Examination of aminophenol-containing compounds designed as antiproliferative agents and potential atypical retinoids

Retinoic acid (RA, 1), an oxidized form of vitamin A, binds to retinoic acid receptors (RAR) and retinoid X receptors (RXR) to regulate gene expression and has important functions such as cell proliferation and differentiation. Synthetic ligands regarding RAR and RXR have been devised for the treatment of various diseases, particularly promyelocytic leukemia, but their side effects have led to the development of new, less toxic therapeutic agents. Fenretinide (4-HPR, 2), an aminophenol derivative of RA, exhibits potent antiproliferative activity without binding to RAR/RXR, but its clinical trial was discontinued due to side effects of impaired dark adaptation. Assuming that the cyclohexene ring of 4-HPR is the cause of the side effects, methylaminophenol was discovered through structure–activity relationship research, and p-dodecylaminophenol (p-DDAP, 3), which has no side effects or toxicity and is effective against a wide range of cancers, was developed. Therefore, we thought that introducing the motif carboxylic acid found in retinoids, could potentially enhance the anti-proliferative effects. Introducing chain terminal carboxylic functionality into potent p-alkylaminophenols significantly attenuated antiproliferative potencies, while a similar structural modification of weakly potent p-acylaminophenols enhanced growth inhibitory potencies. However, conversion of the carboxylic acid moieties to their methyl esters completely abolished the cell growth inhibitory effects of both series. Insertion of a carboxylic acid moiety, which is important for binding to RA receptors, abolishes the action of p-alkylaminophenols, but enhances the action of p-acylaminophenols. This suggests that the amido functionality may be important for the growth inhibitory effects of the carboxylic acids.


Introduction 1.Background
Retinol, also known as vitamin A, is a polyisoprenoid natural product that plays important roles in a variety of physiological processes.Oxidative transformation of its chain-terminating primary hydroxyl group yields the corresponding aldehyde retinal, which serves a central role in vision.All-trans retinoic acid (RA, 1, Fig. 1) is an oxidized form of retinol that binds to retinoic acid receptors (RARs), resulting in modulation of gene expression related to vital physiological processes, including cell growth, differentiation, survival, and death.In contrast, the isomeric RA variant, 9-cis retinoic acid binds to retinoid X receptors (RXRs), which can exist as heterodimers with RARs or other nuclear receptors. 1etinoids (synthetic ligands that engage RARs) and rexinoids (RXR-binding compounds) have therapeutic value against a variety of metabolic diseases and cancer, particularly for the treatment of promyelocytic leukemia.Because retinoids, and to a lesser extent rexinoids, exert unwanted side-effects, there is a need to develop new therapeutic agents that are less toxic."Atypical retinoids" are synthetic analogs that bind and transactivate RARs and have therapeutic promise as anticancer drugs. 2 Fenretinide, [N-(4-hydroxyphenyl)retinamide, 4-HPR, 2, Fig. 1], is an atypical retinoid that has chemo-preventative and anti-proliferative properties, but these are accompanied by side effects that include night blindness and ocular toxicity.Despite its structural similarity to RA, 4-HPR does not bind to RARs and it is a poor inducer of differentiation.Its antiproliferative activity is thought to be related to its ability to induce apoptosis by a mechanism that is RAR-independent and that might be related to the generation of reactive oxygen species (ROS). 3This is somewhat paradoxical, since 4-HPR functions as an antioxidant and accordingly, it might participate in redox cycling reactions. 4RA and retinoids are typically composed of three structural components; a hydrophobic region that is often composed of ring structures; a linker region and polar functionality at the terminus of the linker segment (Fig. 1). 2,5While sharing with RA the first two of these structural elements, 4-HPR includes a neutral 4-hydroxyamido group at the terminus of the linker, rather than the characteristic polar functionality.We have previously reported antiproliferative compounds that contain the 4-aminophenol moiety found in 4-HPR as well as long-chain N-alkyl groups that are somewhat reminiscent of the extended polyene chains found in 4-HPR and other retinoids. 6We found that antiproliferative potencies of the alkylaminophenols are related to alkyl chain lengths.Optimum antiproliferative potencies were obtained with the 12-methylene-long chaincontaining p-dodecylaminophenol (p-DDAP, 3, Fig. 1), which may be viewed as structurally simplified variant of 4-HPR. 7We have shown that p-DDAP is more potent than RA in suppressing the growth of breast, 8 prostate, 9 promyelocytic leukemia, 7 neuroblastoma, [10][11] pancreatic, 12 and cholangiocarcinoma 12 cells in culture.Furthermore, we have found that p-DDAP has antitumor effects on prostate cancer xenograft-bearing mice, while not affecting blood retinol concentrations, which is an undesirable side effect of treatment with 4-HPR and which can lead to reduced night vision. 9We wondered whether it might be possible to enhance antiproliferative potency in compounds structurally related to p-DDAP by introducing motifs found in retinoids and rexinoids.

