Synthesis and Biological Activity of 2',3'-iso-Aryl-abscisic Acid Analogs.

2',3'-iso-Benzoabscisic acid (iso-PhABA), an excellent selective abscisic acid (ABA) analog, was developed in our previous work. In order to find its more structure-activity information, some structural modifications were completed in this paper, including the substitution of phenyl ring and replacing the ring with heterocycles. Thus, 16 novel analogs of iso-PhABA were synthesized and screened with three bioassays, Arabidopsis and lettuce seed germination and rice seedling elongation. Some of them, i.e., 2',3'-iso-pyridoabscisic acid (iso-PyABA) and 2',3'-iso-franoabscisic acid (iso-FrABA), displayed good bioactivities that closed to iso-PhABA and natural (+)-ABA. Some others, for instance, substituted-iso-PhABA, exhibited certain selectivity to different physiological process when compared to iso-PhABA or (+)-ABA. These analogs not only provided new candidates of ABA-like synthetic plant growth regulators (PGRs) for practical application, but also new potential selective agonist/antagonist for probing the specific function of ABA receptors.

1. Introduction

The crucial role of abscisic acid (S-(+)-ABA 1, Figure 1) as a phytohormone in a wide variety of physiological processes of plants, including inhibiting or promoting growth, maintaining bud and seed dormancy, affecting flowering and sex differentiation, as well as conducting environmental stresses response [1,2,3,4,5,6,7,8], motivates numerous scientific investigations into the agricultural application of ABA. However, the widely using of abscisic acid (ABA) has been hindered by its rapid metabolism in plants and light isomerization in vitro [9,10,11,12,13,14,15,16,17]. Focusing on the development of metabolism resistant analogs, significant efforts were devoted by different researchers. For example, methoxyl [18], alkynyl [19], or fluorine atoms [20,21] at the 8′ or 9′-positionm and halogen atoms [22,23] at the 3′-position had been introduced to avoid 8′-hydroxylation and Michael addition, and the corresponding analogs displayed excellent ABA-like activities. Nonetheless, they are too expensive to practical use in field due to their complicated synthetic routes.

Figure 1

Abscisic acid (ABA) and ABA analogs.

A successful case of 8′-hydroxylation resistant analog was 2′,3′-benzoabscisic acid 2 [24,25] (Figure 1), which had excellent bioactivity and relatively efficient synthetic route. Inspired by this compound and iso-ABA 3 [26], in our previous works, we had developed 2′,3′-iso-Benzoabscisic acid (iso-PhABA) 4a [27], which is an excellent and easier preparation ABA analog. The deeply investigations of bioactivity and agonist-receptor interactions for 4a suggested that it is a state-of-art ABA-like regulator and a selective ABA agonist, i.e., PYL10 has the highest inhibitory effect on the phosphatase activity of HAB1 in the presence of (+)-iso-PhABA. Meanwhile, the analysis on the complex crystal structure of iso-PhABA with PYL10 revealed the detailed hydrogen bonds and multiple hydrophobic interactions. Thus, it provides a new and robust precursor for the design of ABA receptor agonists/antagonists. Consequently, in this work, we aimed at the further studies of the analogs of 2′,3′-iso-PhABA 4a to obtain more compounds with good ABA-like bioactivity and selective agonist/antagonists-receptor interactions. Here, we reported the synthesis and biological activity of substituted-iso-PhABA analogs 4b–4d and heterocyclic analogs 5 and 6 (see Figure 2). Their structure-activity relationship (SAR) was also discussed.

Figure 2

Design strategy of 2′,3′-iso-Benzoabscisic acid (iso-PhABA) 4a analogs.

2. Results and Discussion

2.1. Chemistry

As shown in Scheme 1, to obtain target compounds substituted-iso-PhABA (4b–4d) and heterocyclic analogs 2′,3′-iso-heterocylicABA (iso-HetABA, 5a–5d, 6), four different pathways (a–d in Scheme 1) for the preparation of dicarbonyl intermediates 9b–9d, 13a–13d, and 17 were established, respectively. Firstly, the preparation of intermediates 9b–9d (Scheme 1a) were followed the similar pathway of the synthesis of 2′,3′-iso-PhABA 4 [25,27], comprising the vicinal methylation of the carbonyl of subtituted-1-tetralones (step i in Scheme 1a) to obtain 8b–8d and the oxidation of the benzyl carbon with Co(acac)2/t-BuOOH system [28] (step ii in Scheme 1a). Then, for the preparations of pyridine analogs 13a–13d, two different methods were applied for the intermediates 12a–12d. The reaction of 5,5-dimethylcyclohexane-1,3-dione with ammonium acetate afforded 11 (step iii in Scheme 1b), and then intermediate 11 reacted with commercially available 1,1,3,3-tetraethoxylpropane and catalytic amount of p-toluenesulfonic acid to afford 12a (step iv in Scheme 1b), which can oxide by Co(acac)2/t-BuOOH system to afford the dione 13a (step ii in Scheme 1b) [29,30]. The preparation of the diones 13b–13d, on the other hand, were conducted by three steps, including the condensation of methyl ketones with dimethylformamide dimethylacetal to afford 15b–15d (step v in Scheme 1c), the pyridine ring closure between enamino ketones and 5,5-dimethylcyclohexane-1,3-dione in refluxing acetic acid to afford 12b–12d (step vi in Scheme 1c), and the oxidation to afford diones 13b–13d (step ii in Scheme 1c) [31]. Moreover, 2 steps, comprising the furan ring closure between 5,5-dimethylcyclohexane-1,3-dione and ClCH2CHO to afford 16 (step vii in Scheme 1d) and the oxidation to afford dione 17 (step ii in Scheme 1d), were applied for the preparation of furan analog 6 [32,33].

Scheme 1

Synthesis of substitueted-iso-PhABA analogs 5 and heterocyclic analogs substituted-iso-HetABA 6. Reagents and conditions: (i) MeI, NaH, THF; (ii) Co(acac)2, t-BuOOH, acetone, r.t.; (iii) NH4OAc, toluene, reflux; (iv) 1,1,3,3-tetraethoxylpropane, p-toluenesulfonic acid, DMF, reflux; (v) dimethylformamide dimethylacetal, xylene, reflux; (vi) 5,5-dimethylcyclohexane-1,3-dione, HOAc, reflux; (vii) ClCH2CHO, KOH, KI, H2O, r.t.; (viii) n-BuLi, (Z)-3-methylpent-2-en-4-yn-1-ol, THF, −78 °C; (ix) Red-Al reagent, THF, 0 °C; x. (a) Dess-Martin periodinane, CH2Cl2, r.t.; (b) NaClO2, 2-methyl-2-butene, NaH2PO4, t-BuOH/H2O (3:1 in volume), r.t.

With the diones in hands, the synthesis of the title compounds, i.e., substituted-iso-PhABA (4b–4d) and iso-HetABA analogs (5a–5d, 6), were performed following an efficient region-selective nucleophilic addition of alkynyl lithium, which was produced in situ by n-BuLi and (Z)-3-methylpent-2-en-4-yn-1-ol, to the less-steric 4-carbonyl of intermediates 18b–18d, 19a–19d, and 20 (step viii in Scheme 1) [34]. Then, the Red-Al was employed for the trans-selective reduction of alkynol to give enol 21b–21d, 22a–22d, and 23 (step ix in Scheme 1). The final products 4b–4d, 5a–5d, and 6 were yielded by a two steps oxidation (step x in Scheme 1), comprising Dess-Martin oxidation to form the aldehydes and then Lindgren oxidation to give the acids 5 and 6. Furthermore, the alkynol intermediates 18–20 were also followed the two steps oxidation to give the acetylenic acid analogs 24b–24d, 25a, 25b, 25d, and 26 (except 2′-n-Pr-iso-PyABA), since they are good materials for the screening of structural requirement of the trans-double bond of ABA analogs. Meanwhile, the acetylenic iso-PhABA 24a was synthesized following the similar pathway of 24b–24d, with the known intermediate (Z)-(1′-hydroxy-3′,3′-dimethyl-4′-oxo-tetrahydronaphthalene-one-yl)-3-methyl-pentyl-2-em-4-yn-1-ol [27].

