Synthesis and Antiproliferative Activity of Triphenylphosphonium Derivatives of Natural Allylpolyalkoxybenzenes

Abstract

Derivatives of natural allylpolyalkoxybenzenes conjugated to triphenylphosphonium (TPP) cations by aliphatic linkers of three, six, seven, and eight atoms were synthesized to examine the role of the polyalkoxybenzene pharmacophore, TPP fragment, and linker length in antiproliferative activities. The key synthetic procedures included (i) hydroboration–oxidation of apiol, dillapiol, myristicin, and allyltetramethoxybenzene; (ii) acylation of polyalkoxybenzyl alcohols or amines; and (iii) condensation of polyalkoxybenzaldehydes followed by hydrogenation and cyclopropyl–homoallyl rearrangement. The targeted TPP conjugates as well as the starting allylbenzenes, the corresponding alkylpolyalkoxybenzenes, and the respective alkyl-TPP salts were evaluated for cytotoxicity in a panel of human cancer cell lines using MTT and Click-iT-EdU assays and in a sea urchin embryo model. The linker of three carbon atoms was identified as favorable for selective cancer cell growth inhibition. Although the propyl-TPP salt was cytotoxic at low micromolar concentrations, the introduction of a polyalkoxybenzene moiety significantly potentiated inhibition of both cell growth and de novo DNA synthesis in several human cancer cell lines, HST-116 colon cancer, A375 melanoma, PC-3 prostate cancer, and T-47D breast carcinoma cells, while it failed to produce any developmental abnormalities in the sea urchin embryos.

Introduction

Crop plants of family Apiaceae, dill Anethum graveolens L., parsley Petroselinum sativum Hoffm., fennel Foeniculum vulgare Mill., and parsnip Pastinaca sativa L., contain considerable amounts of diverse allylpolyalkoxybenzenes (1a–d, Figure 1).¹ Apiol (1a), dillapiol (1b), myristicin (1c), and allyltetramethoxybenzene (ATMB, 1d) exhibited a wide range of biological potencies, including antiproliferative, anti-inflammatory, antioxidant, antimicrobial, antifungal, and insecticidal activities, along with effects on nervous, cardiovascular, and genitourinary systems.¹⁻⁴ Apiol (1a) and myristicin (1c) were described as calcium channel antagonists.^5,6 In multidrug-resistant cells, apiol (1a) and dillapiol (1b) enhanced the efficacy of anticancer drugs, suggesting their potential application as chemosensitizers for cancer chemotherapy.^7,8 Recently, synergistic cytotoxic effects of apiol (1a) (100 μM) with doxorubicin and vincristine associated with blocking the P-glycoprotein transmembrane efflux pump were reported in OVCAR-3 and NCI/ADR-RES ovarian adenocarcinoma cells, along with direct cytotoxicity of apiol (1a) (IC₅₀ values of 200–700 μM) against a panel of human cancer cell lines.⁸ In addition, allylpolyalkoxybenzenes themselves inhibited human cancer cell growth and induced apoptosis, albeit at relatively high concentrations. Particularly, myristicin (1c) suppressed the growth of Caco-2 colon adenocarcinoma cells with an IC₅₀ value of 760 μM for 24 h treatment.⁹ Apiol (1a) and myristicin (1c) displayed cytotoxicity against K-562 leukemia, NCI-H460 lung carcinoma, MCF-7 breast adenocarcinoma, and COLO-205 colon adenocarcinoma cells with IC₅₀ values of 83–368 μM.^10,11 Besides, apiol (1a) at 25–50 μM concentrations induced G0/G1 cell cycle arrest; caspase-3, -8, and -9 activation; and apoptosis in COLO-205 human colon adenocarcinoma cells without affecting nonmalignant epithelial cells in vitro and demonstrated tumor growth inhibition in a mouse COLO-205 xenograft in vivo model.^12,13 The cytotoxic effect of dillapiol (1b) against MDA-MB-231 breast adenocarcinoma and three melanoma cell lines, SK-MEL-28, MEL-5, and Sbcl2, with IC₅₀ values of 25–28 μM was demonstrated. In MDA-MB-231 cells, dillapiol (1b) stimulated apoptosis, caused disintegration of actin filaments, and inhibited cell migration.¹⁴ Similar dillapiol (1b) cytotoxicity (IC₅₀ = 22 μM) was reported against 3T3 mouse fibroblasts.¹⁵ ATMB inhibited the growth of A549 lung carcinoma, MCF-7 breast adenocarcinoma, and HT-29 colon adenocarcinoma cells with IC₅₀ values of 121.7, 193, and 298 μM, respectively.² A natural apiol-related compound SY-1 (2a, Figure 1), isolated from parasitic fungus Antrodia cinnamomea Chang & Chou (Polyporaceae, Aphyllophorales), inhibited the growth of human cancer cells in vitro by induction of G0/G1 cell cycle arrest and apoptosis at 50–150 μM concentration range.^16,17 Intraperitoneal injection of SY-1 (2a) resulted in significant inhibition of COLO-205 tumor xenografts in athymic mice.¹⁸

Figure 1.

Structures of natural polyalkoxybenzenes 1a–d and 2a, TPP conjugates of selected bioactive molecules, and selected alkyl-TPP salts.

The efficiency and selectivity of anticancer agents can be enhanced by the attachment of lipophilic triphenylphosphonium (TPP) cations to biologically active molecules through various carbonic linkers. TPP provides the selective delivery of the desired compounds into the mitochondria driven by the inner negative transmembrane potential.¹⁹⁻²¹ Hyperpolarization of the mitochondrial membrane potential in malignant cells compared to that in normal cells allows for the preferential mitochondrial accumulation of cytotoxic pharmacophores conjugated with TPP cations,¹⁹⁻²¹ which may finally increase the therapeutic window. The triphenylphosphonium-based modification of dihydrobetulinic acids,²² isosteviol,²³ and curcumin²⁴ markedly potentiated their antiproliferative effects. Resveratrol derivatives combined with TPP cations (Figure 1) effectively inhibited the growth of murine cancer cells, whereas resveratrol and methyl-TPP (MTPP, Figure 1) were inactive at the same concentrations.²⁵ Curcumin–TPP and resveratrol–TPP conjugates (Figure 1) accumulated in mitochondria, yielding the generation of reactive oxygen species, loss of the mitochondrial membrane potential, and finally necrotic/apoptotic cancer cell death.^24,25

Cytotoxicity of allylpolyalkoxybenzenes against cancer cells is also associated with the induction of mitochondrial dysfunction. Specifically, dillapiol (1b) (25–50 μM) initiated mitochondrial depolarization and permeability transition in MDA-MB-231 breast carcinoma cells, inducing mitochondria-dependent apoptosis.¹⁴ Myristicin (1c) (≥50 μM) altered the mitochondrial membrane potential in K-562 human leukemia cells.¹¹ Hence, introducing TPP cations into allylpolyalkoxybenzenes would lead to their preferential mitochondrial accumulation, serving as an approach to improve their antiproliferative potency toward cancer cells.

It is worth noting that mitochondria-targeted antioxidants featuring TTP moieties acquire the ability to inhibit effectively and selectively malignant cell growth.^20,26 For example, a coenzyme Q₁₀–TPP conjugate, mitoquinone (Figure 1), was significantly more cytotoxic to human breast cancer cells than the respective nonmalignant epithelial cells. It induced rapid mitochondrial membrane depolarization and cytochrome c release, G1/S cell cycle arrest, checkpoint kinase activation, and cell death predominantly by autophagy and to a lesser degree by caspase-independent apoptosis. In contrast, coenzyme Q₁₀ exhibited markedly lower cytotoxicity against the same cancer cell lines.²⁷ Since allylbenzenes 1a–d were shown to exhibit antioxidant effects,⁴ conjugation with TPP cations would expectedly increase their cytotoxic activity.

Generally, the presence of TPP cations is not sufficient for the rather prominent cytotoxicity of a molecule. Medium-scale screening data demonstrated that only 33 compounds among 700 structures featuring TPP moieties inhibited cancer cell growth with IC₅₀ values less than 10 μM.²⁸ On the other hand, tetraphenylphosphonium chloride as well as TPP derivatives with only simple alkyl chains, such as MTPP and butyl-TPP chlorides, significantly affected the mitochondrial bioenergetics in cancer cells and inhibited cancer cell growth at the nanomolar and low micromolar concentration range. In contrast, their cytotoxicity toward nonmalignant cells was significantly low.²⁹ In MCF-7 human breast carcinoma cells, dodecyl-TPP impaired mitochondrial function and exhibited cytotoxicity at 250 nM concentration, whereas it possessed approximately five times lower cytotoxicity against nonmalignant human fibroblasts.³⁰

The key role of the TPP cations’ hydrophobicity in the membrane potential-dependent accumulation in mitochondria and subsequent mitochondrial dysfunction was well established.^20,31 In a series of alkyltriphenylphosphonium bromide salts (propyl-, heptyl-, decyl-, and dodecyl-TPPs), the inhibition of the respiratory chain complexes and ATP synthesis in mitochondria as well as mitochondrial membrane potential alteration enhanced with increasing hydrophobicity of the cation.³² Another structure–activity relationship study of TPP derivatives with the aliphatic chains ranging from 5 to 16 carbon atoms unequivocally demonstrated the increase of thiol-dependent metabolic oxidative stress, disruption of mitochondrial metabolism, and killing of human melanoma cells with the elongation of the hydrophobic alkyl chain.³³

The length- and structure-related hydrophobicity of the linker may affect the overall activity of TPP-conjugated molecules. In a series of metformin analogues connected to TPP cations with 2, 6, 10, and 12 methylene groups (Mito-Met_nFigure 1), the cellular uptake, inhibition of mitochondrial respiration, and selective antiproliferative activity toward cancer cells increased significantly with the elongation of the carbonic linker, with Mito-Met₁₀ being the most potent.³⁴ In contrast, only modest differences in the antimitotic activity and embryotoxicity were observed for isosteviol–TPP conjugates containing polymethylene and poly(ethylene glycol) chains of various lengths.²³

Following the above literature evidence, the present study aimed at developing synthetic protocols for preparation of natural allylpolyalkoxybenzene derivatives coupled with TPP moieties using aliphatic chains of different lengths and structures, including ester and amide fragments. Our study was based on the following considerations: (i) allylbenzenes 1a–d were easily accessible in large amounts from commercial dill and parsley seed extracts;^35,36 (ii) compounds 1a–d exhibited moderate cytotoxicity against cancer cells likely associated with mitochondrial impairment; (iii) the reported antioxidant activity of allylbenzenes 1a–d suggested that their conjugation with TPP cations may increase their selectivity toward malignant cells, as observed for mitoquinone.²⁷ The comparative cytotoxic activity of the starting allylbenzenes 1a–d, the corresponding alkylpolyalkoxybenzenes, their TPP conjugates, and the respective alkyl-TPP salts was tested using a panel of human cancer cell lines and a sea urchin embryo model. Our study may provide new opportunities for the further development of novel, simple, and effective natural product-based molecules for cancer therapy.