Analog design
Structure-activity relationship studies conducted on 4-HPR have revealed that the 4aminophenol moiety is key to its ability to suppresses the growth of cancer cells. 13Among a series of 4-alkylaminophenols (4a -6a), antiproliferative potencies have been shown to be enhanced by longer chain lengths.Increasing alkylaminophenol chain length also increases superoxide trapping properties and reduces lipid peroxidation. 7However, we found that conversion of the amines to their corresponding amides (4b -6b), significantly decreases growth inhibitory potencies and ability to function as lipid peroxidation inhibitors (Fig. 2). 7,11In our current study we present the design, synthesis and biological evaluation of a series of analogs that incorporate elements of 4-HPR or RA into our alkylaminophenol platforms that explore this possibility.We designed analogs focused on the incorporation of terminal carboxylic acid functionality that would introduce key chain-terminal polar groups present in parent RA.We prepared carboxylic acid-containing variants of the aminoalkyls (4a -6a) to yield the corresponding acids (4c -6c) and the amidoalkyls (4b -6b) to yield the corresponding acids (4d -6d).Finally, we prepared the methyl esters (4e -6e, 4f -6f) of all acids to potentially facilitate cellular uptake (Fig. 2).

Chemicals
RA, ethylenediaminetetraacetic acid (EDTA) and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical (St.Louis, MO, USA).All other chemicals were of reagent grade.

Cell lines and culture conditions
Human breast cancer cell line MCF-7 and human prostate cancer cell line PC-3 were obtained from the RIKEN Cell Bank (Tsukuba, Ibaraki, Japan).MCF-7 and PC-3 cells were maintained in RPMI 1640 Medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES, GIBCO).All cells described above were incubated at 37 °C in a humidified atmosphere of 5% CO 2 in air.Cell number was estimated using an electric particle counter (Coulter Electronics, Hialeah, FL, USA).

Cell culture
Cells (1 ~ 4 × 10 4 cells/mL) were incubated at 37 °C in a humidified atmosphere of 5% CO 2 in air.After 24 h, various concentrations of compounds were added to the cultures, and then were incubated for 72 h.Control cells were treated with 0.1% DMSO.

In vitro RARα and RXRα reporter gene assays
Compound 6d was investigated with commercially available assays for human RARα (product number: IB02201-32) and RXRα (product number: IB00801-32; INDIGO Biosciences, Inc., State College, PA, USA; Purchased from Cosmo Bio Co., LTD, Tokyo, Japan).RAR and RXR assay kits included RARα and RXRα reporter cells, cell recovery medium, compound screening medium, positive control (10 mM 9-cis-retinoic acid in DMSO, reference agonist for RARα or RXRα), detection substrate, detection buffer, snapin 8-well strips, and plate frame were conducted as described the manufacturer-supplied protocols.
Briefly, for RARα and RXRα assays, cells were seeded into well strips (100 μL/well) and then culture media with test compound was added (100 μL/well) and incubated at 37 °C for 24 h.The media was removed, luciferase detection reagent (detection substrate dissolved in detection buffer) was added (100 μL/well), and light emission was quantified using a 2030 multilabel reader ARVO ™ X2 (PerkinElmer, Waltham County, MA, USA).Each assay strip included three replicate wells per vehicle control.Compound was initially tested across a range of doses including 0.5, 1, 4, 10, and 20 μM on the active dose response range.Positive control (9-cis-retinoic acid) was run across a range of doses as specified by the manufacturer protocols (0.5, 5, 50, and 500 nM).

