou, T. ADMET evaluation in drug discovery. 12. Development of binary classification models for prediction of hERG potassium channel blockage. Mol. Pharmaceutics 2012, 9, 996−1010. (21) (a) Garcia-Vallvé, S.; Guasch, L.; Tomas-Hernández, S.; del Bas, J. M.; Ollendorff, V.; Arola, L.; Pujadas, G.; Mulero, M. Peroxisome proliferator-activated receptor γ (PPARγ) and ligand choreography: Newcomers take the stage. J. Med. Chem. 2015, 58, 5381−5394. (b) Aidhen, I. S.; Mukkamala, R.; Weidner, C.; Sauer, S. A Common building block for the syntheses of amorfrutin and cajaninstilbene acid libraries toward efficient binding with peroxisome proliferatoractivated receptors. Org. Lett. 2015, 17, 194−197. (c) Laclef, S.; Anderson, K.; White, A. J. P.; Barrett, A. G. M. Total synthesis of amorfrutin A via a palladium-catalyzed migratory prenylation− aromatization sequence. Tetrahedron Lett. 2012, 53, 225−227. (22) Kharasch, M. S.; Brown, H. C. Chlorinations with sulfuryl chloride. II. The peroxide-catalyzed reaction of sulfuryl chloride with ethylenic compounds. J. Am. Chem. Soc. 1939, 61, 3432−3434. (23) Saimoto, H.; Hiyama, T. A general highly efficient access to prenylated phenolic natural products. Synthesis of colletochlorins B and D. Tetrahedron Lett. 1986, 27, 597−600. (24) Gulberti, S.; Jacquinet, J. C.; Chabel, M.; Ramalanjaona, N.; Magdalou, J.; Netter, P.; Coughtrie, M. W.; Ouzzine, M.; FournelGigleux, S. Chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) involved in chondroitin sulfate initiation: Impact of sulfation on activity and specificity. Glycobiology 2012, 22, 561−571.

AUTHOR INFORMATION

Corresponding Authors

*For Z.-R.L.: phone, +86-10-63027185; fax, +86-10-6301-7302; E-mail, [email protected]. *For Z.-G.P.: phone, +86-10-63166129; fax, +86-10-63017302; E-mail, [email protected]. Author Contributions †

X.-Y.J. and J.-H.C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the grants from the National Natural Science Foundation of China (81402827, 81322050, 81321004), Ministry of Education, China (NCET-12-0072), and CAMS Initiative for Innovative Medicine (2016-I2M-1010).

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ABBREVIATION USED DAAs, direct-acting antiviral agents; HTAs, host-targeting antivirals; ALV, alisporivir; CSGalNAcT-1, N-acetylgalactosaminyltransferase 1; DIC, diisopropylcarbodiimide; CS, chondroitin sulfate; CD36, cluster of differentiation 36; Hsc70, heatstress cognate 70; CI, combination index; DRI, dose reduction index

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REFERENCES

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Design and Synthesis of Cajanine Analogues against Hepatitis C

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