Results and Discussion

Chemistry

Synthesis of TPP Conjugates of Natural Allylpolyalkoxybenzenes

Methyl- and propyl-substituted derivatives of allylpolyalkoxybenzenes (2a–d and 3a–d, Scheme 1) were prepared from starting natural products 1a–dvia the synthetic procedure published earlier.³⁷ The key step in synthesis of TPP-conjugated polyalkoxyphenyl propanes 7a–d comprised the hydroboration of allylbenzenes 1a–d followed by H₂O₂ oxidation to afford corresponding tetraalkoxyphenyl-1-propanoles 4a–d in high yields (Scheme 1). However, the detailed examination of hydroboration–oxidation products of apiol (1a) revealed negligible amounts of the concomitant propylbenzene 3a and arylpropan-2-ol 5a, which were isolated from the reaction mixture. The separation of byproducts 3a and 5a from the reaction mixture favored the development of 4a purification procedure after hydroboration. As a result, compound 4a with >99% purity was obtained. The respective byproducts of hydroboration–oxidation under the same reaction conditions were also found by NMR spectroscopy for the other allylpolyalkoxybenzenes 1b–d. The replacement of the hydroxy group in 4a–d with an iodine atom was carried out under mild conditions using methyl iodide and carbonyldiimidazole (CDI), according to the published protocol.³⁸ The resulting iodinated intermediates 6a–d were refluxed with triphenylphosphine in CH₃CN to afford targeted TPP conjugates 7a–d.

Scheme 1. Synthesis of Alkylpolyalkoxybenzenes 2–4 and TPP-Conjugated Polyalkoxyphenyl Propanes 7a–d.

To analyze the importance of linker length and structure for antiproliferative activity, we developed synthetic procedures for the preparation of TPP-conjugated polyalkoxybenzene derivatives 11, 16, 17, and 22 with chain lengths of 6–8 atoms including ether, esteric, or amide groups from the corresponding aldehydes 8, intermediate phenol 9,³⁹ benzyl alcohol 12,⁴⁰ or benzyl amide 13(41) (Schemes 2 and 3). To obtain apiol–TPP conjugate 11 with the ether linker of seven atoms, we introduced a synthetic protocol that included alkylation of phenol 9 with an excess of dibromohexane followed by Ph₃P-quaternization of intermediate bromide 10 (Scheme 2). Apiol–TPP derivatives featuring linkers of eight atoms with esteric (16) or amide (17) groups were synthesized by acylation of corresponding benzyl alcohol 12 or benzylamine 13 with 6-bromohexanoic acid followed by Ph₃P-quaternization of intermediates 14 and 15 (Scheme 2).

Scheme 2. Synthesis of Apiol–TPP Conjugates 11, 16, and 17 with Linker Lengths of 7–8 Atoms.

Reagents and conditions for the synthesis of compound 8 (ref (37)) are presented in Scheme 1.

Scheme 3. Synthesis of Polyalkoxybenzene–TPP Conjugates 22 with Hex-3-ene Linkers.

Reagents and conditions for the synthesis of compounds 8 (ref (37)) are presented in Scheme 1.

Synthesis of polyalkoxybenzene–TPP conjugates 22 with hex-3-ene linkers included cyclopropyl–homoallyl rearrangement of the corresponding cyclopropyl carbinoles 20 with HBr followed by a conjugation reaction of the resulting bromide 21 with triphenylphosphine. The starting cyclopropyl carbinoles 20 were obtained from polyalkoxybenzaldehydes 8 by a three-step reaction through intermediate chalcones 18 and ketones 19.

Biology

The impact of allylbenzenes 1a–d; alkylated polyalkoxybenzenes 2a–d, 3a–d, and 4a–d; and TPP conjugates 7a–d, 11, 16, and 17 on the viability and proliferation (newly synthesized DNA) of seven human cancer cell lines was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Click-iT-EdU assays. In the viability assay, MTT is mainly reduced by coenzyme NAD(P)H and glycolytic enzymes of the endoplasmic reticulum.^42,43 Thus, cellular MTT reduction measures the rate of glycolytic NAD(P)H production. The Click-iT-EdU assay, a superior alternative to BrdU incorporation assays, based on the incorporation of thymidine analogue EdU into newly synthesized DNA, quantifies the dividing cells using high-content imaging and analysis in the microplate format.^44,45 Compounds 4a–d, 7a–d, 16, and 17 were also selected for NCI60 cytotoxicity screening on 60 human cancer cell lines, which is based on the sulforodamin B (SRB) assay. This method relies on the property of SRB to bind stoichiometrically to proteins under mildly acidic conditions. Then, SRB can be extracted by a basic medium and the amount of the bound dye is considered a proxy for cell mass, thus estimating the rate of cell proliferation. Since this method does not measure the metabolic activity (e.g., MTT assay), the steps required to optimize the protocol for a specific cell line are substantially simplified.

Since fast-growing cells, both cancerous and nonmalignant, were markedly more sensitive to TPP-conjugated resveratrol derivatives than the respective slow-growing fibroblasts,²⁵ we performed a comparative study of the compounds in a sea urchin embryo model⁴⁶ to assess any developmental abnormalities. In this simple organism model, frequently dividing blastomeres at the cleavage stage completed the first mitotic cycle after ∼75 min followed by blastomere division every 35–40 min, resembling fast-growing nonmalignant cells.

To estimate the contribution of the alkyl-TPP fragment to the cytotoxicity of TPP conjugates, the effects of alkyl-TPP salts, MTPP, propyl-TPP (PrTPP), nonyl-TPP (NTPP), hexadecyl-TPP (HDTPP), and C10-isoprenoid-TPP (C10-ITPP) (Figure 1), were studied as well. The results are summarized in Table 1. Out of natural products 1a–d and 2a, only compounds 1a–c slightly decreased the viability of T-47D cells. Methyl-substituted derivatives of dillapiol (2b) and ATMB (2d) failed to inhibit cancer cell growth, whereas the respective myristicin derivative 2c exhibited an antiproliferative effect on A549 lung carcinoma, HCT-116 colon cancer, PC-3 prostate cancer, and T-47D breast carcinoma cells. Propylpolyalkoxybenzenes 3a–d mainly were inactive or displayed negligible activity, and propanol-containing molecules 4a–d were inactive in either assay as well as in NCI60 cytotoxicity screening (Figures S1–S4, Supporting Information). All molecules 1–4 failed to inhibit de novo DNA synthesis up to 250 μM concentration.

Table 1. Effects of Polyalkoxybenzenes, Their TPP Conjugates, and Alkyl-TPP Salts on Sea Urchin Embryos and Human Cancer Cells^a.

	cancer cell growth inhibition, GI50, μMb							DNA synthesis inhibition, GI50, μMc		NCI60 screend
compd	A549	HCT-116	A375	SK-OV-3	PC-3	DU145	T-47D	HCT-116	T-47D	mean GI50, μM	mean GI, %	cleavage alteration, EC, μMe
tubercidin	0.044 ± 0.003	0.08 ± 0.004	0.043 ± 0.004	0.131 ± 0.002	0.076 ± 0.001	0.256 ± 0.09	0.013 ± 0.001	0.0307 ± 0.002	0.044 ± 0.006	0.331f		NDg
1a	>250	>250	>250	>250	>250	>250	70.7 ± 9.9	>250	>250	NDg		>80
1b	>250	>250	>250	>250	>250	>250	80.5 ± 4.0	>250	>250	NDg		>80
1c	>250	>250	>250	>250	>250	>250	113 ± 12.4	>250	>250	NDg		>8
1d	>250	>250	>250	>250	>250	>250	>250	>250	>250	NDg		>100
2a	>250	>250	>250	>250	>250	>250	>250	>250	>250	NDg		>8
2b	>250	>250	>250	>250	>250	>250	>250	>250	>250	NDg		>8
2c	12.8 ± 1.3	32.9 ± 2.3	>250	>250	3.51 ± 0.11	>250	12.5 ± 1.5	>250	>250	NDg		NDg
2d	>250	>250	>250	>250	>250	>250	>250	>250	>250	NDg		>8
3a	>250	112 ± 23.4	167 ± 27	>250	>250	>250	90.2 ± 13.5	>250	>250	NDg		>8
3b	>250	193 ± 33	109 ± 18.5	>250	>250	>250	116 ± 9.0	>250	>250	NDg		NDg
3c	>250	113 ± 8.0	116 ± 12	>250	>250	>250	100 ± 13	>250	>250	NDg		NDg
3d	>250	>250	>250	>250	>250	>250	13.3 ± 0.8	>250	>250	NDg		NDg
4a	>250	>250	>250	>250	>250	>250	>250	>250	>250	NDg	0	>4
4b	>250	>250	>250	>250	>250	>250	>250	>250	>250	NDg	0	>4
4c	>250	>250	>250	>250	>250	>250	>250	>250	>250	NDg	0	>4
4d	>250	>250	>250	>250	>250	>250	>250	>250	>250	NDg	0	>4
7a	3.00 ± 0.21	0.263 ± 0.033	0.627 ± 0.085	2.42 ± 0.43	0.552 ± 0.03	2.86 ± 0.4	0.089 ± 0.008	0.367 ± 0.05	0.104 ± 0.007	1.66		>4
7b	4.64 ± 0.37	0.229 ± 0.027	0.491 ± 0.019	6.62 ± 0.13	0.555 ± 0.049	3.26 ± 0.36	0.107 ± 0.004	0.405 ± 0.036	0.118 ± 0.007	1.58		>4
7c	12.2 ± 0.61	0.331 ± 0.039	1.08 ± 0.06	11.0 ± 1.4	0.41 ± 0.04	5.12 ± 0.66	0.01 ± 0.0005	0.22 ± 0.03	0.025 ± 0.002	NDg	63.9	>4
7d	17.9 ± 0.36	0.218 ± 0.024	2.35 ± 0.24	21.6 ± 1.53	3.88 ± 0.55	8.13 ± 0.15	0.50 ± 0.07	0.681 ± 0.09	0.613 ± 0.025	NDg	32.9	>4
11	0.463 ± 0.042	1.95 ± 0.21	1.93 ± 0.077	24.6 ± 2.0	0.258 ± 0.026	20.8 ± 1.3	0.421 ± 0.013	2.83 ± 0.37	0.606 ± 0.06	NDg		4
16	18.5 ± 0.67	1.07 ± 0.02	3.43 ± 0.08	10.2 ± 0.19	4.18 ± 0.13	14.1 ± 0.37	0.43 ± 0.88	NDg	NDg	NDg	49.2	>4
17	9.96 ± 0.22	1.05 ± 0.01	2.78 ± 0.17	18.9 ± 2.86	2.15 ± 0.040	11.3 ± 0.75	0.256 ± 0.08	NDg	NDg	NDg	35.8	>4
MTPP	1.32 ± 0.23	2.32 ± 0.70	33.3 ± 4.7	>250	5.01 ± 0.30	1.56 ± 0.03	3.33 ± 0.47	13.7 ± 2.2	6.99 ± 0.84	NDg		>4
PrTPP	0.971 ± 0.068	1.5 ± 0.17	10.2 ± 0.51	4.08 ± 0.61	1.83 ± 0.21	1.06 ± 0.13	2.39 ± 0.26	9.53 ± 1.4	3.45 ± 0.24	NDg		>8
NTPP	0.171 ± 0.022	0.077 ± 0.006	0.254 ± 0.007	0.647 ± 0.029	0.109 ± 0.005	1.23 ± 0.04	0.023 ± 0.002	0.092 ± 0.002	0.047 ± 0.006	NDg		0.5; 1h
HDTPP	0.728 ± 0.066	0.451 ± 0.054	0.52 ± 0.031	13.8 ± 1.4	0.25 ± 0.02	1.11 ± 0.033	0.268 ± 0.005	3.11 ± 0.34	0.398 ± 0.035	NDg		>4; 2i
C10-ITPP	0.58 ± 0.015	0.079 ± 0.0010.72j	0.221 ± 0.073	1.27 ± 0.89	0.296 ± 0.0471.2j	0.932 ± 0.031	0.053 ± 0.002	NDg	NDg	NDg		1; 2h

^aA549, lung carcinoma; HCT-116, colon cancer; A375, melanoma; SK-OV-3, ovarian carcinoma; PC-3 and DU145, prostate cancer; T-47D, breast carcinoma.