Flow cytometry
MCF-7 and PC-3 cells (2 × 10 4 cells/mL) were incubated at 37 °C in a humidified atmosphere of 5% CO 2 in air.After 24 h, cells were treated with 10 μM 6d or DMSO for 24 h.Cells were harvested and fixed in 70% ethanol at 4 °C overnight.Before the analysis, cells were washed with phosphate buffered saline (PBS) twice, treated with 100 μg/mL RNase A at 37 °C for 30 min, and then stained with 10 μg/mL propidium iodide (PI).Cell cycle analysis was performed using a FACSVerse flow cytometer (Becton Dickinson, San Jose, CA, USA).

General synthetic methods
All reactions involving moisture-sensitive compounds were conducted under anhydrous conditions (positive argon pressure) using standard syringe, cannula, and septa apparatus.Commercial reagents such as all the methoxy acids, 4-(benzyloxy)aniline hydrochloride, N,N'-diisopropylcarbodiimide, diisopropylethylamine, sodium borohydride, iodine, lithium hydroxide, palladium on carbon (10%), as were purchased from Sigma, TCI America, Acros, Aapptec, or Chem-Impex and used as received.All solvents were purchased in anhydrous form (Aldrich) and used directly.HPLC-grade hexanes, EtOAc, CH 2 Cl 2 , and MeOH were used in silica gel Combi-Flash chromatography employing a Teledyne CombiFlash R f 200i instrument with either hexane/EtOAc or CH 2 Cl 2 /MeOH gradients with 1% AcOH for purification of compounds with terminal carboxylic acid groups.Analytical thin-layer chromatography (TLC) was performed using Analtech precoated plates (Uniplate, silica gel GHLF, 250 nm) containing a fluorescence indicator.NMR spectra were recorded using a Varian Inova 500 MHz spectrometer.Coupling constants are reported in Hertz, and peak shifts are reported in δ (ppm) relative to CDCl 3 1 H 7.26 ppm; 13 C 77.16 ppm), CD 3 OD 1 H 3.31 ppm; 13 C 49.00 ppm), or dimethyl sulfoxide (DMSO)-d 6 1 H 2.50 ppm, 13 C 39.52 ppm).Low-resolution mass spectra (ESI) were measured with either an Agilent 260 1200 LC/MSD-SL system or a Shimadzu 2020 LC/MS system.High-resolution mass spectra (HRMS) were obtained by positive ion, ESI analysis on a Thermo Scientific LTQ-XL Orbitrap mass spectrometer with HPLC sample introduction using a short narrow-bore C 18 reversed-phase column with MeCN/H 2 O gradients.Preparative HPLC of final products was performed using a Waters 2545 binary pump equipped with a reverse-phase Gemini C 18 column (Phenomenex Inc., pore size: 110 Å, particle size: 10 μm, 250 mm × 21.2 mm) with a gradient of 5-99% MeCN/H 2 O containing 0.1% TFA over 30 min at a flow rate of 10 mL/minutes and monitored with a UV detector at 214 and 254 nm.Semi-preparative HPLC purification was performed using an Agilent 1200 series quaternary pump (MeCN/H 2 O gradient containing 0.1% TFA) with a reverse phase Phenomenex Kinetix-C 18 column (pore size: 110 Å, particle size: 5 μm, 250 mm × 10.0 mm) at a flow rate 3 mL/minutes and monitored with a UV detection at 210 nm.Fractions containing pure compounds were combined and lyophilized to obtain a white powder.Analytical HPLC of final peptides was performed using an Agilent 1200 series quaternary pump (MeCN/H 2 O gradient containing 0.1% TFA) with a Phenomenex Gemini-C 18 column (pore size: 110 Å, particle size:5 μm, 250 × 4 mm), at a flow rate of 1 mL/min, and UV detection at 210 nm.