2.2. Structure-Activity Relationship

The Arabidopsis, lettuce seed germination and the rice seedling elongation inhibiting tests in vivo were conducted to gain insights into the bioactivity of these iso-PhABA analogs. The (±)-iso-PhABA 4a and (+)-ABA 1 were used as the control agents. The corresponding data of bioactivities were summarized in Table 1. For the comparison between the bioactivities of substituted-iso-PhABA analogs, the overall bioactivity of 4b–4d were very close to those of each other, indicating that the effects of electron-donating and withdrawing groups, as well as their substituted-positions (7′ and 6′) were not crucial for their bioactivity. However, the bioactivities of all the substituted-iso-PhABA 4b–4d were worse than those of the control agents 4a and 1, suggesting that a substituent in the benzene ring of iso-PhABA had a significant impact on the bioactivity. Furthermore, there were some preferences of substituted-iso-PhABA to different physiologic process. Compound 4b–4d observed the highest IC50 values for the rice seedlings elongation, which was the lowest values for both the two control agents 4a and 1, indicating their different preferential nature of bioactivity compared to iso-PhABA.

Table 1

Inhibitory bioactivities of title compounds.

Compound Name	IC50 (μM) a of Bioassays
	Arabidopsis Seed Germination	Lettuce Seed Germination	Rice Seedlings Elongation
(+)-ABA, 1	0.39 ± 0.12	0.93 ± 0.06	0.12 ± 0.02
(±)-iso-PhABA, 4a	0.48 ± 0.04	0.65 ± 0.01	0.20 ± 0.03
7′-OMe-iso-PhABA, 4b	2.17 ± 0.52	1.99 ± 0.25	4.71 ± 0.74
6′-OMe-iso-PhABA, 4c	2.11 ± 0.31	3.03 ± 0.28	5.96 ± 0.36
6′-Br-iso-PhABA, 4d	2.81 ± 0.26	3.42 ± 0.46	3.61 ± 0.05
iso-PyABA, 5a	0.57 ± 0.10	1.83 ± 0.35	0.68 ± 0.23
2′-Me-iso-PyABA, 5b	0.91 ± 0.19	9.28 ± 0.62	1.99 ± 0.27
2′-Pr-iso-PyABA, 5c	4.48 ± 0.29	8.22 ± 0.09	>10 b
2′-Ph-iso-PyABA, 5d	>10 b	14.02 ± 3.77	>10 b
iso-FrABA, 6	0.56 ± 0.06	2.77 ± 0.15	0.63 ± 0.14
acetylenic iso-PhABA, 24a	1.38 ± 0.23	7.44 ± 0.53	4.48 ± 0.52
acetylenic 7′-OMe-iso-PhABA, 24b	>10 b	6.50 ± 0.60	5.75 ± 0.28
acetylenic 6′-OMe-iso-PhABA, 24c	7.87 ± 0.65	8.23 ± 0.44	4.41 ± 0.23
acetylenic 6′-Br-iso-PhABA, 24d	6.51 ± 0.51	8.90 ± 0.62	>10 b
acetylenic iso-PyABA, 25a	7.70 ± 0.18	19.69 ± 0.85	9.80 ± 0.50
acetylenic 2′-Me-iso-PyABA, 25b	3.03 ± 0.13	5.97 ± 0.63	8.94 ± 0.66
acetylenic 2′-Ph-iso-PyABA, 25d	>10 b	23.25 ± 5.07	>10 b
acetylenic iso-FrABA, 26	3.45 ± 0.50	8.08 ± 0.02	4.32 ± 0.28

a Required concentrations to inhibit the germinations or seedling elongation by 50%. b ‘>10’ means that the calculated IC50 values are greater than the highest tested concentration, i.e., 10 μM.

For the bioactivity of the heterocyclic analogs iso-HetABA, the compounds without substituent, i.e., 2′,3′-iso-pyridoabscisic acid (iso-PyABA) 5a and 2′,3′-iso-franoabscisic acid (iso-FrABA) 6, had good overall bioactivities, which were close to the control agents 4a and 1, suggesting the introducing of heteroatom (N for 5a and O for 6) in the aryl ring of iso-PhABA can mostly keep the bioactivity. To study the effect of the size of the substituted group on the bioactivity, compound 5b–5d were designed and synthesized for the bioassays, too. Obviously, the IC50 values of these compounds are decreasing with the increasing size of the substituted, i.e., –Me 5b to –n-Pr 5c to –Ph 5d. Therefore, this evidence validated that a big group in the aryl ring is not preferential for the bioactivity.

In order to acquire more analogs for the discussion of structure-activity relationship, the acetylenic acids 24, 25, and 26 were also synthesized and tested with three bioassays. As shown in Table 1, their bioactivities were significantly worse than those of their corresponding alkene acids 4, 5, and 6, suggesting the importance of the double-bond for the bioactivity. Besides, by the comparison between the acetylenic acids, acetylenic iso-PhABA 24a had the best activity for Arabidopsis seed germination (IC50 = 1.38 μM), acetylenic 2′-Me-iso-PyABA 25b had the best activity for lettuce seed germination (IC50 = 5.97 μM), and acetylenic iso-FrABA 26 had the lowest IC50 value (4.32 μM) for rice seedlings elongation. Meanwhile, like the substituted-iso-PhABA analogs, there were obvious preferences of some acetylenic acids to different physiological process. For example, acetylenic 7′-OMe-iso-PhABA 24b showed a weakest inhibitor on Arabidopsis seed germination (IC50 > 10 μM) in three bioassays; acetylenic 6′-Br-iso-PhABA 24d, on the other hand, had a lowest bioactivity on rice seedlings elongation (IC50 > 10 μM). Hence, these results implied that there might be some difference in ABA signaling transduction between different acetylenic acids, as well as the acetylenic acids and alkene acids.

3. Materials and Methods

3.1. Materials and Instruments

Column chromatography was performed using silica gel (100–200 mesh) (Qindao Haiyang Co., Ltd., Qingdao, China). TLC was performed on GF254 silica gel plates (Qingdao Haiyang Co., Ltd., Qingdao, China). NMR spectra were recorded on a Bruker Avance DPX300 spectrometer (Bruker Corporation, Vienna, Austria) with TMS as the internal standard. HR-MS data were obtained on a Thermo FisherScientific LTQ Orbitrap (Thermo Fisher Scientific, Bremen, Germany) instrcument.

3.2. Synthesis

3.2.1. Synthesis of 9b–9d (Pathway a in Scheme 1, Use 9b as Example)

6-Methoxy-2,2-dimethyl-3,4-dihydronaphthalen-1(2H)-one (8b) [25,27]: To a stirred solution of 6-methoxy-3,4-dihydronaphthalen-1(2H)-one 7b (10.0 g, 56.8 mmol) dissolved in dry tetrahydrofuran (THF, 150 mL) in a 500 mL round bottomed flask was added NaH (11.4 g, 284 mmol, 60% in oil). After stirring the mixture for 1 h under ice-water bath, methyl iodide (32.3 g, 227 mmol) in dry THF (50 mL) was added slowly. The mixture was allowed to stir at r.t. for 16 h. The reaction was quenched by addition of water (slowly and dropwise). The mixture was then extracted with ethyl acetate (3 × 100 mL), washed with water (2 × 100 mL), and dried over anhydrous Na2SO4. Evaporation of the solvent yielded a yellow oil. The residue was subjected to silica gel chromatography using petroleum ether (PE) and ethyl acetate (EtOAc) (10:1) as eluent to afford 6-methoxy-2,2-dimethyl-3,4-dihydronaphthalen-1(2H)-one 8b (10.4 g, yield 90%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.99 (d, J = 8.7 Hz, 1H), 6.79 (dd, J = 8.8, 2.6 Hz, 1H), 6.65 (d, J = 2.5 Hz, 1H), 3.81 (s, 3H), 2.92 (t, J = 6.4 Hz, 2H), 2.00–1.85 (m, 2H), 1.19 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 200.95, 162.86, 145.36, 129.76, 124.49, 112.84, 111.83, 54.84, 40.78, 36.30, 25.65, 24.06.

7-Methoxy-2,2-dimethyl-3,4-dihydronaphthalen-1(2H)-one 8c (yield 93%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.52 (d, J = 2.8 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.04 (dd, J = 8.4, 2.8 Hz, 1H), 3.83 (s, 3H), 2.91 (t, J = 6.3 Hz, 2H), 1.96 (t, J = 6.3 Hz, 2H), 1.21 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 202.71, 158.28, 135.82, 132.10, 129.78, 121.33, 109.77, 55.35, 41.36, 36.76, 30.80, 24.82, 24.28.