^bGI₅₀, concentration required for 50% inhibition of cancer cell growth determined by the MTT assay.

^cGI₅₀, concentration required for 50% inhibition of de novo DNA synthesis in the Click-iT-EdU Alexa Fluor 488 cell proliferation assay.

^dScreening on a panel of 60 human cancer cell lines using the SRB assay. GI₅₀, μM, concentration required for 50% inhibition of cancer cell growth in the five-dose test; GI, %, single-dose inhibition of cell growth at 10 μM concentration.

^eThe sea urchin embryo assay was conducted as described previously.⁴⁶ Fertilized eggs were exposed to twofold decreasing concentrations of compounds. Duplicate measurements showed no differences in effective threshold concentration (EC) values.

^fNCI60 screen data, NSC 115712.

^gND, not determined.

^hCleavage arrest.

ⁱEmbryo death at the early gastrula stage at 2 μM concentration.

^jData from ref (28).

MTPP and TPP alkyl derivatives with 3 (PrTPP), 9 (NTPP), 16 (HDTPP), and 10 (C10-ITPP) carbon aliphatic chains showed pronounced cytotoxicity against all cancer cell lines with GI₅₀ values within the nanomolar–low micromolar concentration range in both MTT and Click-iT-EdU assays. The elongation of the alkyl moiety to nine carbon atoms markedly inhibited cancer cell growth (MTPP < PrTPP < NTPP), which was consistent with the reported pivotal role of TPP cation hydrophobicity in the antiproliferative effect.^33,34 NTPP with an alkyl chain of 9 carbon atoms was the most potent, C10-ITPP displayed similar activity, whereas the aliphatic chain elongation to 16 carbon atoms (HDTPP) resulted in a decrease in cytotoxicity. The ability of C10-ITPP to inhibit cancer cell growth in the nanomolar concentration range was described previously.²⁸ MTPP and PrTPP failed to produce any developmental abnormalities in the sea urchin embryo model. In contrast, TPP cations with longer aliphatic chains, NTPP and C10-ITPP, caused cleavage alteration/arrest followed by embryo death and exhibited a toxic effect when applied at the blastula stage after hatching. Similar to cell-based methods, NTPP produced the highest effect in the sea urchin embryo assay, similar to cell-based methods. HDTPP did not affect embryo development during the cleavage stage, albeit it induced embryo death further at early gastrula (Table 1).

Since NTPP, HDTPP, and C10-ITPP, TPP cations with long aliphatic chains of 9–16 carbon atoms, were toxic for normal sea urchin embryo cells, the preparation and the cytotoxicity assessment of polyalkoxybenzenes 7a–d connected to TPP cations by trimethylene linkers was our prime concern. The hydrophobic character of allylpolyalkoxybenzenes 1a–d, exemplified by dillapiol (1b),^14,15 could be considered as a contribution to the overall hydrophobicity-dependent potency of the corresponding TPP-containing derivatives 7a–d without a particular need for linker elongation.

TPP-conjugated polyalkoxybenzenes 7a–d demonstrated different cytotoxicity against various human cancer cell lines in both MTT and Click-iT-EdU assays. In particular, the T-47D breast carcinoma cell line was the most sensitive (GI₅₀ values of 0.01–0.5 μM), whereas A549 lung carcinoma, SK-OV-3 ovarian carcinoma, and DU145 prostate cancer cell lines were less sensitive to these compounds, with the highest GI₅₀ values ranging from 2.42 to 21.6 μM in the MTT assay (Table 1). Structure–activity relationship (SAR) analysis of compounds 7a–c featuring the methylenedioxy moiety did not reveal any obvious trends, while the respective tetramethoxy-substituted derivative 7d displayed the lowest effect. Our results for 7a–d correlated well with NCI60 screening data (Table 1; Figures S5–S10, Supporting Information). Although compounds 7c and 7d were evaluated in NCI60 screening only at 10 μM concentration, they considerably suppressed the proliferation of selected cancer cell lines. Namely, 7c at this concentration inhibited the growth of 18 cancer cell lines by more than 80% and caused a lethal effect on SK-MEL-5 melanoma and MDA-MB-468 breast cancer cells (Figure S9, Supporting Information). ATMB-TPP conjugate 7d was less active, inducing ∼100% growth inhibition of MDA-MB-435 melanoma and A498 renal cancer cells only (Figure S10, Supporting Information). Of note, in both MTT and Click-iT-EdU tests, cytotoxicity of compounds 7a–d against the most sensitive cell lines HCT-116, A375, PC-3, and T-47D (except for the effect of 7d on PC-3 cells in MTT assay) significantly exceeded the effect of the corresponding PrTPP (Table 1), suggesting the contribution of the polyalkoxybenzene pharmacophore to the overall potency of TPP conjugates. In particular, in the T-47D cell line, the activity of TPP conjugates decreased in the following order: 7c > 7a > 7b > 7d in both cell viability (MTT) and proliferation (Click-iT-EdU) assays: 239, 26.9, 22.3, and 4.8 times (MTT test) and 138, 33.2, 29.2, and 5.6 times (Click-iT-EdU test), respectively, more potent than PrTPP. Myristicin derivative 7c exhibited an effect comparable to tubercidin, the positive control. In contrast, whereas the Click-iT-EdU assay of the HCT-166 cell line demonstrated the same activity order as that for T-47D cells, the MTT assay of the same cells revealed reverse activity order 7d > 7b > 7a > 7c, suggesting the cell-type-specific features of glycolytic NAD(P)H production. Such suggestion is consistent with the fact that the HCT-116 cell line is highly glycolytic and has reduced pyruvate dehydrogenase activity and the oxidative pentose-phosphate pathway as the default cytosolic NADPH production pathway.⁴⁷⁻⁴⁹

Considering the reported correlation between the aliphatic linker pattern and antiproliferative potencies of TPP conjugates,^34,50 we conducted a cytotoxicity study of apiol–TPP derivatives 11, 16, and 17 featuring alkyl chains of different structures and lengths (Table 1) and did not find any apparent SAR for these molecules on seven cancer cell lines. In single-dose NCI60 screening, TPP conjugates 16 and 17 at 10 μM concentration caused 80–100% growth inhibition of nine and three cancer cell lines, respectively (Figures S11 and S12, Supporting Information). Generally, the introduction of ether, esteric, or amide linkers with chain lengths of 7–8 atoms yielded less active compounds than the apiol–TPP conjugate 7a featuring a C3 linker. However, ether 11 was more potent than 7a on A549 and, to a lesser extent, on PC-3 cells, together with a moderate antiproliferative effect on the sea urchin embryos. Noteworthy, compounds 11, 16, and 17 showed pronounced cytotoxicity against the most sensitive T-47D breast carcinoma cell line with GI₅₀ values within the nanomolar concentration range. Unfortunately, biological evaluation of compounds 22a,c,e–g was unavailable due to their insufficient solubility in culture media and seawater.

Conclusions

In summary, synthetic routes toward TPP-conjugated polyalkoxybenzenes were developed, which relied on hydroboration–oxidation of available natural allylbenzenes and acylation of polyalkoxybenzyl alcohols or amines. The targeted molecules exhibited pronounced cytotoxicity against seven human cancer cell lines with GI₅₀ values within the nanomolar–low micromolar concentration range. A three-carbon-atom linker seems to favor the selective cancer cell growth inhibition since TPP cations (NTPP, HDTPP, and C10-ITPP) with long aliphatic chains of 9–16 carbon atoms markedly suppressed the division of nontransformed cells, sea urchin embryo blastomeres. The introduction of a polyalkoxybenzene pharmacophore augmented the cytotoxic effect on the most sensitive human cancer cell lines, HST-116, A375, PC-3, and T-47D, as revealed by a comparison of cell growth inhibition caused by TPP-C3-linked conjugates 7a–d and the respective PrTPP.

Experimental Section

General Experimental Procedures

Melting points were measured on a Boetius melting point apparatus and were uncorrected. Reaction mixtures were stirred magnetically. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker DRX-500 instrument [working frequencies of 500.13 MHz (¹H) and 125.76 MHz (¹³C)]. Chemical shifts were stated in parts per million (ppm) and referenced to the appropriate NMR solvent peaks. Spin–spin coupling constants (J) were reported in hertz (Hz). NMR spectra (Supporting Information) were prepared using original software designed at N.D. Zelinsky Institute of Organic Chemistry RAS (Moscow, Russian Federation) (http://nmr.ioc.ac.ru:8080/SDF2PDF.kl1). Low-resolution mass spectra (m/z) were recorded on a Finnigan MAT/INCOS 50 mass spectrometer at 70 eV using direct probe injection. High-resolution mass spectra (HRMS) were measured on a Bruker micrOTOF II instrument using electrospray ionization (ESI).

Elemental analysis was performed on an automated PerkinElmer 2400 CHN microanalyzer. Flash chromatography was carried out on silica gel (Acros, 0.035–0.070 mm, 60 Å). Thin-layer chromatography (TLC) was performed on Merck 60 F₂₅₄ plates. Nonanhydrous solvents and reagents of the highest commercial quality were purchased from Acros Organics (Belgium) and used as received. Methyl-TPP bromide (MTPP) was purchased from Sigma-Aldrich (Germany). Nonyl-TPP bromide (NTPP), hexadecyl-TPP iodide (HDTPP), and 2,6-dimethylocta-2,6-diene-TPP bromide (C10-ITPP) were synthesized according to literature procedures.^51,52

Synthesis of Propyltriphenylphosphonium Methanesulphonate (PrTPP)

Propyl methanesulfonate (0.20 g, 1.45 mmol) and PPh₃ (0.455 g, 1.74 mmol) were refluxed in n-decane (3 mL) for 6 h. The reaction mixture was cooled to room temperature, and the solvent was decanted. The residual solid was dissolved in EtOH (1 mL), and the product was precipitated by slow addition of ether (5 mL). The precipitate was filtered off, washed with ether (3 mL), and dried to afford PrTPP (0.325 g, 56% yield) as white crystals: mp 261–263 °C (lit.⁵³ 263–264 °C); ¹H NMR (300 MHz, DMSO-d₆) δ 7.71–7.96 (15H, m, Ph), 3.57 (2H, m, CH₂), 2.30 (3H, s, SCH₃), 1.56 (2H, m, CH₂), 1.08 (3H, t, J = 7.5 Hz, CH₃).