General method A: anhydride-based synthesis of amides 4b -6b
To a cold solution of 4-aminophenol (0.109 g, 1.00 mmol) in dry THF (6.9 mL) at 0 °C under N 2 was added anhydride 7a-7c (1.10 mmol) and the mixture was allowed to come to room temperature with stirring (16 h).The solvent was removed under reduced pressure and the residue was washed with CHCl 3 (6.9mL) and stirred (1 h).The resulting solid was collected and washed to yield 4b-6b as white crystals (Scheme 1).

General Method B: Reduction of Amides to Amines 4a, 5a and 6a
To a solution of p-alkyl-amidophenol (4b-6b) (0.05 g, 0.172 mmol) in anhydrous THF (3 mL, 0.33 M) at 0 °C under N 2 was added NaBH 4 (0.016 g, 0.429 mmol, 2.5 eq.) and the reaction mixture was stirred.The suspension was treated dropwise with I 2 (0.044 g, 0.172 mmol) in anhydrous THF and the reaction mixture was refluxed (3 h) and monitored by TLC then cooled to room temperature.The reaction mixture was treated with 3 M aqueous HCl (2.00 mL, 65.8 mmol), neutralized with 1 M NaOH and extracted with EtOAc.The combined organic extract was washed with H 2 O and brine and dried over Na 2 SO 4 .The solvent was removed under vacuum to provide a crude product, which was purified by silica gel chromatography by elution with EtOAc and hexanes (gradient: 5-30% over 25 min) to yield 4a, 5a and 6a as white solids (Scheme 1).

Results
Breast and prostate cancers are cancer types with very high prevalence in women and men.Currently, chemotherapy is often given in addition to surgical resection as treatment for both cancers.Cyclophosphamide, doxorubicin, and fluorouracil are used as therapeutic agents for these cancers, and docetaxel is used for prostate cancer.However, it is known that treatment with these anticancer agents causes side effects such as myelosuppression that increases in the risk of infectious diseases.In order to potentially address these problems, we evaluated the growth inhibitory effects of the new compound against these two types of cancer cells.

Effects on growth of human breast and prostate cancer cells inserting carboxylic acid functionality into p-alkylaminophenols
First, we investigated the growth-inhibitory effects against MCF-7 cells of three p-alkylaminophenols (4a, 5a, and 6a) with different carbon chain-lengths.All palkylaminophenols significantly suppressed the growth of MCF-7 cells in a concentrationdependent manner as compared to the control (Fig. 3).Among the three palkylaminophenols, analog 6a having the longest carbon chain length, had the greatest growth inhibitory potency.The inhibition percentages at 4 μM and 10 μM concentrations were approximately 61% and 73% for 6a, 31% and 51% for 5a, and 14% and 45% for 4a, with 4 μM RA exhibiting approximately 62%.Therefore, at 4 μM 6a showed a cell proliferation inhibitory potency that was equal to RA.In contrast, insertion of a carboxylic acid moiety at the terminus of the polymethylene chains of 4a, 5a, and 6a to give the corresponding acids 4c, 5c, and 6c uniformly abrogated growth inhibitory potencies (Fig. 3).
The growth inhibitory effects of synthetic compounds against PC-3 cells were investigated.The three parent p-alkylaminophenols (4a, 5a, and 6a) significantly suppressed cell proliferation in concentration-dependent manner as compared to controls (Fig. 4).At a concentration of 1 μM, 6a inhibited cell proliferation by approximately 91%, 5a by 65%, and 4a by 33%.Similar to the results seen with MCF-7 cells, compound 6a, which has the longest chain length, showed the greatest potency.In contrast, the growth inhibitory potency of 4 μM RA of approximately 18%, was significantly weaker effect than the p-alkylaminophenols.Similar to what was observed with MCF-7 breast cancer cells, against PC-3 cells, compounds 4c, 5c, and 6c, which have terminal carboxyl groups, did not show significant growth inhibitory effects.We also found that the parent p-alkylaminophenols (4a -6a) more potently inhibited the proliferation of PC-3 cells than was observed with MCF-7 cells.These results suggest that the p-alkylaminophenols exhibit cell proliferation inhibitory effects against both MCF-7 and PC-3 cells with potencies that are in the order 6a > 5a > 4a.In contrast insertion of a chain-terminating carboxylic acid group abrogated the growth inhibitory effects against both cell lines.