7-Br-2,2-dimethyl-3,4-dihydronaphthalen-1(2H)-one 8d (yield 86%) as a red oil. 1H-NMR (300 MHz, CDCl3) δ 7.99 (d, J = 2.2 Hz, 1H), 7.37 (dd, J = 8.2, 2.2 Hz, 1H), 6.96 (d, J = 8.2 Hz, 1H), 2.78 (t, J = 6.3 Hz, 2H), 1.82 (t, J = 6.4 Hz, 2H),, 1.07 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 200.09, 140.92, 134.57, 131.85, 129.54, 129.49, 119.49, 40.32, 35.18, 24.12, 23.11.

6-Methoxy-2,2-dimethyl-2,3-dihydronaphthalene-1,4-dione (9b) [27,28]: To a stirred solution of 6-methoxy-2,2-dimethyl-3,4-dihydronaphthalen-1(2H)-one 8b (10.0 g, 49 mmol), peroxy-t-butanol (t-BuOOH, 62.8 g, 490 mmol, 70% aqueous) and Co(acac)2 (1.26 g, 4.9 mmol) in 150 mL acetone at r.t. for 24 h. The mixture was then extracted with ethyl acetate (3 × 50 mL), washed with water (2 × 50 mL) and dried over anhydrous Na2SO4. Evaporation of the solvent yielded a brown oil. The residue was subjected to silica gel chromatography using PE and EtOAc (5:1) as eluent to afford 6-methoxy-2,2-dimethyl-2,3-dihydronaphthalene-1,4-dione 9b (7.7 g, 72%) as a colorless solid. 1H-NMR (300 MHz, CDCl3) δ 8.04 (d, J = 8.7 Hz, 1H), 7.42 (d, J = 2.7 Hz, 1H), 7.23 (dd, J = 8.7, 2.7 Hz, 1H), 3.94 (s, 3H), 2.93 (s, 2H), 1.31 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 200.14, 196.37, 164.01, 136.94, 129.89, 127.07, 121.61, 108.31, 55.82, 52.21, 45.21, 25.90.

7-Methoxy-2,2-dimethyl-2,3-dihydronaphthalene-1,4-dione 9c (74%) as a colorless solid. 1H-NMR (300 MHz, CDCl3) δ 7.99 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 2.7 Hz, 1H), 7.21 (dd, J = 8.6, 2.7 Hz, 1H), 3.94 (s, 3H), 2.89 (s, 2H), 1.31 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 201.43, 194.85, 164.42, 135.81, 128.53, 128.41, 121.05, 109.85, 55.81, 51.66, 45.60, 30.84, 25.77.

7-Br-2,2-dimethyl-2,3-dihydronaphthalene-1,4-dione 9d (69%) as a red solid. 1H-NMR (300 MHz, CDCl3) δ 8.20 (dd, J = 1.9, 0.6 Hz, 1H), 8.00–7.69 (m, 2H), 2.92 (s, 2H), 1.31 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 199.96, 195.16, 137.01, 134.84, 133.50, 130.59, 129.89, 127.96, 51.77, 45.77, 25.68.

3.2.2. Synthesis of 13a (Pathway b in Scheme 1)

3-Amino-5,5-dimethylcyclohex-2-enone (11) [29]: 5,5-dimethylcyclohexane-1,3-dione 10 (7.0 g, 50 mmol) was added to a mixture of ammonium acetate (3.85 g, 50 mmol) in dry toluene (100 mL). The mixture was heated for 5 h under reflux using a Dean–Stark water separator. The red oily layer formed was then separated and recrystallized with ethyl acetate to afford 3-amino-5,5-dimethylcyclohex-2-enone 11 (5.9 g, yield 85%) as a yellow solid. 1H-NMR (300 MHz, DMSO-d6) δ 6.67 (s, 2H), 4.90 (s, 1H), 2.11 (s, 2H), 1.90 (s, 2H), 0.96 (s, 6H).

7,7-Dimethyl-7,8-dihydroquinolin-5(6H)-one (12a) [29]: A solution of 1,1,3,3-tetraethoxylpropane (10 mL, 40 mmol), 3-amino-5,5-dimethylcyclohex-2-enone 11 (5.6 g, 40 mmol) and a catalytical amount of p-toluenesulfonic acid hydrate in DMF (50 mL) was heated under reflux for 18 h (monitored by TLC). The solvent was distilled in vacuo, the residue neutralized with NaHCO3, extracted with EtOAc (3 × 100 mL) and dried (anhydrous Na2SO4). Column chromatography using PE and EtOAc (6:1) as eluent to afford 7,7-dimethyl-7,8-dihydroquinolin-5(6H)-one 12a (2.1 g, yield 30%) as a yellow solid. 1H-NMR (300 MHz, CDCl3) δ 8.71 (d, J = 4.7 Hz, 1H), 8.25 (d, J = 7.8 Hz, 1H), 7.30 (dd, J = 7.8, 4.8 Hz, 1H), 3.06 (s, 2H), 2.56 (s, 2H), 1.12 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 197.41, 161.82, 153.36, 134.25, 126.90, 121.86, 51.67, 45.94, 32.60, 27.94.

7,7-Dimethyl-6,7-dihydroquinoline-5,8-dione (13a): The synthesis of compound 13a was followed the same method as the synthesis of 9b using intermediate 12a as substrate.

7,7-Dimethyl-6,7-dihydroquinoline-5,8-dione 13a (yield 42%) as a yellow solid. 1H-NMR (300 MHz, CDCl3) δ 9.08 (dd, J = 4.6, 1.7 Hz, 1H), 8.37 (dd, J = 7.9, 1.8 Hz, 1H), 7.67 (dd, J = 7.9, 4.6 Hz, 1H), 3.02 (s, 2H), 1.37 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 199.22, 195.03, 155.53, 149.62, 134.68, 131.69, 127.66, 51.84, 45.49, 25.53.

3.2.3. Synthesis of 13b–13d (Pathway c in Scheme 1, Use 13b as Example)

4-(Dimethylamino)but-3-en-2-one (15b) [31]: To a mixture of acetone 14b (5.8 g, 100 mmol), in xylene (200 mL), was added dimethylformamide dimethylacetal (DMF-DMA, 11.9 g, 100 mmol) and the reaction refluxed for 15 h (monitored by TLC). The xylene was distilled off and the resulting solid residue was purified by column chromatography using PE and EtOAc (6:1) as eluent to afford 4-(dimethylamino)but-3-en-2-one 15b (4.3 g, 38%) as a yellow oil. 1H-NMR (300 MHz, Chloroform-d) δ 7.48 (d, J = 12.8 Hz, 1H), 5.05 (d, J = 12.8 Hz, 1H), 2.95 (d, J = 55.7 Hz, 6H), 2.09 (s, 3H).

1-(Dimethylamino)hex-1-en-3-one 15c (25%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.19 (d, J = 12.7 Hz, 1H), 4.72 (d, J = 12.7 Hz, 1H), 2.62 (d, J = 27.6 Hz, 6H), 2.08–1.92 (m, 2H), 1.31 (h, J = 7.4 Hz, 2H), 0.61 (t, J = 7.4 Hz, 3H).

3-(Dimethylamino)-1-phenylprop-2-en-1-one 15d (81%) as a yellow solid. 1H-NMR (300 MHz, CDCl3) δ 7.90 (dd, J = 7.9, 1.7 Hz, 2H), 7.80 (d, J = 12.4 Hz, 1H), 7.54–7.35 (m, 3H), 5.72 (d, J = 12.4 Hz, 1H), 3.13 (s, 3H), 2.93 (s, 3H).

2,7,7-Trimethyl-7,8-dihydroquinolin-5(6H)-one (12b) [31]: To a mixture of 4-(dimethylamino)but-3-en-2-one 15b (2.5 g, 22 mmol), 5,5-dimethylcyclohexane-1,3-dione (3.1 g, 22 mmol),ammonium acetate (NH4Oac, 3.4 g, 44 mmol) in 50 mL of AcOH refluxed for 15 h (monitored by TLC). The resulting mixture was cooled to r.t., the solution was concentrated on rotary evaporator and then purified by column chromatography using PE and EtOAc (4:1) as eluent to afford 2,7,7-trimethyl-7,8-dihydroquinolin-5(6H)-one 12b (3.4 g, 81%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.15 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 3.00 (s, 2H), 2.60 (s, 3H), 2.52 (s, 2H), 1.11 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 197.57, 163.46, 161.66, 134.66, 124.71, 121.77, 51.84, 46.26, 32.79, 28.13, 24.80.