Isolation of Plant Allylpolyalkoxybenzenes 1a–d

Liquid CO₂ extraction of parsley and dill seeds was carried out by the company Karavan Ltd. (Krasnodar, Russia).^35,36 Allylpolyalkoxybenzenes 1a–d with 98–99% purity were obtained by high-efficiency distillation of CO₂ extracts using a pilot plant device at N.D. Zelinsky Institute of Organic Chemistry RAS (Moscow, Russia). The seed essential oils of parsley varieties cultivated in Russia contained 70–75% apiol (1a) (var. Sakharnaya), 40–46% myristicin (1c) (var. Astra), and 21% allyltetramethoxybenzene (1d) (var. Slavyanovskaya). Indian dill seeds were purchased from Vremya & Co. (St. Petersburg, Russia). The dill seed essential oil contained 30–33% dillapiol (1b).

General Procedure for the Synthesis of 1-Arylpropan-1-ols (4a–d)

A cold solution (0–5 °C) of I₂ (25 mmol, 6.4 g) in tetrahydrofuran (THF, 75 mL) was added dropwise to the solution of NaBH₄ (95 mmol, 3.6 g) in dry THF at 0–5 °C under Ar, monitoring iodine decolorization. Then, the solution was stirred for 10 min, and a cold solution (0–5 °C) of allylbenzene (90 mmol) in THF (50 mL) was added. The resulting mixture was stirred for 1 h at room temperature, cooled to 7–10 °C, and diluted gradually with water (25 mL) to obtain a clear solution. Then, a cooled solution (7–10 °C) of 3 M NaOH (25 mL) was added followed by careful addition of 30% hydrogen peroxide (9 mL), maintaining the temperature below 30 °C, taking into account the strong exothermic effect. The resulting mixture was stirred for 1 h at room temperature, the organic layer was separated, the aqueous layer was washed with THF. The remaining water solution was diluted with water (15 mL), saturated with NaCl (5 mL), extracted with EtOAc (100 mL), and dried with MgSO₄. All organic layers (THF and EtOAc) were combined and evaporated. The resulting oil was collected to afford target 3-arylpropanoles 4a–d, which could contain 1–2% of 3-aryl-propanol-2 (5a) and 3–5% of starting allylbenzenes 1a–d according to NMR spectra. Pure 4a–d were obtained from the CH₂Cl₂ solution of arylpropanols by reprecipitation with hexane.

3-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)propan-1-ol (4a)

20.5 g, 92% yield; white solid; mp 49–51 °C; ¹H NMR (500 MHz, CDCl₃) δ 6.31 (s, 1H, H_Ar), 5.95 (s, 2H, OCH₂O), 3.90 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 3.60 (t, J = 6.1 Hz, 2H, CH₂), 2.65 (t, J = 7.3 Hz, 2H, CH₂), 1.80 (p, J = 6.7 Hz, 2H, CH₂); ¹³C NMR (126 MHz, CDCl₃) δ 139.1, 138.5, 136.3, 134.9, 127.1, 108.3, 101.4, 61.6, 60.1, 56.8, 33.7, 25.9; anal. C 60.02; H 6.68; calcd for C₁₂H₁₆O₅, C 60.0; H 6.71.

3-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)propan-1-ol (4b)

18.8 g, 87% yield; colorless oil; ¹H NMR (500 MHz, CDCl₃) δ 6.35 (s, 1H, H_Ar), 5.88 (s, 2H OCH₂O), 4.02 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.57 (t, J = 6.1 Hz, 2H, CH₂), 2.63 (t, J = 7.3 Hz, 2H, CH₂), 1.78 (p, J = 6.7 Hz, 2H, CH₂); ¹³C NMR (126 MHz, CDCl₃) δ 144.8, 144.3, 137.4, 135.7, 127.4, 102.6, 101.1, 61.4, 59.9, 33.6, 25.6; anal. C 60.01; H 6.70; calcd for C₁₂H₁₆O₅, C 60.0; H 6.71.

3-(7-Methoxy-1,3-benzodioxol-5-yl)propan-1-ol (4c)

16.4 g, 86% yield; colorless oil; ¹H NMR (500 MHz, CDCl₃) δ 6.39 (d, J = 1.3 Hz, 1H, H_Ar), 6.36 (d, J = 1.4 Hz, 1H, H_Ar), 5.92 (s, 2H, OCH₂O), 3.88 (s, 3H, OCH₃), 3.66 (t, J = 6.4 Hz, 2H, CH₂), 2.62 (t, J = 7.6 Hz, 2H, CH₂), 1.84 (p, J = 6.5 Hz, 2H, CH₂); ¹³C NMR (126 MHz, CDCl₃) δ 149.2, 143.9, 137.0, 133.7, 108.0, 102.9, 101.6, 62.5, 57.0, 34.8, 32.6; anal. C 62.86; H 6.68; calcd for C₁₁H₁₄O₄, C 62.85; H 6.71.

3-(2,3,4,5-Tetramethoxyphenyl)propan-1-ol (4d)

17.3 g, 75% yield; colorless oil; ¹H NMR (500 MHz, CDCl₃) δ 6.45 (s, 1H, H_Ar), 3.93 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 3.82 (s, 6H, OCH₃), 3.59 (t, J = 6.1 Hz, 2H, CH₂), 2.68 (t, J = 7.3 Hz, 2H, CH₂), 1.82 (p, J = 5.8 Hz, 2H, CH₂); ¹³C NMR (126 MHz, CDCl₃) δ 149.5, 146.8, 145.2, 141.1, 129.5, 107.5, 61.5, 61.1, 61.1, 61.0, 56.1, 33.5, 25.8; anal. C 60.88; H 7.85; calcd for C₁₃H₂₀O₅, C 60.92; H 7.87.

1-(4,7-Dimethoxy-2H-1,3-benzodioxol-5-yl)propan-2-ol (5a)

This was isolated by column chromatography (EtOAc): 0.19 g, 1% yield; white solid; mp 55–56 °C; ¹H NMR (DMSO-d₆, 300 MHz) δ 6.41 (1H, s, H_Ar), 5.95 (2H, s, OCH₂O), 4.47 (1H, d, J = 4.8 Hz, OH), 3.75 (7H, m, 2 OCH₃, CHOH), 2.60 (1H, dd, J = 13.0 Hz, J = 6.5 Hz, CH₂), 2.46 (1H, m, CH₂), 1.00 (3H, d, J = 6,1, CH₃); ¹H NMR (CDCl₃, 500 MHz) δ 6.32 (1H, s, H_Ar), 5.95 (2H, s, OCH₂O), 3.97 (1H, m, CHOH), 3.90 (3H, s, OCH₃), 3.85 (3H, s, OCH₃), 2.76 (1H, dd, J = 13.5 Hz, J = 4.3 Hz, CH₂), 2.62 (1H, dd, J = 13.5 Hz, J = 8 Hz, CH₂), 1.22 (3H, d, J = 6.1 Hz, CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ 138.9, 138.6, 136.5, 135.5, 124.0, 109.4, 101.5, 68.3, 59.9, 56.9, 40.2, 22.9; anal. C 59.77; H 6.82; calcd for C₁₂H₁₆O₅, C 60.00; H 6.71.

General Procedure for the Synthesis of (3-Iodopropyl)benzenes (6a–d)

CH₃I (7.1 g, 50 mmol) was added to the solution of 1-arylpropan-1-ol (4a–d) (10 mmol) and CDI (10 mmol) in dry CH₃CN (20 mL); the mixture was stirred for 30 min at room temperature, refluxed for 2 h, and cooled; and then, ether (30 mL) and water (30 mL) were added. The organic layer was separated; washed with aqueous 20% HCl, a saturated aqueous solution of NaHCO₃, and aqueous 30% Na₂S₂O₃, water; and dried. After evaporation, the target product (7–7.5 mmol, 70–75%) was obtained with a purity of ∼90%. It can be used for the next step without further purification. Pure iodopropylbenzenes 6a–d were isolated by column chromatography (EtOAc–petr, 1:5).

5-(3-Iodopropyl)-4,7-dimethoxy-2H-1,3-benzodioxole (6a)

2.6 g, 75% yield; white solid; mp 51–53 °C; ¹H NMR (500 MHz, CDCl₃) δ 6.34 (1H, s, H_Ar), 5.94 (2H, s, OCH₂O), 3.90 (3H, s, OCH₃), 3.86 (3H, s, OCH₃), 3.17 (2H, t, J = 7.0 Hz, CH₂), 2.64 (2H, t, J = 7.2 Hz, CH₂), 2.06 (2H, quintet, J = 7.0 Hz, CH₂); ¹³C NMR (126 MHz, CDCl₃) δ 138.8, 138.6, 136.4, 135.2, 125.9, 108.6, 101.5, 60.0, 57.0, 34.2, 31.0, 6.8; anal. C 41.18; H 4.32; I 36.22; calcd for C₁₂H₁₅IO₄, C 41.16; H 4.32; I 36.24.

6-(3-Iodopropyl)-4,5-dimethoxy-1,3-benzodioxole (6b)

3.0 g, 85% yield; colorless oil; ¹H NMR (500 MHz, CDCl₃) δ 6.36 (1H, s, H_Ar), 5.89 (2H, s, OCH₂O), 4.02 (3H, s, OCH₃), 3.77 (3H, s, OCH₃), 3.18 (2H, t, J = 7.0 Hz, CH₂), 2.62 (2H, t, J = 7.3 Hz, CH₂), 2.06 (2H, quintet, J = 7.0 Hz, CH₂); ¹³C NMR (126 MHz, CDCl₃) δ 145.0, 138.1, 136.4, 126.8, 103.1, 101.6, 61.7, 60.3, 35.0, 31.2, 7.0; anal. C 41.18; H 4.30; I 36.23; calcd for C₁₂H₁₅IO₄, C 41.16; H 4.32; I 36.24.

6-(3-Iodopropyl)-4-methoxy-1,3-benzodioxole (6c)

1.8 g, 55% yield; colorless oil; ¹H NMR (500 MHz, CDCl₃) δ 6.38 (1H, d, J = 1.3 Hz, H_Ar), 6.37 (1H, d, J = 1.3 Hz, H_Ar), 5.93 (2H, s, OCH₂O), 3.89 (3H, s, OCH₃), 3.16 (2H, t, J = 6.8 Hz, CH₂), 2.64 (2H, t, J = 7.2 Hz, CH₂), 2.07 (2H, quintet, J = 6.8 Hz, CH₂); ¹³C NMR (126 MHz, CDCl₃) δ 149.3, 144.0, 135.3, 134.0, 108.3, 103.0, 101.7, 57.0, 36.6, 35.4, 6.7; anal. C 41.33; H 4.22; I 39.83; calcd for C₁₁H₁₃IO₃, C 41.27; H 4.09; I 39.64.