Effects on growth of human breast and prostate cancer cells inserting carboxylic acid functionality into p-amidophenols
Next, we investigated the growth inhibitory effects of p-amidophenols (4b, 5b, and 6b) on MCF-7 and PC-3 cells.We found that against MCF-7 cells, conversion of the alkylamine groups of 4b, 5b, and 6b to the corresponding alkylamido groups, resulted in a significant loss of cell growth inhibitory potencies.In contrast, inserting chain-terminal carboxyl groups into the p-amidophenols to give the corresponding carboxyl-containing amidophenols (4d, 5d, and 6d) resulted in a gain of growth inhibitory potencies (Fig. 5).At a concentration of 4 μM, significant cell-growth inhibitory potencies were observed.When expressed in terms of suppressing percentages as compared to control, growth inhibitory values were approximately 40% for 6d, 28% for 5d, and 30% for 4d, while 4 μM RA showed approximately 52% growth inhibition.
Next, we examined the effects of synthetic compounds on PC-3 cell proliferation.While we found that p-amidophenols 6b, 5b, and 4b did not show significant growth inhibitory effects against MCF-7 cells, they did show moderate growth inhibitory effects against PC-3 cells as compared to controls (Fig. 6).At a concentration of 10 μM, compound 6b showed cell-suppressing effect as compared to the control at levels of 24%, 5b 43%, and 4b 45%, with the potencies being in the order 6b < 5b < 4b (Fig. 6).Inserting a terminal carobxyl group into 6b to yield 6d, enhanced cell growth inhibitory potency (approximately 54% inhibition at 4 μM and 70% inhibition at 10 μM).In contrast, similar structural modifications to 5b, and 4b to yield 5d and 4d attenuated growth inhibitory potencies (Fig. 6).Under the assay conditions, treatment with 4 μM RA inhibited PC-3 cell growth by approximately 19% (data not shown).
As mentioned above, while the p-amidophenols 6b, 5b, and 4b did not significantly inhibit the proliferation of MCF-7 cells, they did have antiproliferative effects against PC-3 cells.Proliferation of PC-3 cells were suppressed by 5b and 4b with short chains greater than by 6b with a longer chain.Compounds 4d, 5d, and 6d having a carboxyl group appended onto the terminal chain of the p-amidophenols 4b, 5b and 6b, showed enhanced growth inhibitory potencies against MCF-7 cells.However, against PC-3 cells, the inhibitory potencies of 4d and 5d were reduced, while the potency of 6d was enhanced.These results indicate that the p-amidophenols suppressed the proliferation of PC-3 cells in the order 6b < 5b < 4b but did not significantly affect MCF-7 cells.Furthermore, against MCF-7 cell, the variants 4d, 5d, and 6d having chain-terminal carboxyl groups showed enhanced inhibitory potencies, while against PC-3 cells the compounds showed enhanced (6d) or attenuated potencies (4d and 5d) as compared to the non-carboxyl-containing congeners.

Growth inhibitory effects of methyl ester-containing p-aminophenols 4e -6e and p-amidophenols 4f -6f.
Next, we examined the effects of converting the carboxyl-containing amidophenols 4b -6b to their corresponding methyl esters (4f -6f, Figure 2).We examined compounds 4f, 5f and 6f having chain-terminal carboxyl groups appended onto amidophenols 4b, 5b and 6b and compounds 4e, 5e and 6e, which represent variants of the p-alkylaminophenols 4a, 5a and 6a having chain-terminal methyl esters.None of the methyl ester-containing analogs exhibited significant growth inhibitory potencies against MCF-7 cells (Fig. 7).In this assay, 4 μM RA inhibited MCF-7 cell growth by approximately 37%.The growth inhibitory effects were also minimal against PC-3 cells.While 6f and 6e showed minimal effects (approximately 10% inhibition), the growth inhibitory effects of 6b and 6d were abrogated by conversion to 6f (Fig. 8).In this assay the growth inhibitory potency of 4 μM RA was approximately 20% (data not shown).This data indicates that the carboxyl group is important for the growth inhibitory effects of p-amidophenols (4d, 5d and 6d) but not for p-alkylaminophenols (4c, 5c and 6c).The data also suggest the methyl esters are not converted to the corresponding carboxylic acids within the cells.