7,7-Dimethyl-2-propyl-7,8-dihydroquinolin-5(6H)-one 12c (63%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.79 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 2.66 (s, 2H), 2.52–2.37 (m, 2H), 2.16 (s, 2H), 1.43 (h, J = 7.4 Hz, 2H), 0.75 (s, 6H), 0.63 (t, J = 7.4 Hz, 3H). 13C-NMR (75 MHz, CDCl3) δ 196.59, 166.65, 161.07, 133.91, 124.30, 120.52, 51.29, 45.89, 40.12, 32.19, 27.63, 22.17, 13.25.

7,7-Dimethyl-2-phenyl-7,8-dihydroquinolin-5(6H)-one 12d (92%) as a yellow solid. 1H-NMR (300 MHz, CDCl3) δ 8.31 (d, J = 8.2 Hz, 1H), 8.06 (dd, J = 7.8, 1.7 Hz, 2H), 7.71 (d, J = 8.2 Hz, 1H), 7.51–7.47 (m, 3H), 3.11 (s, 2H), 2.57 (s, 2H), 1.14 (s, 6H).

2,7,7-Trimethyl-6,7-dihydroquinoline-5,8-dione (13b): The synthesis of compound 13b–13d was followed the same method as the synthesis of 9b using intermediate 12b–12d as substrate.

2,7,7-Trimethyl-6,7-dihydroquinoline-5,8-dione 13b (yield 57%) as a yellow solid. 1H-NMR (300 MHz, CDCl3) δ 8.24 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 2.99 (s, 2H), 2.77 (s, 3H), 1.36 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 199.54, 194.92, 165.77, 149.03, 134.68, 129.39, 127.65, 51.64, 45.37, 25.45, 25.21.

7,7-Dimethyl-2-propyl-6,7-dihydroquinoline-5,8-dione 13c (yield 61%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.04 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 2.90–2.67 (m, 4H), 1.62 (h, J = 7.4 Hz, 2H), 1.15 (s, 6H), 0.79 (t, J = 7.3 Hz, 3H). 13C-NMR (75 MHz, CDCl3) δ 199.01, 194.49, 169.05, 148.73, 134.24, 129.20, 126.56, 51.24, 44.93, 40.34, 25.06, 22.37, 13.34.

7,7-Dimethyl-2-phenyl-6,7-dihydroquinoline-5,8-dione 13d (yield 51%) as a yellow solid. 1H-NMR (300 MHz, CDCl3) δ 8.40 (d, J = 8.3 Hz, 1H), 8.22–8.15 (m, 2H), 8.08 (d, J = 8.3 Hz, 1H), 7.52 (dd, J = 5.1, 1.9 Hz, 3H), 3.02 (s, 2H), 1.39 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 199.27, 194.78, 162.64, 149.54, 137.25, 135.39, 130.57, 129.81, 129.76, 128.82, 128.41, 127.62, 123.89, 51.71, 45.43, 25.40.

3.2.4. Synthesis of 17 (Pathway d in Scheme 1)

6,6-Dimethyl-6,7-dihydrobenzofuran-4(5H)-one (16) [32,33]: To a stirred ice-cooled suspension of 5,5-dimethylcyclohexane-1,3-dione 10 (14 g, 100 mmol) in water (100 mL) was added dropwise a solution of KOH (7 g, 125 mmol) in water (100 mL). Then, KI (0.3 g, 20 mmol) was added to the resulting clear solution followed by the dropwise addition of 40% aqueous chloroacetaldehyde (ClCH2CHO, 20 mL) over 25 min. The reaction mixture was allowed to warm to reflux and stir overnight. The reaction was quenched by the dropwise addition of 2 M HCl until acid to pH paper. The solution was extracted by EtOAc (3 × 100 mL). The combined organic layers were dried (anhydrous Na2SO4), concentrated, and vacuum distilled to afford 6,6-dimethyl-6,7-dihydrobenzofuran-4(5H)-one 16 (12.5 g, 76%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.25 (d, J = 2.0 Hz, 1H), 6.57 (d, J = 2.0 Hz, 1H), 2.66 (s, 2H), 2.29 (s, 2H), 1.05 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 193.53, 166.03, 142.70, 119.66, 106.07, 51.86, 37.13, 35.03, 28.31.

6,6-Dimethyl-5,6-dihydrobenzofuran-4,7-dione (17): The synthesis of compound 17 was followed the same method as the synthesis of 9b using intermediate 16 as substrate.

6,6-Dimethyl-5,6-dihydrobenzofuran-4,7-dione 17 (yield 52%) as a white solid. 1H-NMR (300 MHz, CDCl3) δ 7.72 (d, J = 1.9 Hz, 1H), 6.80 (d, J = 1.9 Hz, 1H), 2.90 (s, 2H), 1.35 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 192.11, 190.88, 153.06, 148.64, 132.08, 107.55, 53.99, 46.59, 26.23.

3.2.5. General Procedure for the Preparation of Intermediate 18b–18d, 19a–19d and 20 (Use 18b as Example) [34]

To a stirred solution of (Z)-3-methylpent-2-en-4-yn-1-ol (0.88 g, 9.2 mmol) in dry THF (20 mL) was cooled to −78 °C under an atmosphere of argon. n-Butyl lithium (7.7 mL, 18.4 mmol, 2.4 M in hexane) was then added slowly, via syringe. The mixture was allowed to stir at −78 °C for 1 h, after which, 6-methoxy-2,2-dimethyl-2,3-dihydronaphthalene-1,4-dione 9b (2 g, 9.2 mmol), dissolved in dry THF (10 mL) was added. The mixture was stirred for a further 1 h at −78 °C, and then the cold bath was removed. The reaction mixture was stirred at r.t. for a further 16 h. The reaction was quenched by addition of a saturated aqueous solution of NH4Cl. The mixture was stirred for 10 min and extracted with ethyl acetate (3 × 50 mL), washed with water (2 × 30 mL) and dried over anhydrous Na2SO4. Evaporation of the solvent yielded the desired alcohol as a yellowish oil. The residue was subjected to silica gel chromatography using PE and EtOAc (4:1) as eluent to afford (Z)-(1′-hydroxy-3′,3′-dimethyl-4′-oxo-7′-methoxy-tetrahydronaphthalene-one-yl)-3-methyl-pentyl-2-em-4-yn-1-ol 18b (2.37 g, 82%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.80 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 2.8 Hz, 1H), 7.13 (dd, J = 8.6, 2.9 Hz, 1H), 5.88 (t, J = 7.5 Hz, 1H), 4.27 (d, J = 6.7 Hz, 2H), 3.83 (s, 3H), 2.92 (d, J = 16.9 Hz, 1H), 2.73 (s, 1H), 2.58 (d, J = 17.4 Hz, 1H), 1.89 (s, 3H), 1.21 (s, 3H), 1.13 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 197.49, 159.63, 136.82, 136.36, 131.11, 129.04, 121.66, 120.16, 109.42, 94.54, 85.58, 74.29, 61.03, 55.54, 48.38, 41.60, 25.02, 23.31, 23.02.

(Z)-(1′-Hydroxy-3′,3′-dimethyl-4′-oxo-6′-methoxy-tetrahydronaphthalene-one-yl)-3-methyl-pentyl-2-em-4-yn-1-ol 18c (75%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.96 (d, J = 8.7 Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 5.89 (t, J = 6.4 Hz, 1H), 4.27 (d, J = 6.4 Hz, 2H), 3.87 (s, 3H), 3.01–2.41 (m, 3H), 1.88 (s, 3H), 1.16 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 196.35, 164.42, 147.09, 136.57, 129.46, 123.57, 120.04, 114.19, 111.84, 94.29, 85.76, 74.60, 60.98, 55.52, 48.48, 41.51, 30.86, 24.94, 22.97.

(Z)-(1′-Hydroxy-3′,3′-dimethyl-4′-oxo-6′-Br-tetrahydronaphthalene-one-yl)-3-methyl-pentyl-2-em-4-yn-1-ol 18d (70%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.06 (d, J = 1.9 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.56 (dd, J = 8.3, 1.9 Hz, 1H), 5.92 (t, J = 6.8 Hz, 1H), 4.27 (d, J = 6.8 Hz, 2H), 4.03 (s, 1H), 2.85 (d, J = 18.7 Hz, 1H), 2.64 (d, J = 23.1 Hz, 2H), 1.89 (s, 3H), 1.17 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 196.68, 146.17, 136.66, 131.84, 130.46, 129.55, 128.80, 128.58, 120.18, 93.72, 86.38, 74.11, 60.94, 48.57, 41.62, 24.92, 22.95.