1-(3-Iodopropyl)-2,3,4,5-tetramethoxybenzene (6d)

2.7 g; 75% yield; colorless oil; ¹H NMR (500 MHz, CDCl₃) δ 6.47 (1H, s, H_Ar), 3.92 (3H, s, OCH₃), 3.87 (3H, s, OCH₃), 3.83 (3H, s, OCH₃), 3.81 (3H, s, OCH₃), 3.21 (2H, t, J = 7.0 Hz, CH₂), 2.67 (2H, t, J = 7.4 Hz, CH₂), 2.10 (2H, quintet, J = 7.0 Hz, CH₂); ¹³C NMR (126 MHz, CDCl₃) δ 149.2, 147.1, 145.4, 141.4, 128.2, 107.8, 61.2, 61.1, 61.0, 56.3, 34.3, 31.0, 6.8; anal. C 42.65; H 5.24; I 34.65; calcd for C₁₃H₁₉IO₄, C 42.64; H 5.23; I 34.66.

General Procedure for the Synthesis of Triphenyl(propyl)phosphonium Conjugates (7a–d)

A solution of (3-iodopropyl)benzene (6a–d) (2.86 mmol) and triphenylphosphine (2.85 mmol) in dry toluene (10 mL) was refluxed for 7 h, cooled overnight in a refrigerator, and filtered to afford target 7a–d, 1.6–2.6 mmol (57–92%).

[3-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)propyl](triphenyl)phosphonium Iodide (7a)

1.0 g, 57% yield; white solid; mp 208–210 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.04–7.60 (15H, m, PPh₃), 6.41 (1H, s, H_Ar), 5.95 (2H, s, OCH₂O), 3.75 (3H, s, OCH₃), 3.72 (3H, s, OCH₃), 3.64–3.52 (2H, m, CH₂), 2.69 (2H, t, J = 7.6 Hz, CH₂), 1.88–1.70 (2H, m, CH₂); ¹³C NMR (126 MHz, DMSO-d₆) δ 138.5, 138.2, 135.3 (d, ¹J_C,P = 100.8 Hz), 134.8, 133.5 (d, ²J_C,P = 9.9 Hz), 130.1 (d, ³J_C,P = 12.4 Hz), 125.3, 118.7, 118.1, 108.6, 101.3, 59.6, 56.6, 30.5 (d, ³J_C,P = 17.0 Hz), 23.0 (d, ²J_C,P = 3.1 Hz), 20.0 (d, ¹J_C,P = 50.9 Hz); anal. C 59.01; H 4.82; I 20.74, P 4.89; calcd for C₃₀H₃₀IO₄P, C 58.83; H 4.94; I 20.72, P 5.06.

[3-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)propyl](triphenyl)phosphonium Iodide (7b)

1.3 g, 72% yield; white solid; mp 172–174 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.19–7.65 (15H, m, PPh₃), 6.47 (1H, s, H_Ar), 5.94 (2H, s, OCH₂O), 3.90 (3H, s, OCH₃), 3.68–3.60 (2H, m, CH₂), 3.58 (3H, s, OCH₃), 2.68 (2H, t, J = 7.8 Hz, CH₂), 1.75 (qiuntet, J = 8.5 Hz, 1H); ¹³C NMR (126 MHz, DMSO-d₆) δ 144.2, 144.1, 136.6 (d, ¹J_C,P = 139.1 Hz), 134.9, 133.6 (d, ²J_C,P = 10.4 Hz), 130.3 (d, ³J_C,P = 12.4 Hz), 125.9, 118.86, 118.2, 102.7, 101.2, 60.9, 59.7, 30.2 (d, ³J_C,P = 17.5 Hz), 23.3 (d, ²J_C,P = 3.8 Hz), 20.2 (d, ¹J_C,P = 50.0 Hz); anal. C 58.95; H 4.96; I 20.64, P 5.25; calcd for C₃₀H₃₀IO₄P, C 58.83; H 4.94; I 20.72, P 5.06.

[3-(7-Methoxy-1,3-benzodioxol-5-yl)propyl](trimethyl)phosphonium Iodide (7c)

1.2 g, 75% yield; white amorphous solid; ¹H NMR (500 MHz, DMSO-d₆) δ 8.04–7.56 (15H, m, PPh₃), 6.46 (1H, s, H_Ar), 6.45 (1H, s, H_Ar), 5.94 (2H, s, OCH₂O), 3.79 (3H, s, OCH₃), 3.68–3.51 (2H, m, CH₂), 2.70 (t, J = 7.4 Hz, 2H, CH₂), 1.82 (quintet, J = 7.8 Hz, 1H); ¹³C NMR (126 MHz, DMSO-d₆) δ 148.4, 143.1, 133.8 (d, ¹J_C,P = 192.9 Hz) 133.7 (d, ²J_C,P = 10.5 Hz), 130.3 (d, ³J_C,P = 12.5 Hz), 118.9, 118.2, 108.04, 102.4, 101.01, 56.4, 35.6, 35.6 (d, ³J_C,P = 17.1 Hz), 24.1 (d, ²J_C,P = 3.9 Hz) 19.9 (d, ¹J_C,P = 50.3 Hz); anal. C 59.66.; H 4.83; I 21.95, P 5.23; calcd for C₂₉H₂₈IO₃P, C 59.81; H 4.85; I 21.79, P 5.32.

[3-(2,3,4,5-Tetramethoxyphenyl)propyl](triphenyl)phosphonium Iodide (7d)

1.7 g, 92% yield; white solid; mp 138–141 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.40–7.52 (15H, m, PPh₃), 6.53 (1H, s, H_Ar), 3.77 (3H, s, OCH₃), 3.71 (3H, s, OCH₃), 3.70 (3H, s, OCH₃), 3.66–3.61 (2H, m, CH₂), 3.59 (3H, s, OCH₃), 2.70 (2H, t, J = 7.7 Hz, CH₂), 1.79 (quintet, J = 7.9, 7.2 Hz, 1H); ¹³C NMR (126 MHz, DMSO-d₆) δ 149.0, 145.7 (d, ¹J_C,P = 217.6 Hz), 141.1, 135.0, 133.7 (d, ²J_C,P = 10.3 Hz), 130.3 (d, ³J_C,P = 12.5 Hz), 127.8, 118.9, 118.2, 108.2, 60.9, 60.8, 60.6, 56.1, 30.5 (d, ³J_C,P = 17.4 Hz), 23.2 (d, ²J_C,P = 3.8 Hz), 20.2 (d, ¹J_C,P = 50.6 Hz); anal. C 59.36.; H 5.29; I 20.34, P 4.82; calcd for C₃₁H₃₄IO₄P, C 59.24; H 5.45; I 20.19, P 4.93.

5-[(6-Bromohexyl)oxy]-4,7-dimethoxy-2H-1,3-benzodioxole (10)

Phenol 9 (5.1 mmol, 1.0 g) was added to the solution of Na (5.46 mmol, 0,13 g) in abs. EtOH (10 mL); the mixture was refluxed for 1.5 h, then dibromohexane (15.3 mmol, 3.7 g) was added, and the solution was refluxed for 6 h. Then, the reaction mixture was evaporated, diluted with CH₂Cl₂ (30 mL), washed with water, and dried. The target bromide 10 was separated by column chromatography (SiO₂, benzene–EtOAc, grad. 1:40–1:15 R_f = 0.5): 0.9 g, 51% yield, mp 61–62 °C (petr. ether); ¹H NMR (500 MHz, CDCl₃) δ 6.11 (1H, s, H_Ar), 5.91 (2H, s, OCH₂O), 3.93 (2H, t, J = 6.5 Hz, OCH₂), 3.89 (3H, s, OCH₃), 3.86 (3H, s, OCH₃), 3.43 (2H, t, J = 6.8 Hz, BrCH₂), 1.90 (2H, quintet, J = 6.9 Hz, CH₂), 1.80 (2H, quintet, J = 6.7 Hz, CH₂), 1.51 (4H, m, 2 CH₂); anal. C 50.05; H 5.99; Br 21.81; calcd for C₁₅H₂₁BrO₅, C 49.87; H 5.86; Br 22.12.

{6-[(4,7-Dimethoxy-2H-1,3-benzodioxol-5-yl)oxy]hexyl}(triphenyl)phosphonium Bromide (11)

The solution of bromide 10 (0.94 mmol, 0.34 g), PPh₃ (0.94 mmol, 0.25 g), and KI (0.3 mmol, 0.05 g) in abs. toluene (7 mL) was refluxed for 48 h and cooled. Toluene was decanted to afford 0.38 g (85% yield) of target phosphonium bromide 11 with 90% purity. Further recrystallization from acetone resulted in 11 as white crystals: 0.17 g, 34% yield, mp 122–123 °C (acetone); ¹H NMR (500 MHz, CDCl₃) δ 7.90 (3H, m, H_Ph), 7.84–7.75 (12H, m, H_Ph), 6.24 (1H, s, H_Ar), 5.92 (2H, s, OCH₂O), 3.88 (2H, t, J = 6.4 Hz, OCH₂), 3.77 (3H, s, OCH₃), 3.70 (3H, s, OCH₃), 3.59 (2H, m, PCH₂), 1.68-1.40 (8H, m, 4 CH₂); anal. C 63.66; H 5.80; Br 13.16; calcd for C₃₃H₃₆BrO₅P, C 63.57; H 5.82; Br 12.82.

(4,7-Dimethoxy-2H-1,3-benzodioxol-5-yl)methyl 6-Bromohexanoate (14)

SOCl₂ (1.19 g, 10 mmol, 0.72 mL) was added to 6-bromohexanoic acid (0.641 g, 3.3 mmol) and stirred at 60 °C for 1 h. HCl, SO₂, and the excess of SOCl₂ were evaporated using a water jet pump and then evaporated with abs. benzene (3 × 0.5 mL). The resulting oil was dissolved in abs. benzene (2 mL), added to the cold solution (4 °C) of benzyl alcohol (12) (0.678 g, 3.2 mmol) in abs. pyridine (2.2 mL), and stirred at room temperature overnight. The reaction mixture was diluted with ice water (50 mL) and extracted with CH₂Cl₂ (2 × 25 mL). The extract was washed with 10% aq. HBr (10 mL), water, 5% aq. NaHCO₃ (10 mL), and water until neutral pH; dried by filtration through a cotton plug; and concentrated in vacuo upon heating. The resulting liquid (1.06 g) was purified by column chromatography (benzene–EtOAc, 19:1) to afford 14 (0.938 g, 73% yield) as a yellowish liquid that was used for the subsequent synthetic step without further purification: ¹H NMR (500 MHz, CDCl₃) δ 6.51 (1H, s, H_Ar), 6.00 (2H, s, OCH₂O), 5.05 (2H, s, OCH₂), 3.94 (3H, s, OCH₃), 3.86 (3H, s, OCH₃), 3.40 (2H, t, J = 7.4 Hz, CH₂), 2.33 (2H, t, J = 7.3 Hz, CH₂), 1.86 (2H, m, CH₂), 1.66 (2H, m, CH₂), 1.47 (2H, m, CH₂). The corresponding chloro-derivative (8%) was identified in 14 by ¹H NMR analysis.