Mechanisms of growth inhibition by 6d having a chain-terminal carboxyl group in human breast and prostate cancer cells
Compound 6d, having a chain-terminal carboxyl group, showed enhanced growth inhibitory potencies against MCF-7 cells (Fig. 5) and PC-3 cells (Fig. 6).Previous reports have shown that RARα is involved in retinoid-mediated signaling and that it is important for the antiproliferative effects of RA in MCF-7 cells. 16In PC-3 cells, ligand binding to RARα and RXRα RA receptors has also been investigated. 17Therefore, in order to investigate whether the antiproliferative effects of 6d against both cell lines are mediated by RAR or RXR or whether 6d binds to RARα and RXRα and activates both receptors to exert its actions, we performed Luciferase-Reporter Assays on RARα and RXRα.In these assays, 9-cis-retinoic acid, an agonist of RARα and RXRα, was used as positive control.This increased luciferase activity in a concentration-dependent manner (0 ~ 500 nM), whereas 6d had almost no effect at the control level (Fig. 9).These results suggest that 6d did not bind to RARα and RXRα and that the cell growth inhibitory action of 6d is not mediated by RARα and RXRα.
Since 6d was found to exert its effects through mechanisms not mediated by the RA receptor, we investigated the effects of 6d on other pathways (cell cycle arrest, induction of apoptosis (Sub-G1, BCL-2, BCL-xL), and phosphorylation inhibition of ERK).First, we examined the population of each cell cycle in 6d-treated MCF-7 cells and PC-3 cells and the cell population of the Sub-G1 stage, which is an apoptosis index (data not shown).6d treatment on MCF-7 and PC-3 cells had no effect on the cell cycle and had no significant effect on the Sub-G1 population, an indicator of apoptosis.This data shows that 6d does not induce cell cycle arrest.In addition, the effects of 6d on expression of the apoptosisinhibitory Bcl-2 and Bcl-xL proteins were investigated.The data showed that 6d did not affect the expression of either protein.In addition, phosphorylated ERK levels involved in cell proliferation were not affected by 6d treatment.These results suggest that 6d does not induce apoptosis and inhibit ERK phosphorylation.
Next, we examined the involvement of autophagy, one non-apoptotic cause of cell death.Treatment of MCF-7 and PC-3 cells with 6d significantly increased by approximately 1.6-fold and 1.5-fold, respectively the expression of LC3-II (the lipid adduct of LC3-I), which is indicator of autophagy (Fig. 10).This suggests that 6d induces autophagy in both cells.Finally, we investigated the effect of 6d on the PI3K/Akt pathway, which is a survival signal.We found that phosphorylated Akt levels (p-Akt/Akt) decreased significantly by approximately 35% in PC-3 cells, while phosphorylated Akt levels tended to decrease in MCF-7 cells (Fig. 11).Collectively, this data suggests that 6d suppresses the survival signal by Akt in PC-3 cells.Since no Sub-G1 population was observed and apoptosis was not induced, this suggests that 6d suppresses the proliferation of MCF-7 and PC-3 cells by inducing autophagic cell death, which is non-apoptotic cell death.