(Z)-(5′-Hydroxy-7′,7′-dimethyl-8′-oxo-tetrahydroquinolin-one-yl)-3-methyl-pentyl-2-em-4-yn-1-ol 19a (yield 79%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.77 (dd, J = 4.8, 1.8 Hz, 1H), 8.30 (dd, J = 7.8, 1.8 Hz, 1H), 7.45 (dd, J = 7.8, 4.8 Hz, 1H), 5.85 (t, J = 6.6 Hz, 1H), 5.48 (s, 1H), 4.14 (d, J = 6.5 Hz, 2H), 3.00 (d, J = 17.4 Hz, 2H), 2.68 (d, J = 17.4 Hz, 1H), 1.79 (s, 3H), 1.39 (s, 3H), 1.02 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 196.02, 161.46, 153.11, 136.96, 135.23, 124.98, 123.67, 119.37, 93.94, 85.72, 73.93, 60.68, 50.01, 40.70, 24.67, 22.63, 21.14.

(Z)-(5′-Hydroxy-2′,7′,7′-trimethyl-8′-oxo-tetrahydroquinolin-one-yl)-3-methyl-pentyl-2-em-4-yn-1-ol 19b (yield 85%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.17 (d, J = 7.9 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 5.84 (t, J = 6.6 Hz, 1H), 5.35 (s, 1H), 4.16 (d, J = 6.6 Hz, 2H), 3.00 (d, J = 17.5 Hz, 1H), 2.66 (s, 3H), 2.59 (d, J = 17.5 Hz, 2H), 1.80 (s, 3H), 1.40 (s, 3H), 0.97 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 195.87, 163.50, 160.80, 136.79, 135.44, 123.43, 122.57, 119.43, 94.30, 85.23, 73.63, 60.86, 50.24, 40.79, 24.67, 24.66, 22.68, 20.93.

(Z)-(5′-Hydroxy-7′,7′-dimethyl-2′-propyl-8′-oxo-tetrahydroquinolin-one-yl)-3-methyl-pentyl-2-em-4-yn-1-ol 19c (yield 73%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.10 (d, J = 7.9 Hz, 1H), 7.19 (d, J = 7.9 Hz, 1H), 5.75 (t, J = 6.1 Hz, 1H), 5.31 (s, 1H), 4.09 (d, J = 6.3 Hz, 2H), 2.93 (d, J = 17.4 Hz, 1H), 2.80 (d, J = 7.3 Hz, 2H), 2.50 (d, J = 17.5 Hz, 1H), 1.81–1.65 (m, 5H), 1.32 (s, 3H), 0.92–0.86 (m, 6H). 13C-NMR (75 MHz, CDCl3) δ 195.60, 166.92, 160.52, 136.72, 135.12, 122.56, 122.38, 118.79, 94.04, 84.93, 73.34, 60.55, 50.03, 40.45, 39.82, 24.42, 22.39, 21.98, 20.59, 13.30.

(Z)-(5′-Hydroxy-7′,7′-dimethyl-2′-phenyl-8′-oxo-tetrahydroquinolin-one-yl)-3-methyl-pentyl-2-em-4-yn-1-ol 19d (yield 79%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.35 (d, J = 8.2 Hz, 1H), 8.20–8.02 (m, 2H), 7.87 (d, J = 8.2 Hz, 1H), 7.53 (qd, J = 4.6, 1.6 Hz, 3H), 5.82 (t, J = 7.4 Hz, 1H), 5.34 (s, 1H), 4.17 (s, 2H), 3.08 (d, J = 17.7 Hz, 1H), 2.62 (d, J = 17.6 Hz, 1H), 1.80 (s, 3H), 1.45 (s, 3H), 1.00 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 195.74, 161.32, 160.49, 137.29, 136.59, 136.36, 130.62, 128.99, 127.50, 123.46, 120.24, 119.88, 94.60, 85.30, 73.89, 61.28, 50.61, 40.97, 24.81, 22.87, 21.00.

(Z)-(4′-Hydroxy-6′,6′-dimethyl-7′-oxo-tetrahydrobenzofuran-one-yl)-3-methyl-pentyl-2-em-4-yn-1-ol 20 (yield 90%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.42 (d, J = 2.0 Hz, 1H), 6.68 (d, J = 2.0 Hz, 1H), 5.95 (t, J = 6.8 Hz, 1H), 4.31 (d, J = 6.7 Hz, 2H), 2.80 (d, J = 16.4 Hz, 1H), 2.45 (d, J = 16.5 Hz, 1H), 1.88 (s, 3H), 1.21 (d, J = 12.0 Hz, 6H). 13C-NMR (75 MHz, CDCl3) δ 193.65, 163.80, 143.91, 137.11, 119.66, 119.21, 106.44, 90.38, 85.67, 70.16, 60.77, 49.40, 43.91, 24.79, 22.67, 22.55.

3.2.6. General Procedure for the Preparation of Intermediate 21b−21d, 22a−22d and 23 (Use 21b as Example)

To a stirred solution of (Z)-(1′-hydroxy-3′,3′-dimethyl-4′-oxo-7′-methoxy-tetrahydronaphthalene-one-yl)-3-methyl-pentyl-2-em-4-yn-1-ol 18b (1 g, 3.2 mmol) in dry THF (20 mL) was cooled to 0 °C and Red-Al reagent (2.9 mL, 9.6 mmol, 3.3 M in toluene) added dropwise via syringe. After 4 h stirring at r.t., the reaction was quenched by slow addition of saturate brine (10 mL) and extracted with diethyl ether (3 × 30 mL). The organic phase was washed with water (2 × 20 mL) and dried over anhydrous Na2SO4 and filtered, filtrate concentrated under reduced pressure. The residue was subjected to silica gel chromatography using CH2Cl2 and methanol (25:1) as eluent to afford (1E,3Z)-(1′,4′-dihydroxy-3′,3′-dimethyl-7′-methoxy-tetraloneyl)-3-methyl-pentyl-2-ene-4-yn-1-ol 21b (0.57 g, 56%) as a white solid. 1H-NMR (300 MHz, MeOD) δ 7.32 (d, J = 8.7 Hz, 1H), 7.06 (d, J = 2.5 Hz, 1H), 6.81 (dd, J = 8.7, 2.7 Hz, 1H), 6.31 (d, J = 15.6 Hz, 1H), 6.05 (d, J = 15.7 Hz, 1H), 5.44 (t, J = 6.9 Hz, 1H), 4.76 (dd, J = 9.9, 6.8 Hz, 1H), 4.09 (d, J = 7.1 Hz, 2H), 3.79 (s, 3H), 1.91 (dd, J = 13.3, 6.7 Hz, 1H), 1.82 (d, J = 13.3 Hz, 4H), 0.99 (d, J = 5.8 Hz, 6H). 13C-NMR (75 MHz, MeOD) δ 160.23, 141.14, 138.07, 134.97, 134.39, 130.26, 129.55, 126.56, 115.18, 111.87, 79.59, 67.32, 58.48, 55.63, 44.95, 39.99, 25.70, 23.24, 20.82.

(1E,3Z)-(1′,4′-Dihydroxy-3′,3′-dimethyl-6′-methoxy-tetraloneyl)-3-methyl-pentyl-2-ene-4-yn-1-ol 21c (49%) as a white solid. 1H-NMR (300 MHz, MeOD) δ 7.42 (d, J = 8.1 Hz, 1H), 6.98 (d, J = 2.7 Hz, 1H), 6.83 (dd, J = 8.6, 2.7 Hz, 1H), 6.37 (d, J = 15.7 Hz, 1H), 6.06 (d, J = 15.5 Hz, 1H), 5.46 (t, J = 6.8 Hz, 1H), 4.75 (dd, J = 9.7, 7.0 Hz, 1H), 4.10 (dd, J = 6.5, 2.8 Hz, 2H), 3.75 (s, 3H), 1.91 (dd, J = 13.4, 6.9 Hz, 1H), 1.87–1.72 (m, 4H), 0.99 (d, J = 10.5 Hz, 6H). 13C-NMR (75 MHz, MeOD) δ 160.48, 143.82, 137.72, 134.99, 132.00, 129.69, 129.51, 126.76, 114.75, 112.80, 79.89, 67.00, 58.47, 55.61, 45.06, 40.26, 25.65, 23.10, 20.79.