6-Bromo-N-[(4,7-dimethoxy-2H-1,3-benzodioxol-5-yl)methyl]hexanamide (15)

SOCl₂ (10 mmol, 1.19 g, 0.72 mL) was added to 6-bromohexanoic acid (3.3 mmol, 0.641 g) and stirred at 60 °C for 1 h. The excess of SOCl₂, HCl, and SO₂ was evaporated using a water jet pump and then evaporated with abs. benzene (3 × 0.5 mL). The resulting oil was dissolved in abs. benzene (2 mL), added to the solution of benzylamine·HCl (13) (2.5 mmol, 0.619 g) in abs. pyridine (1.5 mL), and stirred at room temperature overnight. The reaction mixture was diluted with ice water (50 mL) and extracted twice with CH₂Cl₂ (70 mL and 30 mL). The extract was washed with 10% aq. HBr (15 mL), ice water (2 × 20 mL), 5% aq. NaHCO₃ (15 mL), and water until neutral pH; dried through cotton wool; and evaporated in vacuo. The resulting crystals were purified by column chromatography (benzene–EtOAc, 9:1, 4:1, 1:1) to afford 15 as yellowish crystals used for the next step without further purification: 0.667 g, 69% yield; mp 102–105 °C; ¹H NMR (500 MHz, CDCl₃) δ 6.48 (1H, s, H_Ar), 5.97 (2H, s, OCH₂O), 5.87 (1H, br.s, NH), 4.33 (2H, d, J = 5.8 Hz, NCH₂), 3.95 (3H, s, OCH₃), 3.84 (3H, s, OCH₃), 3.39 (2H, t, J = 6.8 Hz, BrCH₂), 2.18 (2H, t, J = 7.5 Hz, CH₂), 1.86 (2H, quintet, J = 7.1 Hz, CH₂), 1.66 (2H, quintet, J = 7.6 Hz, CH₂), 1.45 (2H, m, CH₂). The corresponding chloro-derivative (11%) was identified in 15 by ¹H NMR analysis.

{6-[(4,7-Dimethoxy-2H-1,3-benzodioxol-5-yl)methoxy]-6-oxohexyl}(triphenyl)phosphonium Bromide (16)

The solution of 6-bromohexanoate 14 (0.937 g, 2.4 mmol) and Ph₃P (0.67 g, 2.9 mmol) in dry CH₃CN (8 mL) was refluxed for 36 h and evaporated. The residue was dissolved in CH₂Cl₂ (50 mL) and stirred with 20% of aq. KBr (25 mL) for 15 min. The organic layer was washed with water (3 × 25 mL) and filtered through cotton wool, and the resulting dark oil (1.56 g) was purified by column chromatography (benzene–EtOAc, 9:1, and then CHCl₃–MeOH, 4:1) to afford 16 (1.186 g, 1.8 mmol, 76% yield) as a pure white foamy solid: ¹H NMR (500 MHz, CDCl₃) δ 7.86 (6H, m, H_Ph), 7.79 (3H, m, H_Ph), 7.70 (6H, td, J = 7.7 Hz, J = 3.3 Hz, H_Ph), 6.50 (1H, s, H_Ar), 5.97 (2H, s, OCH₂O), 5.00 (2H, s, OCH₂), 3.89 (5H, m, PCH₂ and OCH₃), 3.85 (3H, s, OCH₃), 2.31 (2H, t, J = 7.3 Hz, CH₂), 1.73 (2H, m, CH₂), 1.63 (4H, m, 2 CH₂); anal. C 63.02; H 5.45; Br 12.20; calcd for C₃₄H₃₆BrO₆P, C 62.68; H 5.57; Br 12.26.

(6-{[(4,7-Dimethoxy-2H-1,3-benzodioxol-5-yl)methyl]amino}-6-oxohexyl)(triphenyl)phosphonium Bromide (17)

The solution of 6-bromohexanamide 15 (0.233 g, 0.6 mmol) and Ph₃P (0.168 g, 0.63 mmol) in dry CH₃CN (2.5 mL) was kept under a pressure of 5000 bar at 80 °C for 5 h and then at room temperature overnight and evaporated. The residue was dissolved in CH₂Cl₂ (70 mL) and stirred with 20% aq. KBr (10 mL) for 15 min. The organic layer was washed with water (3 × 10 mL) and filtered through a cotton plug. The resulting oil (0.48 g) was purified by column chromatography (benzene–EtOAc, 4:1, 1:1; and then CHCl₃–MeOH, 4:1, 3:1) to afford 17 (0.158 g, 0.24 mmol, 40% yield) as a pure white foamy solid: ¹H NMR (500 MHz, CDCl₃) δ 7.81 (9H, m, H_Ph), 7.70 (6H, m, H_Ph), 7.47 (1H, m, NH), 6.63 (1H, s, H_Ar), 5.91 (2H, s, OCH₂O), 4.34 (2H, d, J = 6.0 Hz, NCH₂), 3.93 (3H, s, OCH₃), 3.84 (3H, s, OCH₃), 3.73 (2H, m, PCH₂), 2.32 (2H, t, J = 7.4 Hz, CH₂), 1.70–1.55 (6H, m, 3 CH₂); anal. C 63.09; H 5.80; N 2.02; Br 12.00; calcd for C₃₄H₃₇BrNO₅P, C 62.77; H 5.73; N 2.15; Br 12.28.

General Procedure for the Synthesis of Chalcones 18

Aqueous NaOH (20%, 0.8 mL) was added to a mixture of the respective aldehyde 8 (5 mmol) and cyclopropyl methyl ketone (5 mmol) in EtOH (3 mL) at room temperature upon stirring, and the reaction mixture was stirred for 8 h. The resulting precipitate was filtered off, washed with water (2 × 10 mL) and 50% aq. EtOH (5 mL), and dried to yield chalcones 18a,c,e–g as colorless crystals. Chalcones 18f (75% yield) and 18g (99% yield) were described earlier.⁵⁴

1-Cyclopropyl-3-(4,7-dimethoxybenzo[d][1,3]dioxol-5-yl)prop-2-en-1-one (18a)

1.24 g, 90% yield; white solid; mp 81–83 °C (EtOH); ¹H NMR (300 MHz, DMSO-d₆) δ 7.72 (1H, d, J = 16.0 Hz, =CH), 7.10 (1H, s, H_Ar), 7.06 (1H, d, J = 16.0 Hz, =CH), 6.10 (2H, s, OCH₂O), 3.90 (3H, s, OCH₃), 3.86 (3H, s, OCH₃), 2.35 (1H, m, H_cyclo-Pr), 0.96 (4H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 277.1065 (calcd for 277.1071); anal. calcd for C₁₅H₁₆O₅, C 65.21, H 5.84; found: C 65.30, H 5.77.

1-Cyclopropyl-3-(7-methoxybenzo[d][1,3]dioxol-5-yl)prop-2-en-1-one (18c)

1.16 g, 94% yield; white solid; mp 88–89 °C (EtOH); ¹H NMR (500 MHz, DMSO-d₆) δ 7.58 (1H, d, J = 16.1 Hz, =CH), 7.09 (2H, s, H_Ar), 6.96 (1H, d, J = 16.1 Hz, =CH), 6.08 (2H, s, OCH₂O), 3.87 (3H, s, OCH₃), 2.38 (1H, m, H_cyclo-Pr), 0.94 (4H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 247.0959 (calcd for 247.0965); anal. calcd for C₁₄H₁₄O₄, C 68.28, H 5.73; found: C 68.05, H 5.79.

1-Cyclopropyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (18e)

1.15 g, 88% yield; white solid; mp 121–122 °C (EtOH). ¹H NMR (300 MHz, DMSO-d₆) δ 7.64 (1H, d, J = 16.0 Hz, =CH), 7.10 (2H, s, H_Ar), 7.05 (1H, d, J = 16.0 Hz, =CH), 3.84 (6H, s, 2 OCH₃), 3.70 (3H, s, OCH₃), 2.44 (m, 1H, H_cyclo-Pr), 0.97 (4H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 263.1280 (calcd for 263.1278); anal. calcd for C₁₅H₁₈O₄, C 68.68, H 6.92; found: C 68.85, H 6.81.

General Procedure for the Synthesis of Ketones 19

NaBH₄ (0.76 g, 20 mmol) was added portionwise to a mixture of the respective chalcone 18 (2 mmol) and NiCl₂·6H₂O (4.76 g, 20 mmol) in MeOH (10 mL) for 30 min while stirring at room temperature. The mixture was stirred for additional 1 h and diluted with MeOH (10 mL), and the precipitate was filtered off. Then, the filtrate was evaporated to dryness, the residue was extracted with CH₂Cl₂ (3 × 20 mL), and the solvent was removed in vacuo to yield compounds 19 used for the subsequent synthetic step without further purification.

1-Cyclopropyl-3-(4,7-dimethoxybenzo[d][1,3]dioxol-5-yl)propan-1-one (19a)

0.53 g, 95% yield; ¹H NMR (500 MHz, DMSO-d₆) δ 6.44 (1H, s, H_Ar), 5.93 (2H, s, OCH₂O), 3.79 (3H, s, OCH₃), 3.74 (3H, s, OCH₃), 2.76 (2H, m, CH₂), 2.70 (2H, m, CH₂), 2.02 (1H, m, H_cyclo-Pr), 0.83 (4H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 279.1222 (calcd for 279.1227).

1-Cyclopropyl-3-(7-methoxybenzo[d][1,3]dioxol-5-yl)propan-1-one (19c)

0.46 g, 93% yield; ¹H NMR (500 MHz, DMSO-d₆) δ 6.51 (1H, s, H_Ar), 6.48 (1H, s, H_Ar), 5.92 (2H, s, OCH₂O), 3.70 (3H, s, OCH₃), 2.85 (2H, t, J = 7.5 Hz, CH₂), 2.72 (2H, t, J = 7.5 Hz, CH₂), 2.05 (1H, m, H_cyclo-Pr), 0.85 (4H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 249.1126 (calcd for 249.1121).

1-Cyclopropyl-3-(3,4,5-trimethoxyphenyl)propan-1-one (19e)

0.52 g, 99% yield; ¹H NMR (300 MHz, DMSO-d₆) δ 6.55 (2H, s, H_Ar), 3.78 (6H, s, 2 OCH₃), 3.62 (3H, s, OCH₃), 2.87 (2H, m, CH₂), 2.76 (2H, m, CH₂), 2.07 (1H, m, H_cyclo-Pr), 0.86 (4H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 265.1435 (calcd for 265.1434).

1-Cyclopropyl-3-(3,4-dimethoxyphenyl)propan-1-one (19f)

0.46 g, 99% yield; ¹H NMR (300 MHz, DMSO-d₆) δ 6.85 (2H, m, H_Ar), 6.70 (1H, d, J = 8.3 Hz, H_Ar), 3.74 (3H, s, OCH₃), 3.72 (3H, s, OCH₃), 2.86 (2H, m, CH₂), 2.75 (2H, m, CH₂), 2.08 (1H, m, H_cyclo-Pr), 0.86 (4H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 235.1332 (calcd for 235.1329).