Discussion
RA (1) is an active form of vitamin A that exhibits a variety of actions in vivo.It is used in the treatment of APL (acute promyelocytic leukemia) due to its effectiveness in inducing differentiation of leukemia cells.RA is believed to exert many of its actions by binding to RA receptors (RAR and RXR) and regulating gene expression.In addition, the terminal carboxylic acid group of RA has been reported to be important when RA binds to the RA receptors.To alleviate the side effects of retinoids and rexinoids, 4-HPR (2) was developed as an atypical retinoid.While 4-HPR has potent antiproliferative properties, it exhibits side effects, such as night blindness and ocular toxicity.4-HPR represents a variant of RA in which the carboxylic acid group has been converted to an p-amidophenol group (Fig. 1).4-HPR does not bind to RAR receptors, and it does not induce cell differentiation.The anti-proliferative activity of 4-HPR results from induction of apoptosis through the generation of ROS rather than through binding to RARs. 3 In order to overcome the side effects of 4-HPR, a structure-activity relationship study was conducted that examined long-chain N-alkyl p-aminophenols. 8,13][10] By analogy to RA, the focus of our current study was to examine the effects of introducing terminal carboxylic acid groups into long-chain N-alkyl p-alkylaminophenols.We found that modifying the parent palkylaminophenols (4a -6a) in this fashion (4c -6c) resulted in a loss of growth inhibitory potency against both MCF-7 cells (Fig. 3) and PC-3 cells (Fig. 4).In contrast, while against MCF-7 cells p-amidophenols (4b -6b) did not suppress proliferation (Fig. 5), the introduction of chain-terminal carboxyl groups onto the side chains of these p-amidophenols to yield 4d -6d, enhanced antiproliferative potencies to values similar to what was found with RA.Against PC-3 cells p-amidophenols with shorter carbon chains inhibited cell growth more strongly (4b and 5b, Fig. 6).The introduction of terminal carboxyl groups into the side chains of p-amidophenols enhanced the inhibitory effects on cell growth analogs having a longer chain (C12, 6d), but attenuated the potencies of analogs having the shorter chains (C10 and C8, 5d and 4d).Against MCF-7 cells (Figure 7) and PC-3 cells (Fig. 8), the introduction of terminal carboxyl moieties into the side chains of p-alkylaminophenols resulted in a loss of effects on cell growth (4e -6e), relative to the corresponding analogs having free carboxylic acids (4c -6c).In contrast, conversion of the carboxylic acids in the amidophenol series (4d -6d) to their methyl esters (4f -6f) abolished the antiproliferative enhancement incurred by the original introduction of carboxylic acid functionality.The proliferation of MCF-7 cells was inhibited by approximately 60% by 4 μM RA, but that of PC-3 cells was inhibited by only approximately 20%.The p-alkylaminophenols and pamidophenols were more effective than RA against PC-3 cells than to MCF-7.Introduction of carboxylic acid functionality enhanced antiproliferative potency against both cell lines with the exception of 5d and 6d against PC-3 cells.
While the introduction of carboxylic acid and methyl ester moieties into palkylaminophenols, which had originally been shown to have strong antiproliferative activities, significantly attenuated growth inhibitory effects, the introduction of carboxylic acid groups into the side chains of p-amidophenols, which had only a weak antiproliferative potencies, enhanced their potencies.However, the conversion of these acids to their corresponding methyl esters completely abolished the effects.While a carboxylic acid group is important for the binding of RA to the RA receptor, introduction of this functionality into p-alkylaminophenols abolishes antiproliferative potency, while enhancing the potency of p-amidophenols.Conversion of the carboxylic acids to their methyl esters completely abolished antiproliferative potency, in spite of the fact that this should have increased lipophilicity and cellular uptake.This suggests that the methyl esters are not converted to their active free acids by esterases in the cytoplasm.