(1E,3Z)-(1′,4′-Dihydroxy-3′,3′-dimethyl-6′-Br-tetraloneyl)-3-methyl-pentyl-2-ene-4-yn-1-ol 21d (45%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.59–7.50 (m, 1H), 7.50–7.39 (m, 1H), 7.36–7.26 (m, 2H), 6.29 (d, J = 15.7 Hz, 1H), 6.03 (d, J = 15.7 Hz, 1H), 5.51 (t, J = 7.1 Hz, 1H), 4.95–4.77 (m, 1H), 4.06 (d, J = 7.8 Hz, 2H), 2.17 (s, 2H), 2.03 (q, J = 6.9 Hz, 2H), 1.81 (s, 3H), 1.01 (d, J = 15.2 Hz, 6H). 13C-NMR (75 MHz, CDCl3) δ 140.59, 138.16, 135.73, 135.22, 128.17, 128.03, 127.62, 127.31, 127.24, 126.71, 78.87, 66.64, 57.97, 43.85, 39.11, 25.05, 22.51, 20.48.

(1E,3Z)-(5′,8′-Dihydroxy-7′,7′-dimethyl-tetrahyroquinolinyl)-3-methyl-pentyl-2-ene-4-yn-1-ol 22a (yield 12%) as a white solid. 1H-NMR (300 MHz, CDCl3) δ 8.54–8.45 (m, 1H), 7.95 (dd, J = 7.8, 1.4 Hz, 1H), 7.33–7.23 (m, 1H), 6.06 (m, 2H), 5.51 (t, J = 7.7 Hz, 1H), 4.99–4.67 (m, 1H), 4.02 (dd, J = 7.2, 3.9 Hz, 2H), 3.00 (d, J = 16.9 Hz, 1H), 2.65 (d, J = 17.5 Hz, 1H), 1.80 (s, 3H), 1.10 (s, 3H), 0.97 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 157.52, 152.85, 148.02, 135.90, 135.14, 132.74, 127.71, 126.82, 122.41, 78.03, 65.54, 57.50, 43.22, 38.94, 24.60, 21.88, 20.01.

(1E,3Z)-(5′,8′-Dihydroxy-2′,7′,7′-trimethyl-tetrahyroquinolin-yl)-3-methyl-pentyl-2-ene-4-yn-1-ol 22b (yield 43%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.77 (d, J = 7.9 Hz, 1H), 7.11 (d, J = 7.9 Hz, 1H), 6.45 (d, J = 15.6 Hz, 1H), 5.87 (d, J = 15.6 Hz, 1H), 5.51 (t, J = 7.0 Hz, 1H), 4.86 (dd, J = 6.9, 3.2 Hz, 1H), 4.18 (dd, J = 6.6, 3.2 Hz, 2H), 2.51 (s, 3H), 2.20 (dd, J = 14.9, 6.9 Hz, 1H), 1.88–1.82 (m, 1H), 1.79 (s, 3H), 1.02 (d, J = 8.3 Hz, 6H). 13C-NMR (75 MHz, CDCl3) δ 157.03, 156.50, 136.95, 134.22, 134.16, 128.87, 128.01, 125.70, 122.30, 77.53, 64.78, 57.87, 50.20, 42.65, 36.27, 24.22, 23.74, 20.32.

(1E,3Z)-(5′,8′-Dihydroxy-7′,7′-dimethyl-2′-propyl-tetrahyroquinolin-yl)-3-methyl-pentyl-2-ene-4-yn-1-ol 22c (yield 26%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.82 (d, J = 7.9 Hz, 1H), 7.10 (d, J = 7.9 Hz, 1H), 6.15–5.91 (m, 2H), 5.51 (t, J = 7.3 Hz, 1H), 4.89 (s, 1H), 4.86–4.75 (m, 1H), 4.01 (d, J = 8.8 Hz, 2H), 2.77–2.72 (m, 2H), 2.03–1.84 (m, 2H), 1.81 (s, 3H), 1.77–1.69 (m, 2H), 1.11 (s, 3H), 0.94 (d, J = 7.3 Hz, 6H). 13C-NMR (75 MHz, CDCl3) δ 160.78, 156.37, 136.29, 135.98, 135.36, 129.53, 127.44, 126.81, 121.49, 77.78, 65.59, 57.52, 43.41, 39.39, 39.04, 24.60, 22.35, 21.85, 20.09, 13.38.

(1E,3Z)-(5′,8′-Dihydroxy-7′,7′-dimethyl-2′-phenyl-tetrahyroquinolin-yl)-3-methyl-pentyl-2-ene-4-yn-1-ol 22d (yield 65%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.98 (d, J = 7.9 Hz, 3H), 7.69 (d, J = 8.1 Hz, 1H), 7.43 (m, 3H), 6.08 (s, 2H), 5.48 (t, J = 7.1 Hz, 1H), 4.93 (s, 1H), 4.90–4.77 (m, 1H), 3.99 (dd, J = 6.9, 3.6 Hz, 2H), 2.29 (t, J = 6.0 Hz, 1H), 1.97 (m, 3H), 1.79 (s, 3H), 1.13 (s, 3H), 0.99 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 160.43, 157.42, 155.70, 138.38, 137.04, 136.46, 135.70, 131.33, 129.21, 128.68, 127.80, 127.31, 126.80, 125.06, 119.37, 78.31, 65.87, 57.89, 43.64, 39.48, 24.88, 22.22, 20.34.

(1E,3Z)-(4′,7′-Dihydroxy-6′,6′-dimethyl-tetrahyrobenzofuran-yl)-3-methyl-pentyl-2-ene-4-yn-1-ol 23 (yield 39%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.35 (d, J = 1.9 Hz, 1H), 6.47 (d, J = 1.9 Hz, 1H), 6.30 (d, J = 15.8 Hz, 1H), 5.91 (d, J = 15.7 Hz, 1H), 5.57 (t, J = 7.2 Hz, 1H), 4.74 (dd, J = 8.3, 5.8 Hz, 1H), 4.12 (qd, J = 12.6, 6.9 Hz, 2H), 2.53 (s, 1H), 1.95 (dd, J = 13.5, 5.8 Hz, 1H), 1.85 (s, 3H), 1.76–1.55 (m, 2H), 1.03 (d, J = 5.1 Hz, 6H). 13C-NMR (75 MHz, CDCl3) δ 151.59, 143.03, 134.79, 132.52, 128.70, 127.27, 121.86, 108.80, 76.08, 63.34, 57.96, 45.40, 41.37, 24.29, 22.29, 20.46.

3.2.7. General Procedure for the Preparation of Title Compounds 4b–4d, 5a–5d, 6, 24a–24d, 25a, 25b, 25d and 26 (Use 4b as Example)

To a stirred solution of (1E,3Z)-(1′,4′-dihydroxy-3′,3′-dimethyl-7′-methoxy-tetraloneyl)-3-methyl-pentyl-2-ene-4-yn-1-ol 21b (0.5 g, 1.57 mmol), Dess-Martin periodinane (DMP, 1.32 g, 3.14 mmol) in 30 mL CH2Cl2 at r.t. for 2 h. After added 5 mL aqueous Na2S2O3 solution and 10 mL aqueous NaHCO3 solution, the resulting mixture, which was stirred for 20 min, was extracted repeatedly with CH2Cl2 (3 × 30 mL). The collected organic extracts were washed with saturate aqueous brine solution, dried, and then concentrated. The crude aldehyde was dissolved in 20 mL solvent (t-BuOH:H2O = 3:1 in volume), stirred with 2-methyl-2-butene (2.2 g, 31.4 mmol, 90%), NaClO2 (1.67 g, 15.7 mmol, 85%) and NaH2PO4·2H2O (0.97 g, 6.28 mmol) at r.t. for 30 min. Extracted repeatedly with EtOAc (3 × 30 mL). The collected organic extracts were washed with saturate aqueous brine solution, dried, and concentrated under reduced pressure afford crude product. The residue was subjected to silica gel chromatography using CH2Cl2 and methanol (20:1) as eluent to afford 7′-OMe-iso-PhABA 4b (0.42 g, yield 81% over two steps) as a white solid. 1H-NMR (300 MHz, MeOD) δ 7.65 (d, J = 16.4 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.45 (d, J = 2.8 Hz, 1H), 7.20 (dd, J = 8.7, 2.9 Hz, 1H), 6.48 (d, J = 16.0 Hz, 1H), 5.71 (s, 1H), 3.85 (s, 3H), 2.72 (d, J = 16.8 Hz, 1H), 2.59 (d, J = 17.5 Hz, 1H), 2.04 (d, J = 1.2 Hz, 3H), 1.08 (s, 3H), 1.04 (s, 3H). 13C-NMR (75 MHz, MeOD) δ 199.70, 169.46, 160.69, 151.21, 140.82, 140.37, 133.46, 130.84, 129.57, 122.49, 119.23, 109.90, 78.80, 55.92, 50.91, 42.38, 24.68, 23.95, 21.31. HR-MS (ESI) calcd. for C19H22O5 (M + NH4)+ 348.18055, measured 348.18057.