1-Cyclopropyl-3-(4-methoxyphenyl)propan-1-one (19g)⁵⁵

0.34 g, 83% yield; ¹H NMR (300 MHz, DMSO-d₆) δ 7.14 (2H, d, J = 8.4 Hz, H_Ar), 6.83 (2H, d, J = 8.4 Hz, H_Ar), 3.70 (3H, s, OCH₃), 2.82 (2H, m, CH₂), 2.69 (2H, m, CH₂), 2.05 (1H, m, H_cyclo-Pr), 0.80 (4H, m, H_cyclo-Pr).

General Procedure for the Synthesis of Alcohols 20

NaBH₄ (0.10 g, 2.6 mmol) was added to a mixture of the respective crude ketone 19 (2 mmol) in MeOH (10 mL) while stirring at room temperature. The mixture was stirred for additional 0.5–1.5 h (TLC-control), and the solvent was removed in vacuo. The residue was dissolved in water (10 mL) and extracted with CH₂Cl₂ (3 × 10 mL), and the combined extracts were evaporated to dryness affording alcohols 20 used for the subsequent synthetic step without further purification.

1-Cyclopropyl-3-(4,7-dimethoxybenzo[d][1,3]dioxol-5-yl)propan-1-ol (20a)

0.54 g, 97% yield; ¹H NMR (500 MHz, DMSO-d₆) δ 6.40 (1H, s, H_Ar), 5.96 (2H, s, OCH₂O), 4.43 (1H, d, J = 4.8 Hz, OH), 3.79 (3H, s, OCH₃), 3.77 (3H, s, OCH₃), 2.85 (1H, m, CHOH), 2.64 (1H, m, CH₂); 2.50 (1H, m, CH₂), 1.65 (2H, m, CH₂), 0.80 (1H, m, H_cyclo-Pr), 0.35 (2H, m, H_cyclo-Pr), 0.23 (m, 1H, H_cyclo-Pr), 0.12 (1H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 281.1381 (calcd for 281.1384).

1-Cyclopropyl-3-(7-methoxybenzo[d][1,3]dioxol-5-yl)propan-1-ol (20c)

0.47 g, 95% yield; ¹H NMR (500 MHz, DMSO-d₆) δ 6.44 (1H, s, H_Ar), 6.42 (1H, s, H_Ar), 5.92 (2H, s, OCH₂O), 4.43 (1H, d, J = 4.8 Hz, OH), 3.81 (3H, s, OCH₃), 2.84 (1H, m, CHOH), 2.64 (1H, m, CH₂); 2.52 (1H, m, CH₂), 1.70 (2H, m, CH₂), 0.80 (1H, m, H_cyclo-Pr), 0.36 (2H, m, H_cyclo-Pr), 0.22 (m, 1H, H_cyclo-Pr), 0.12 (1H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 251.1280 (calcd for 251.1278).

1-Cyclopropyl-3-(3,4,5-trimethoxyphenyl)propan-1-ol (20e)

0.40 g, 76% yield; ¹H NMR (500 MHz, DMSO-d₆) δ 6.47 (2H, s, H_Ar), 4.46 (1H, d, J = 4.8 Hz, OH), 3.75 (3H, s, OCH₃), 3.61 (6H, s, 2 OCH₃), 2.87 (1H, m, CHOH), 2.66 (1H, m, CH₂), 2.56 (1H, m, CH₂), 1.73 (2H, m, CH₂), 0.81 (1H, m, H_cyclo-Pr), 0.36 (2H, m, H_cyclo-Pr), 0.25 (1H, m, H_cyclo-Pr), 0.15 (1H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 267.1596 (calcd for 267.1591).

1-Cyclopropyl-3-(3,4-dimethoxyphenyl)propan-1-ol (20f)

0.43 g, 91% yield; ¹H NMR (500 MHz, DMSO-d₆) δ 6.83 (1H, d, J = 8.1 Hz, H_Ar), 6.78 (1H, s, H_Ar), 6.69 (1H, d, J = 8.1 Hz, H_Ar), 4.43 (1H, d, J = 4.9 Hz, OH), 3.74 (3H, s, OCH₃), 3.72 (3H, s, OCH₃), 2.85 (1H, m, CHOH), 2.67 (1H, m, CH₂), 2.56 (1H, m, CH₂), 1.72 (2H, m, CH₂), 0.81 (1H, m, H_cyclo-Pr), 0.35 (2H, m, H_cyclo-Pr), 0.23 (1H, m, H_cyclo-Pr), 0.13 (1H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 237.1481 (calcd for 237.1485).

1-Cyclopropyl-3-(4-methoxyphenyl)propan-1-ol (20g)⁵⁵

0.38 g, 93% yield; ¹H NMR (300 MHz, DMSO-d₆) δ 7.11 (2H, d, J = 8.4 Hz, H_Ar), 6.83 (2H, d, J = 8.4 Hz, H_Ar), 4.44 (1H, d, J = 4.9 Hz, OH), 3.71 (3H, s, OCH₃), 2.85 (1H, m, CHOH), 2.64 (1H, m, CH₂), 2.55 (1H, m, CH₂), 1.70 (2H, m, CH₂), 0.80 (1H, m, H_cyclo-Pr), 0.34 (2H, m, H_cyclo-Pr), 0.24 (1H, m, H_cyclo-Pr), 0.11 (1H, m, H_cyclo-Pr); HRMS m/z [M + H]⁺ 207.1377 (calcd for 207.1380).

General Procedure for the Synthesis of 6-Arylhex-3-enyl Bromides 21

Crude alcohol 20 (1 mmol) was stirred intensively for 20 min in a solution of 48% aq. HBr (0.5 mL) and AcOH (1 mL). Then, water (5 mL) was added, and the resulting mixture was extracted with CH₂Cl₂ (2 × 3 mL). The organic layer was washed with water (2 × 2 mL) and thoroughly evaporated to dryness. Pure bromides 21 were isolated by column chromatography on silica gel (EtOAc–hexane, 1:3) as colorless oils.

6-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)hex-3-enyl Bromide (21a)

0.31g, 91% yield; colorless oil; ¹H NMR (500 MHz, DMSO-d₆) δ 6.42 (s, 1H, Ar), 5.95 (s, 2H, OCH₂O), 5.56 (td, J = 15.4 Hz, J = 6.7 Hz, 1H, CH=), 5.41 (td, J = 15.3 Hz, J = 6.7 Hz, 1H, CH=), 3.79 (s, 3H, OMe), 3.76 (s, 3H, OMe), 3.48 (t, J = 6.9 Hz, 2H, CH₂), 2.53 (m, 4H, CH₂), 2.19 (q, J = 7.3 Hz, 2H, CH₂); HRMS m/z [M + H]⁺ 343.0545 (calcd for 343.0539); anal. calcd for C₁₅H₁₉BrO₄, C, 52.49; H, 5.58; found: C, 52.35; H, 5.41.

6-(7-Methoxybenzo[d][1,3]dioxol-5-yl)hex-3-enyl Bromide (21c)

0.26 g, 82% yield; colorless oil; ¹H NMR (500 MHz, DMSO-d₆) δ 6.47 (s, 1H, Ar), 6.45 (s, 1H, Ar), 5.92 (s, 2H, OCH₂O), 5.56 (td, J = 15.4 Hz, J = 6.6 Hz, 1H, CH=), 5.41 (td, J = 15.4 Hz, J = 6.7 Hz, 1H, CH=), 3.79 (s, 3H, OMe), 3.47 (t, J = 6.9 Hz, 2H, CH₂), 2.54 (m, 4H, CH₂), 2.24 (q, J = 7.6 Hz, 2H, CH₂); HRMS m/z [M + H]⁺ 313.0436 (calcd for 313.0434); anal. calcd for C₁₄H₁₇BrO₃, C, 53.69; H, 5.47; found: C, 54.01; H, 5.29.

6-(3,4,5-Trimethoxyphenyl)hex-3-enyl Bromide (21e)

0.27 g, 81% yield; colorless oil; ¹H NMR (500 MHz, DMSO-d₆) δ 6.50 (s, 2H, Ar), 5.59 (m, 1H, CH=), 5.44 (m, 1H, CH=), 3.74 (s, 6H, OMe), 3.61 (s, 3H, OMe), 3.48 (t, J = 6.9 Hz, 2H, CH₂), 2.56 (m, 4H, CH₂), 2.29 (q, J = 7.1 Hz, 2H, CH₂); HRMS m/z [M + H]⁺ 329.0747 (calcd for 329.0747); anal. calcd for C₁₅H₂₁BrO₃, C, 54.72; H, 6.43; found: C, 54.38; H, 6.58.

6-(3,4-Dimethoxyphenyl)hex-3-enyl Bromide (21f)

0.20 g, 68% yield; colorless oil; ¹H NMR (500 MHz, DMSO-d₆) δ 6.84 (d, J = 8.1 Hz, 1H, Ar), 6.80 (d, J = 1.6 Hz, 1H, Ar), 6.71 (d, J = 8.1 Hz, 1H, Ar), 5.58 (td, J = 15.3 Hz, J = 6.6 Hz, 1H, CH=), 5.43 (td, J = 15.4 Hz, J = 6.7 Hz, 1H, CH=), 3.74 (s, 3H, OMe), 3.71 (s, 3H, OMe), 3.48 (t, J = 6.8 Hz, 2H, CH₂), 2.57 (m, 4H, CH₂), 2.27 (q, J = 7.5 Hz, 2H, CH₂); HRMS m/z [M + H]⁺ 299.0637 (calcd for 299.0641); anal. calcd for C₁₄H₁₉BrO₂, C, 56.20; H, 6.40; found: C, 56.47; H, 6.49.

6-(4-Methoxyphenyl)hex-3-enyl Bromide (21g)⁵⁵

0.16 g, 59% yield; colorless oil; ¹H NMR (500 MHz, CDCl₃) δ 7.09 (d, J = 8.5 Hz, 2H, Ar), 6.82 (d, J = 8.5 Hz, 2H, Ar), 5.57 (td, J = 15.3 Hz, J = 6.7 Hz, 1H, CH=), 5.41 (td, J = 15.3 Hz, J = 6.7 Hz, 1H, CH=), 3.78 (s, 3H, OMe), 3.34 (t, J = 7.2 Hz, 2H, CH₂), 2.62 (t, J = 7.4 Hz, 2H, CH₂), 2.53 (q, J = 7.1 Hz, 2H, CH₂), 2.30 (q, J = 7.7 Hz, 2H, CH₂); anal. calcd for C₁₃H₁₇BrO, C, 58.01; H, 6.37; found: C, 58.29; H, 6.22.

General Procedure for the Synthesis of (6-Arylhex-3-en-1-yl)triphenylphosphonium Bromides 22

A mixture of the respective 6-arylhex-3-enyl bromide 21 (1 mmol) and PPh₃ (1 mmol) was diluted with CH₃CN (4 mL) and heated at 95–100 °C for 9 h without a condenser. The resulting oily residue was purified with column chromatography (EtOH–EtOAc, from 1:10 to 1:1) to afford 22 as a brownish oil.