Compound 6a is a highly promising compound that is non-toxic and non-inflammatory when applied to the skin of hairless mice in vivo. 18In contrast, RA induces inflammation when applied to hairless mice.In addition, 6a is non-toxic to the non-malignant human epidermal keratinocyte cell line (HaCaT cells) in vitro.Compound 6a, which has a phenolic OH residue, undergoes metabolism by mechanisms that include glucuronidation and sulfate conjugation and it is excreted in the urine.Compound 6a is also considered to be noncarcinogenic because it does not decompose into primary amines.When administered in vivo, 4-HPR (2) an aminophenol derivative of RA, whose clinical trial was discontinued due to side effects (impaired dark adaptation), decreases blood retinol levels, whereas 6a does not reduced retinol levels.Compound 6a eliminates the side effect of impaired dark adaptation, because it has no effect on blood retinol levels. 9No cytotoxic effects of 6a were observed in in vivo studies using prostate cancer cells and neuroblastoma. 9,11mpound 6a was developed based on the speculation that the cyclohexene ring is the cause of the side effects of 4-HPR.The structure of 4-HPR was divided into four parts and structure-activity relationship studies were conducted.These studies determined that the methylaminophenol moiety played a central role and that extending the length of the polymethylene side chain was beneficial. 13,7The phenolic OH moiety is important for the cancer cell growth inhibitory action of compound 6a, and the action is enhanced by elongating the side chain of methylaminophenol. 9It has been reported that 6a does not bind to RARs and RXRs, 18 and that growth inhibitory effects on prostate cancer PC-3 cells are due to induction of apoptosis and cell cycle arrest. 9In our current study, we investigated the mechanism of action of the newly developed 6d and found that 6d does not bind to RARα and RXRα (Fig. 9), arrest cell cycle, induce apoptosis or inhibit the ERK pathway.In contrast we found that 6d induces autophagy and inhibits the Akt pathway, leading to non-apoptotic cell death.These results suggest that the growth suppression of MCF-7 cells and PC-3 cells by 6a and 6d are not mediated by RA receptors and that they have different mechanisms of action.
Our previous studies have examined the inhibitory effects of cinnamic acid derivatives, including caffeic acid phenethyl ester, on the growth of MCF-7 and PC-3 cells. 19The inhibitory effects on cancer cell growth are stronger depending on the number of OH groups on the cinnamic acid aryl ring and that tri-hydroxycinnamic acid is more potent than the mono-and di-hydroxycinnamic acids.These results indicate that the growth inhibitory effects on cancer cells becomes stronger with increasing number of hydroxyl groups on the cinnamic acid aryl ring.Our current study shows that the anti-cancer effects of 6a and 6d have anti-proliferative potencies against MCF-7 and PC-3 cells.These results suggests the potential value of investigating the effects on anti-proliferative potencies of increasing the number of OH groups on the aromatic ring.
In conclusion, we have shown that introduction of chain-terminal carboxylic acid moieties into a series of p-alkylamidophenols enhances growth inhibitory potencies against both MCF-7 and PC-3 cells.The phenolic 4-carboxyamido group was seen as being important for this potency enhancement, since the effect was not observed when similar carboxylic acid functionality was introduced into a corresponding series of p-alkylaminophenols.This may reflect a difference between MCF-7 cells and PC-3 cells in the molecular targets being affected by RA, p-alkylaminophenols and p-alkylamidophenols.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.Transcriptional activity of 6d and 9-cis RA.Cells were treated with 9-cis RA (0.5 ~ 500 nM) or 6d (500 ~ 20000 nM), and luciferase activities were measured as described in "Materials and Methods".Each bar represents the mean ± SD (n = 3).Protein levels of LC-3 in MCF-7 and PC-3 cells.MCF-7 (A) and PC-3 (B) cells (2 × 10 4 cells/ml) were incubated at 37 °C in a humidified atmosphere of 5% CO 2 in air.After 24 h, cells were treated with DMSO (control) or 6d at 10 μM for 72 h.The protein levels of LC3 and β-actin were analyzed by Western blot analysis with specific antibodies against LC3 and β-actin as described in "Materials and Methods".The value of control was defined as 1.0.
Each bar represents the mean ± SD of each group (n = 3).*p < 0.05 compared with control.Protein levels of phosphorylated-Akt, and Akt in MCF-7 and PC-3 cells.MCF-7 (A) and PC-3 (B) cells (2 × 10 4 cells/ml) were incubated at 37 °C in a humidified atmosphere of 5% CO 2 in air.After 24 h, cells were treated with DMSO (control) or 6d at 10 μM for 72 h.The protein levels of phosphorylated-Akt, and Akt were analyzed by Western blot analysis with specific antibodies against phosphorylated-Akt, and Akt as described in "Materials and Methods".The value of control was defined as 1.0.Each bar represents the mean ± SD of each group (n = 3).*p < 0.05 compared with control.
Cell viability was determined by MTT method.Data shown are mean ± SD (n = 3).***p < 0.001 vs Control, 666 p < 0.001 vs the 6b at the same concentration, 555 p < 0.001 vs the 5b at the same concentration, 444 p < 0.001 vs the 4b at the same concentration (Bonferroni's multiple comparisons test).