Synthesis and Antiproliferative Effects of Amino-Modified Perillyl Alcohol Derivatives

Two series of amino-modified derivatives of (S)-perillyl alcohol were designed and synthesized using (S)-perillaldehyde as the starting material. These derivatives showed increased antiproliferative activity in human lung cancer A549 cells, human melanoma A375-S2 cells and human fibrosarcoma HT-1080 cells comparing with that of (S)-perillyl alcohol. Among these derivatives, compounds VI5 and VI7 were the most potent agents, with the IC50s below 100 μM. It was demonstrated that the antiproliferative effect of VI5 was mediated through the induction of apoptosis in A549 cells.


Introduction
In recent decades natural products continue to attract intense attention due to their various bioactivities. Most of the drugs on the clinic market today are inspired by or derived from natural sources [1]. Perillyl alcohol, a naturally occurring monoterpene found in lavender, cherries and mint, has been suggested to be an effective agent against a variety of tumors as a farnesyl transferase (FTase) inhibitor [2][3][4][5][6][7]. Perillyl alcohol has been put into phase II clinical trials in cancer patients and OPEN ACCESS the preliminary results indicate that this agent is well tolerated [8,9]. Since the potency of perillyl alcohol is modest compared to many antitumor agents [10], structural modification of perillyl alcohol has been carried out in recent years, and several kinds of perillyl alcohol derivatives have been synthesized. Among these derivatives, the perillyl alcohol carbamates, which were conjugated compounds of perillyl alcohol with some therapeutic agents, were found to be more active compounds [11], whereas other perillyl alcohol esters [12,13] and glucosides [14] were proved to be less potent than perillyl alcohol in vitro.
Amino-modification has been proved to be an efficient approach to increase water solubility and/or antitumor activity of several natural products, such as that of comptothecin [15,16], β-elemene [17] and limonene [18]. Thus, introduction of an amino moiety into the skeleton of perillyl alcohol might be favorable to improving antitumor activity. In this communication, two series of amino-modified derivatives of perillyl alcohol IV, VI were synthesized. Their activity of inhibiting tumor cell growth and the potential mechanism were studied in a few of cancer cell lines.

Synthesis of (S)-Perillyl Alcohol Derivatives
The synthetic route of the perillyl alcohol derivatives starting with (S)-perillaldehyde is outlined in Scheme 1, where the substituent groups are listed. (S)-Perillyl alcohol (I) was obtained from (S)-perillaldehyde via reduction with sodium borohydride. For a selective chlorination at the terminal allyl group of (S)-perillyl alcohol, acetylation of the hydroxyl group was carried out. The resulted perillyl acetate (II) was reacted with hypochloric acid, affording the intermediate III. The nucleophilic substitution of III with a heterocyclic amine or an aromatic amine and subsequent hydrolysis gave the target compound IV. The selective chlorination from (S)-perillyl alcohol (I) to the intermediate V was achieved in a mild condition via Appel reaction, avoiding the undesired chlorination of olefins by other reagents. The substitution reaction of V with an aliphatic amine or a heterocyclic amine gave the target compound VI.

Antiproliferative Effects in Tumor Cells
The cell growth inhibitory effect of these amino-modified derivatives was measured in A549, A375-S2 and HT-1080 cells using MTT assay. As shown in Table 1, the IC 50 s of (S)-perillyl alcohol in the three cells were more than 1,000 μM (the highest concentration used in this experiment). All the synthesized derivatives except IV 6 displayed much more potent cytotoxicity than (S)-perillyl alcohol. Among them, the two secondary aliphatic amines, VI 5 and VI 7 , were the most effective agents with the IC 50 s below 100 μM in the three tumor cells. Introduction of a substituted piperazinyl moiety (IV 1 -IV 5 , VI 1 -VI 3 ) was of benefit to antiproliferative activity to some extent, but it did not show the same enhanced effect as that in the modification of β-elemene [17] and limonene [18]. It was found that the replacement of the hydroxyl group of (S)-perillyl alcohol with an amino moiety was more favorable to improving cytotoxic activity than the introduction of an amino moiety at the terminal allyl group, by comparing the IC 50 s of the two kinds of derivatives bearing the same substitutes (VI 1 vs. IV 1 , VI 2 vs. IV 2 , VI 4 vs. IV 6 ). To further verify the effect of compound VI 5 , we measured the viability of A549 cells treated with (S)-perillyl alcohol, amantadine, VI 5 or the combination of perillyl alcohol and amantadine (with the same concentrations) for 24 h. It was shown that the IC 50 (152.72 μM) of VI 5 was 6-fold less than the IC 50 (920.75 μM) of the combination of perillyl alcohol and amantadine while A549 cells treated with (S)-perillyl alcohol or amantadine alone didn't reveal significant viability inhibition at concentration below 2,000 μM. The result indicated that the inhibitory effect of VI 5 was not caused by an additive effect or synergistic effect, but the specific structure of VI 5 .