[6-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)hex-3-en-1-yl]triphenylphosphonium Bromide (22a)

0.23 g, 38% yield; brownish oil; ¹H NMR (500 MHz, CDCl₃) δ 7.87 (m, 6H, PPh₃), 7.79 (m, 3H, PPh₃), 7.70 (m, 6H, PPh₃), 6.27 (s, 1H, Ar), 5.92 (s, 2H, OCH₂O), 5.60 (m, 1H, CH=), 5.48 (m, 1H, CH=), 3.93 (m, 2H, CH₂), 3.85 (s, 3H, OMe), 3.84 (s, 3H, OMe), 2.49 (t, J = 7.9 Hz, 2H, CH₂), 2.41 (m, 2H, CH₂), 2.14 (m, 2H, CH₂); anal. calcd for C₃₃H₃₄BrPO₄, C, 65.46; H, 5.66; P, 5.12; found: C, 65.31; H, 5.51; P, 5.21.

[6-(7-Methoxybenzo[d][1,3]dioxol-5-yl)hex-3-en-1-yl]triphenylphosphonium Bromide (22c)

0.34 g, 59% yield; brownish oil; ¹H NMR (500 MHz, CDCl₃) δ 7.87 (m, 6H, PPh₃), 7.79 (m, 3H, PPh₃), 7.70 (m, 6H, PPh₃), 6.31 (s, 1H, OCH₂O), 6.29 (s, 1H, OCH₂O), 5.90 (s, 2H, Ar), 5.63 (m, 1H, CH=), 5.47 (m, 1H, CH=), 3.97 (m, 2H, CH₂), 3.88 (s, 3H, OMe), 2.48 (t, J = 7.7 Hz, 2H, CH₂), 2.41 (m, 2H, CH₂), 2.18 (q, J = 7.3 Hz, 2H, CH₂); anal. calcd for C₃₂H₃₂BrPO₃, C, 66.79; H, 5.61; P, 5.38; found: C, 67.00; H, 5.74; P, 5.09.

[6-(3,4,5-Trimethoxyphenyl)hex-3-en-1-yl]triphenylphosphonium Bromide (22e)

0.27 g, 45% yield; brownish oil; ¹H NMR (500 MHz, CDCl₃) δ 7.87 (m, 6H, PPh₃), 7.79 (m, 3H, PPh₃), 7.70 (m, 6H, PPh₃), 6.37 (s, 2H, Ar), 5.69 (m, 1H, CH=), 5.51 (m, 1H, CH=), 3.98 (m, 2H, CH₂), 3.83 (s, 6H, OMe), 3.81 (s, 3H, OMe), 2.53 (t, J = 7.5 Hz, 2H, CH₂), 2.40 (m, 2H, CH₂), 2.23 (q, J = 7.5 Hz, 2H, CH₂); anal. calcd for C₃₃H₃₆BrPO₃, C, 67.01; H, 6.13; P, 5.24; found: C, 66.72; H, 6.29; P, 5.54.

[6-(3,4-Dimethoxyphenyl)hex-3-en-1-yl]triphenylphosphonium Bromide (22f)

0.17 g, 31% yield; brownish oil; ¹H NMR (500 MHz, CDCl₃) δ 7.87 (m, 6H, PPh₃), 7.79 (m, 3H, PPh₃), 7.70 (m, 6H, PPh₃), 6.77 (d, J = 8.1 Hz, 1H, Ar), 6.66 (m, 2H, Ar), 5.66 (m, 1H, CH=), 5.50 (m, 1H, CH=), 3.97 (m, 2H, CH₂), 3.86 (s, 3H, OMe), 3.84 (s, 3H, OMe), 2.52 (t, J = 7.8 Hz, 2H, CH₂), 2.42 (m, 2H, CH₂), 2.21 (q, J = 7.7 Hz, 2H, CH₂); anal. calcd for C₃₂H₃₄BrPO₂, C, 68.45; H, 6.10; P, 5.52; found: C, 68.23; H, 6.18; P, 5.38.

[6-(4-Methoxyphenyl)hex-3-en-1-yl]triphenylphosphonium Bromide (22g)

0.16 g, 30% yield; brownish oil; ¹H NMR (CDCl₃, 500 MHz) δ 7.85 (m, 6H, PPh₃), 7.79 (m, 3H, PPh₃), 7.70 (m, 6H, PPh₃), 7.03 (d, J = 8.6 Hz, 2H, Ar), 6.80 (d, J = 8.6 Hz, 2H, Ar), 5.58 (m, 1H, CH=), 5.43 (m, 1H, CH=), 3.91 (m, 2H, CH₂), 3.77 (s, 3H, OMe), 2.50 (t, J = 7.4 Hz, 2H, CH₂), 2.41 (m, 2H, CH₂), 2.18 (q, J = 8.0 Hz, 2H, CH₂); anal. calcd for C₃₁H₃₂BrPO, C, 70.06; H, 6.07; P, 5.83; found: C, 70.35; H, 6.21; P, 5.56.

Phenotypic Sea Urchin Embryo Assay⁴⁶

Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected from the Mediterranean Sea on the Cyprus coast and kept in an aerated seawater tank. Gametes were obtained by intracoelomic injection of 0.5 M KCl. Eggs were washed with filtered seawater and fertilized by adding drops of diluted sperm. Embryos were cultured at room temperature under gentle agitation with a motor-driven plastic paddle (60 rpm) in filtered seawater. The embryos were observed with a Biolam light microscope (LOMO, St. Petersburg, Russian Federation). For treatment with the test compounds, 5 mL aliquots of the embryo suspension were transferred to six-well plates and incubated as a monolayer at a concentration of up to 2000 embryos/mL. Stock solutions of tested molecules were prepared in dimethyl sulfoxide (DMSO) at 10–20 mM concentration followed by a tenfold dilution with 96% EtOH. This procedure enhanced the solubility of the test compounds in the salt-containing medium (seawater), as evidenced by microscopic examination of the samples. The maximum tolerated concentrations of DMSO and EtOH in the in vivo assay were determined to be 0.05 and 1%, respectively. Higher concentrations of either DMSO (>0.1%) or EtOH (>1%) caused nonspecific alteration and retardation of the sea urchin embryo development independent of the treatment stage. The antimitotic activity was assessed by exposing fertilized eggs (8–15 min postfertilization, 45–55 min before the first mitotic cycle completion) to twofold decreasing concentrations of the compound. Cleavage alteration and arrest were clearly detected at 2.5–5.5 h after fertilization. The effects were estimated quantitatively as an effective threshold concentration, resulting in cleavage alteration and embryo death before hatching or full mitotic arrest. At these concentrations, all tested compounds caused 100% cleavage alteration and embryo death before hatching, whereas at twofold lower concentrations, the compounds failed to produce any effect. The sea urchin embryo assay data are available free of charge via the Internet at http://www.zelinsky.ru. Experiments with sea urchin embryos fulfill the requirements of biological ethics. Artificial spawning does not cause animal death, embryos develop outside the female organism, and both postspawned adult sea urchins and the excess of intact embryos are returned to the sea, their natural habitat.

Human Cancer Cell Culture

A549 human lung epithelial carcinoma cells (CCL-185) and A375 human skin epithelial malignant melanoma cells (CRL-1619) were cultured in Dulbecco’s modified Eagle’s medium. DU145 human prostate epithelial carcinoma cells derived from the brain metastatic site (HTB-81) were cultured in Eagle’s minimum essential medium. PC-3 human prostate epithelial adenocarcinoma grade IV cells, derived from the bone metastatic site (CRL-1435), were cultured in Ham’s F-12K (Kaighn’s) medium. HCT-116 human colon epithelial colorectal carcinoma cells (CCL-247) and SK-OV-3 human ovary ascites epithelial adenocarcinoma cells (HTB-77) were cultured in McCoy’s 5a modified medium. T-47D human breast carcinoma cells were cultured in RPMI-1640 medium containing human insulin (0.2 Units/mL). All cancer cell cultures were performed in media containing 10% fetal bovine serum, penicillin (100 Units/mL), and streptomycin (100 μg/mL) at 37 °C under a 5% CO₂ humidified atmosphere.

MTT Cell Toxicity Assay

The cells were seeded in 96-well plates (Eppendorf, Cat. No. 0030730119), containing 200 μL of cell suspension in culture media (Gibco) with 10% fetal bovine serum (FBS, Gibco) at a density of 5 × 10³ cells per well. Cells were incubated in a humidified atmosphere with 5% CO₂. Stock solutions of test compounds were prepared in DMSO (Sigma-Aldrich). The next day (overnight incubation), cells were treated for 72 h with test compounds titrated in DMSO (the final concentration of DMSO did not exceed 0.5%). Tubercidin (Sigma-Aldrich, Germany) served as a positive control. The cytotoxicity was determined by the colorimetric cell proliferation assay, the MTT assay (thiazolyl blue tetrazolium bromide, Sigma-Aldrich, Germany). For this, 20 μL of the MTT reagent in Dulbecco’s phosphate-buffered saline was added per well (final concentration of 0.5 mg/mL). The plates were incubated at 37 °C for 2–3 h under a 5% CO₂ humidified atmosphere. The absorbance was recorded at 570 nm using a CLARIO star microplate reader (BMG LABTECH, Ortenberg, Germany). Experiments for all compounds were repeated three times. GI₅₀ values were determined by sigmoidal curve fitting using GraphPad Prism 6 software.

Click-iT-EdU Alexa Fluor 488 Cell Proliferation Assay

The cells were seeded in 384-well plates (Corning, Cat. No. 3712), containing 45 μL of the cell suspension in culture media (Gibco) with 10% fetal bovine serum (FBS, Gibco) at a density of 800 cells per well. Cells were incubated in a humidified atmosphere with 5% CO₂. Stock solutions of test compounds were prepared in DMSO (Sigma-Aldrich, Germany). The next day (overnight incubation), cells were treated for 72 h with test compounds titrated in DMSO (the final concentration of DMSO did not exceed 0.5%). Tubercidin (Sigma-Aldrich) served as a positive control. After 72 h, 5-ethynyl-2′-deoxyuridine (EdU) diluted in PBS was added to cells and incubated for 2.5 h in a 5% CO₂ humidified atmosphere at 37 °C. Cells were then fixed in 4% paraformaldehyde for 30 min at room temperature and stained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA). After rinsing four times with PBS, the Click-iT reaction with Alexa Fluor 488 dye was performed for 1 h at room temperature (protected from light), followed by rinsing four times with PBS for further imaging. Imaging and analysis were performed using an ImageXpress Micro XL High-Content Screening System (Molecular Devices LLC, San Jose, CA). IC₅₀ values were determined by sigmoidal curve fitting using GraphPad Prism 6 software.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c05515.

• NCI60 cytotoxicity graphs for compounds 4a–d, 7c,d, 16, and 17; and ¹H NMR and ¹³C NMR spectra of compounds 4, 5, 7, 11, 16–22, NTTP, HDTTP, C10-ITTP, and Pr-TTP (PDF)

The authors declare no competing financial interest.