6′-OMe-iso-PhABA 4c (yield 83% over two steps) as a white solid. 1H-NMR (300 MHz, CDCl3) δ 8.00 (d, J = 8.7 Hz, 1H), 7.75 (d, J = 15.8 Hz, 1H), 7.06 (d, J = 2.2 Hz, 1H), 6.88 (dd, J = 8.7, 2.5 Hz, 1H), 6.41 (d, J = 16.0 Hz, 1H), 5.72 (s, 1H), 3.83 (s, 3H), 2.70 (d, J = 16.2 Hz, 1H), 2.56 (d, J = 17.3 Hz, 1H), 2.02 (s, 3H), 1.08 (d, J = 11.6 Hz, 6H). 13C-NMR (75 MHz, CDCl3) δ 196.35, 170.87, 164.62, 151.87, 148.60, 139.22, 129.38, 128.33, 124.51, 117.74, 113.90, 111.79, 78.48, 55.51, 49.61, 41.12, 24.25, 23.38, 21.40. HR-MS (ESI) calcd. for C19H22O5 (M + H)+ 331.15400, measured 331.15402.

6′-Br-iso-PhABA 4d (yield 72% over two steps) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.05 (d, J = 9.1 Hz, 1H), 7.58 (dt, J = 13.5, 7.2 Hz, 2H), 7.50–7.36 (m, 1H), 6.94 (d, J = 15.3 Hz, 1H), 6.14 (d, J = 15.8 Hz, 1H), 5.98 (s, 1H), 2.83 (d, J = 17.8 Hz, 1H), 2.60 (d, J = 16.9 Hz, 1H), 2.04 (s, 1H), 1.87 (s, 3H), 1.08 (d, J = 4.4 Hz, 6H). 13C-NMR (75 MHz, CDCl3) δ197.21, 169.75, 145.90, 134.47, 134.44, 133.14, 130.98, 128.18, 127.13, 126.72, 126.35, 116.39, 78.21, 49.79, 40.93, 24.33, 23.40, 18.25. HR-MS (ESI) calcd. for C18H19BrO4 (M + H)+ 396.08050, measured 396.08035.

iso-PyABA 5a (yield 78% over two steps) as colorless oil. 1H-NMR (300 MHz, CDCl3) δ 8.73 (dd, J = 4.8, 1.8 Hz, 1H), 8.39 (dd, J = 7.8, 1.8 Hz, 1H), 7.89 (d, J = 15.6 Hz, 1H), 7.48 (dd, J = 7.8, 4.9 Hz, 1H), 6.39 (d, J = 16.0 Hz, 1H), 5.70 (s, 1H), 2.87 (d, J = 18.7 Hz, 1H), 2.66 (d, J = 17.3 Hz, 1H), 2.01 (s, 3H), 1.15 (s, 3H), 1.11 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 196.24, 169.30, 163.28, 152.65, 150.54, 137.97, 135.20, 128.66, 126.25, 123.29, 117.66, 77.94, 49.44, 39.98, 23.92, 22.94, 20.68. HR-MS (ESI) calcd. for C17H19NO4 (M + H)+ 302.1387, measured 302.1387.

2′-Me-iso-PyABA 5b (yield 71% over two steps) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.23 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 15.8 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 6.38 (d, J = 16.1 Hz, 1H), 5.68 (s, 1H), 2.68 (s, 2H), 2.63 (s, 3H), 1.99 (s, 3H), 1.16 (s, 3H), 1.05 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 196.04, 169.66, 163.45, 162.46, 150.81, 138.72, 135.02, 128.48, 123.65, 123.04, 117.58, 77.62, 49.54, 39.99, 24.26, 23.78, 22.85, 20.71. HR-MS (ESI) calcd. for C18H21NO4 (M + H)+ 316.1543, measured 316.1545.

2′-Pr-iso-PyABA 5c (yield 87% over two steps) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.24 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 15.6 Hz, 1H), 7.27 (d, J = 6.0 Hz, 1H), 6.39 (d, J = 16.1 Hz, 1H), 5.68 (s, 1H), 2.90–2.80 (m, 2H), 2.77–2.56 (m, 2H), 1.99 (s, 3H), 1.83–1.72 (m, 2H), 1.20 (s, 3H), 1.04 (s, 3H), 0.95 (d, J = 7.4 Hz, 3H). 13C-NMR (75 MHz, CDCl3) δ 196.03, 169.79, 167.22, 162.25, 151.00, 139.08, 134.89, 128.34, 123.72, 122.34, 117.47, 77.59, 49.67, 39.97, 39.86, 23.77, 22.83, 22.15, 20.75, 13.37. HR-MS (ESI) calcd. for C20H25NO4 (M + H)+ 344.1856, measured 316.1854.

2′-Ph-iso-PyABA 5d (yield 80% over two steps) as a yellow solid. 1H-NMR (300 MHz, CDCl3) δ 9.06 (s, 1H), 8.40 (d, J = 8.2 Hz, 1H), 8.08 (dd, J = 7.6, 2.0 Hz, 2H), 7.88 (d, J = 8.2 Hz, 1H), 7.57 (d, J = 15.8 Hz, 1H), 7.53–7.40 (m, 3H), 6.44 (d, J = 16.1 Hz, 1H), 5.67 (s, 1H), 5.13 (s, 1H), 2.82 (d, J = 18.2 Hz, 1H), 2.60 (d, J = 18.0 Hz, 1H), 1.99 (s, 3H), 1.26 (s, 3H), 1.08 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 196.03, 170.91, 162.86, 160.65, 151.65, 139.57, 137.36, 135.89, 130.39, 128.82, 128.76, 127.50, 124.52, 119.91, 117.83, 78.17, 50.06, 40.28, 24.03, 23.22, 21.09. HR-MS (ESI) calcd. for C23H23NO4 (M + H)+ 378.17000, measured 378.16980.

iso-FrABA 6 (yield 86% over two steps) as a yellow solid. 1H-NMR (300 MHz, MeOD) δ 7.68 (d, J = 16.4 Hz, 1H), 7.58 (s, 1H), 6.67 (s, 1H), 6.32 (d, J = 16.1 Hz, 1H), 5.75 (s, 1H), 3.34 (s, 1H), 2.62 (d, J = 17.3 Hz, 1H), 2.44 (d, J = 15.9 Hz, 1H), 2.04 (s, 3H), 1.07 (d, J = 11.6 Hz, 6H). 13C-NMR (75 MHz, MeOD) δ 196.03, 168.17, 150.56, 145.84, 137.65, 136.47, 130.40, 121.68, 119.99, 106.84, 76.32, 51.84, 45.09, 24.18, 23.28, 21.11. HR-MS (ESI) calcd. for C16H18O5 (M + Na)+ 313.1046, measured 313.1048.

Acetylenic iso-PhABA 24a (yield 84% over two steps) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.98 (d, J = 7.74 Hz, 2H), 7.60 (t, J = 7.50 Hz, 1H), 7.41 (t, J = 7.44 Hz, 1H), 6.05 (s, 1H), 2.77 (m, 2H), 2.10 (s, 3H), 1.17 (s, 6H). 13C-NMR (75 MHz, CDCl3) δ 197.5, 169.2, 143.7, 136.5, 134.3, 130.0, 128.6, 127.5, 126.8, 124.4, 101.8, 86.1, 74.7, 48.07, 41.5, 25.1, 24.8. HR-MS (ESI) calcd. for C18H18O4 (M + Na)+ 321.1097, measured 321.1096.

Acetylenic 7′-OMe-iso-PhABA 24b (yield 71% over two steps) as a yellow solid. 1H-NMR (300 MHz, CDCl3) δ 7.65 (d, J = 8.7 Hz, 1H), 7.51 (d, J = 2.9 Hz, 1H), 7.18 (dd, J = 8.7, 2.9 Hz, 1H), 6.09 (d, J = 1.5 Hz, 1H), 4.67 (s, 1H), 3.88 (s, 3H), 2.90 (d, J = 18.1 Hz, 1H), 2.60 (d, J = 17.5 Hz, 1H), 2.44 (s, 3H), 1.23 (s, 3H), 1.10 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 196.65, 165.16, 159.67, 156.72, 146.97, 136.16, 133.76, 129.19, 125.58, 121.79, 118.97, 108.31, 83.52, 55.54, 49.81, 43.32, 25.19, 24.72, 18.37. HR-MS (ESI) calcd. for C19H20O5 (M + H)+ 329.13835, measured 329.13821.