Apoptosis in A549 Cells Induced by VI 5
As previously reported, perillyl alcohol could induce apoptosis in A549 cells [7]. Therefore, we examined whether the growth inhibitory effect of VI 5 was mediated through the induction of apoptosis. After treated with VI 5 for 24 h, significant morphologic changes were observed in A549 cells by phase contrast microscopy. Some of the cells showed membrane blebbing: a hallmark of apoptosis ( Figure 1a). Meanwhile, compared with the control group, results of AO staining showed remarkable chromatin condensation and nuclear fragmentation in VI 5 -treated cells ( Figure 1b). Flow cytometric analysis after PI staining revealed that the percentage of subG0/G1 ratio elevated from 1.97% to 45.12%, indicating that VI 5 induce apoptosis in a dose-dependent manner ( Figure 1c). Caspase-3, a member of aspartate-specific cysteine proteases (caspase) family, has been considered as a key mediator of apoptosis [19]. Western blot results showed that the degradation of procaspase-3 increased after treatment with VI 5 , indicating that VI 5 could induce the activation of caspase-3 ( Figure 1d).

General Information
All reagents and solvents (analytical grade) were commercially available and used without further purification. Melting points were determined with a Yanaco micro melting point apparatus and were uncorrected. 1 H-NMR spectra and 13 C-NMR spectrum were recorded in CDCl 3 on a Bruker ARX-300 spectrometer. The coupling constants were recorded in hertz (Hz) and chemical shifts were reported in parts per million (δ, ppm) downfield from tetramethylsilane (TMS). High-resolution mass spectra (HRMS) were recorded on a high-resonance electrospray time-of-flight mass spectrometer LC/MSD QTOF 6520 (Agilent). Specific rotation was measured on a Perkin-Elmer 241 MC polarimeter (path length 1 cm). Column chromatography was performed on silica gel. Analytical TLC was performed on plates precoated with silica gel and iodine vapor was used to develop color on the plates.
The residue was purified on a silica gel column with petroleum ether-ethyl acetate

(S)-(4-(Prop-1-en-2-yl)cyclohex-1-enyl)methyl Acetate (II)
To a solution of compound I (12.1 g, 0.08 mol) in pyridine (25 mL), acetic anhydride (25 mL) was added dropwise. The mixture was stirred at room temperature for 4 h. The reaction was terminated by addition of methanol (2 mL), followed by addition of ethyl acetate (50 mL). The mixture was washed with aqueous sodium bicarbonate solution and brine. The organic layer was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo. The residue was purified on a silica gel column with petroleum ether-ethyl acetate (400:1, R f = 0.41) as eluent to afford compound II as a colorless liquid (14.8 g, yield 95.4%). 1

General Procedure for the Synthesis of Target Compounds IV 1 -IV 7
To a solution of compound III (0.9 g, 4 mmol) in ethanol (10 mL), potassium carbonate (1.10 g, 8 mmol) and amine (4.4 mmol) were added. The mixture was stirred and refluxed for 8-12 h. Then, aqueous sodium hydroxide solution (20%, 2 mL) was added and the resulting mixture was refluxed for another 2 h. The solvent was evaporated in vacuo. Brine (15 mL) was added to the residue and the mixture was extracted with dichloromethane (3 × 10 mL). The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo. The residue was purified on a silica gel column with dichloromethane-methanol (100:1 → 50:1 → 20:1) as eluent to afford the target product.     (4-(3-(4-Benzylpiperazin-1-yl)prop-1-en-2-yl)

General Procedure for the Synthesis of Target Compounds VI 1 -VI 7
To a solution of compound V (0.85 g; 5 mmo1) in acetonitrile (10 mL); potassium carbonate (1.04 g; 7.5 mmol) and amine (5.5 mmol) were added. The mixture was stirred and refluxed for 6-8 h. Then the solvent was evaporated in vacuo. Brine (15 mL) was added to the residue and the mixture was extracted with dichloromethane (3 × 10 mL). The combined organic extracts were washed with brine; dried over anhydrous sodium sulfate; and filtered. The filtrate was concentrated in vacuo. The residue was purified on a silica gel column with dichloromethane-methanol (100:1 → 50:1 → 20:1) as eluent to afford the target product VI.
Acridine orange (AO) staining [23]: A549 cells were treated with 0, 120, 160 and 180 μM VI 5 for 24 h on 24-well flat bottom plates. Then cells were washed with PBS, followed by incubation at room temperature with PBS containing 20 μg/mL AO for 15 min. The fluorescence of cells was observed using fluorescence microscopy.
Flow cytometric analysis using propidium iodide (PI) [24]: A549 cells were treated with 0, 120, 160 and 180 μM VI 5 for 24 h on 6-well flat bottom plates. The cells were harvested and washed by PBS and fixed by 70% cold ethanol at 4 °C for more than 18 h. After stained with 50 μg/mL PI and 1 mg/mL DNaseA-free RNaseA on ice in dark for 1 h, cells were analyzed on FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Statistical assay: All the presented data were confirmed at least three independent experiments. The data were analyzed by ANOVA using Statistics Package for Social Science SPSS software (version 13.0; SPSS, Chicago, IL, USA).

Conclusions
Two series of amino-modified derivatives of (S)-perillyl alcohol were designed and synthesized. The target compounds showed improved antiproliferative activity against A549, A375-S2 and HT-1080 cells. The structure-activity relationships revealed that the replacement of the hydroxyl group of (S)-perillyl alcohol with an amino moiety was more favorable to improving cytotoxic activity than the introduction of an amino moiety at the terminal allyl group. The antiproliferative effect of VI 5 was proved to be mediated through the induction of apoptosis in A549 cells.