Acetylenic 6′-OMe-iso-PhABA 24c (yield 85% over two steps) as a yellow solid. 1H-NMR (300 MHz, CDCl3) δ 7.95 (d, J = 8.7 Hz, 1H), 7.47 (s, 1H), 6.87 (dd, J = 8.7, 2.1 Hz, 1H), 6.05 (s, 1H), 3.87 (s, 3H), 2.82 (d, J = 24.2 Hz, 4H), 2.07 (s, 3H), 1.17 (d, J = 8.6 Hz, 6H). 13C-NMR (75 MHz, CDCl3) δ 196.15, 164.44, 146.40, 129.49, 124.76, 124.70, 123.71, 114.57, 111.89, 111.83, 101.62, 86.20, 74.83, 55.61, 48.52, 41.64, 29.68, 25.12, 24.84. HR-MS (ESI) calcd. for C19H20O5 (M + H)+ 329.13835, measured 329.13828.

Acetylenic 6′-Br-iso-PhABA 24d (yield 79% over two steps) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.13 (s, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.54 (dd, J = 8.3, 1.9 Hz, 1H), 6.05 (d, J = 1.8 Hz, 1H), 2.79 (s, 2H), 2.08 (s, 3H), 1.17 (d, J = 17.4 Hz, 6H). 13C-NMR (75 MHz, CDCl3) δ 195.72, 168.33, 144.62, 135.17, 130.93, 129.60, 128.65, 127.87, 127.63, 123.85, 99.99, 85.67, 73.33, 47.61, 40.80, 24.13, 23.75, 21.66. HR-MS (ESI) calcd. for C18H17BrO4 (M + H)+ 394.06485, measured 394.06485.

Acetylenic iso-PyABA 25a (yield 77% over two steps) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.74 (dd, J = 4.8, 1.8 Hz, 1H), 8.32 (dd, J = 7.8, 1.8 Hz, 1H), 7.45 (dd, J = 7.8, 4.8 Hz, 1H), 5.84 (s, 1H), 5.60 (s, 1H), 5.07 (s, 1H), 2.73 (d, J = 17.4 Hz, 1H), 2.63 (d, J = 17.1 Hz, 1H), 2.15 (s, 3H), 1.28 (s, 3H), 1.05 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 195.96, 167.91, 162.11, 155.35, 153.02, 149.76, 134.68, 126.88, 123.78, 116.39, 113.15, 77.20, 49.64, 41.50, 24.31, 22.68, 11.95. HR-MS (ESI) calcd. for C17H17NO4 (M + H)+ 300.1230, measured 300.1230.

Acetylenic 2′-Me-iso-PyABA 25b (yield 90% over two steps) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.20 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 7.7 Hz, 3H), 5.84 (s, 1H), 5.57 (s, 1H), 5.24 (s, 1H), 2.71 (d, J = 16.9 Hz, 1H), 2.62 (s, 3H), 2.56 (d, J = 17.5 Hz, 1H), 2.13 (s, 3H), 1.28 (s, 3H), 1.03 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 195.83, 168.11, 163.25, 161.47, 155.39, 149.63, 134.89, 124.46, 123.47, 116.33, 113.37, 76.91, 49.68, 41.47, 24.81, 24.31, 22.58, 11.93. HR-MS (ESI) calcd. for C18H19NO4 (M + H)+ 314.13868, measured 314.13858.

Acetylenic 2′-Ph-iso-PyABA 25d (yield 75% over two steps) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 8.35 (d, J = 8.2 Hz, 1H), 8.11 (dd, J = 7.4, 2.0 Hz, 2H), 7.85 (d, J = 8.2 Hz, 1H), 7.63–7.40 (m, 3H), 5.97 (s, 1H), 5.59 (s, 1H), 3.38 (d, J = 17.6 Hz, 1H), 2.59 (d, J = 17.7 Hz, 1H), 2.01 (s, 3H), 1.47 (s, 3H), 0.99 (s, 3H). 13C-NMR (75 MHz, CDCl3) δ 195.97, 168.91, 160.65, 160.24, 137.27, 136.37, 136.16, 130.41, 128.83, 127.44, 124.34, 123.67, 120.23, 101.71, 85.06, 74.04, 50.09, 41.24, 25.03, 24.50, 20.99. HR-MS (ESI) calcd. for C23H21NO4 (M + H)+ 376.15433, measured 376.15414.

Acetylenic iso-FrABA 26 (yield 89% over two steps) as a yellow oil. 1H-NMR (300 MHz, CDCl3) δ 7.46 (d, J = 1.7 Hz, 1H), 6.67 (d, J = 1.7 Hz, 1H), 6.10 (s, 1H), 5.78 (s, 2H), 2.75 (d, J = 16.9 Hz, 1H), 2.58 (d, J = 16.5 Hz, 1H), 2.08 (s, 3H), 1.23 (d, J = 6.1 Hz, 6H). 13C-NMR (75 MHz, CDCl3) δ 193.42, 163.13, 144.19, 141.58, 132.98, 127.89, 119.68, 106.58, 97.64, 86.13, 70.69, 49.85, 44.28, 24.91, 24.62, 22.42. HR-MS (ESI) calcd. for C16H16O5 (M + H)+ 289.1071, measured 289.1073.

3.3. Bioassays

3.3.1. Arabidopsis Seed Germination

Twenty-five seeds of Arabidopsis (Columbia wild type) were sterilized successively with 70% (v/v) ethanol for 30 min and were washed with sterile water. The sterilized seeds were soaked in about 400 μL of a test solution and incubated in the dark for 3 days at 4 °C. The vernalized seeds in the test solution were transferred to plates in which two sheets of filter paper soaked in 2 mL of water had been placed and allowed to germinate under day for 16 h and night for 8 h at 22 °C. The percentage of seeds with an emerged radicle was calculated. All of the tests were conducted thrice. All of the IC50 values were calculated by IBM SPSS Statistics (IBM Corporation, Armonk, NY, USA).

3.3.2. Lettuce Seed Germination

Fifty seeds of lettuce (Lactuca sativa L.cv.) were soaked in about 1 mL of a test solution in the dark for 1 day at 25 °C. Then, the seeds were transferred to plates with two sheets of filter paper, soaked in 2 mL of water, and were allowed to germinate under continuous light for 24 h at 25 °C. The percentage of seeds with an emerged radicle was calculated. All tests were conducted thrice. All the IC50 values were calculated by IBM SPSS Statistics.

3.3.3. Rice Seedling Growth

Seeds of rice (indica) were sterilized with 70% (v/v) ethanol for 5 min and washed with distilled water. The sterilized seeds were soaked in water to germinate for three days at 25 °C. Then, the well germinated seeds were selected to place in a glass tube containing 2 mL of a test solution and grown with the tube sealed with a plastic cap under continuous light at 30 °C. The length of the second leaf sheath was measured when the seedlings were seven days old. All of the tests were conducted thrice. All of the IC50 values were calculated by IBM SPSS Statistics.

4. Conclusions

Collectively, some solid findings were established by the discussion of structure-activity relationship. Introducing of any groups in the benzene ring of iso-PhABA was not preferential for bioactivity, but replacing of the benzene ring of iso-PhABA to a heterocycle ring can mostly keep the bioactivity, i.e., the IC50 values of iso-PyABA 5a, iso-FrABA 6 were close to those of control agents 4a and 1. Furthermore, the bioactivity of substituted-iso-aryl-ABA decreased with an increased size of group in the aryl ring. Also, a double-bond in the 4,5-position of the side chain of iso-PhABA analogs was essential for the bioactivity. For the preference of the different bioassays, some new compounds displayed different preference to physiological process comparing with each other and control agents. For example, substituted-iso-PhABA 4b–4d observed highest IC50 values for the rice seedlings elongation, which was the most efficient assay for the control agents. These results indicated that these analogs might have selectivity and preference to different ABA receptors. As is well known, ABA receptors of plants are multiple gene family [35], and remain unclear for their functions. A selective agonist or antagonist can be used as chemical probe for the discovery of this subject [36]. Thus, those analogs with different preference to bioassays comparing with iso-PhABA and ABA might be potential selective agonist and antagonist for ABA receptors, which behave with a different selectivity for the agonist/antagonist-receptor interaction when compared to iso-PhABA and ABA. Therefore, these finding made a good opportunity to screening more bicyclic ABA analogs for many important applications, including pointing out the direction for structural modification, practical application on the plant protection and probing the responding of different ABA receptors.