Research ArticleExperimental Studies

Synthesis and Structure–Activity Relationships of Tetrahydro-β-carboline Derivatives as Anticancer and Cancer-chemopreventive Agents

MINGMING ZHANG, EUN-JUNG PARK, TAMARA P. KONDRATYUK, JOHN M. PEZZUTO and DIANQING SUN
Anticancer Research August 2018, 38 (8) 4425-4433; DOI: https://doi.org/10.21873/anticanres.12744

Abstract

Background/Aim: There is an unmet clinical need to develop new anticancer and chemopreventive agents. The aim of the present study was to identify β-carboline derivatives with cancer chemopreventive and therapeutic potential. Materials and Methods: Forty-eight tetrahydro-β-carboline derivatives were synthesized and evaluated for their anticancer and chemopreventive activities, through induction of quinone reductase 1 (QR1), aromatase inhibition, as well as inhibition of nitric oxide (NO) production. Results: 2-((1-Bromonaphthalen-2-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole demonstrated the most potent activity in the QR1 induction assay with an induction ratio value of 3.2 (CD=1.3 μM). The R-isomer of the amide derivative (2-((1-bromonaphthalen-2-yl)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-3-yl)(4-methylpiperazin-1-yl)methanone was the most potent inhibitor of NO production with a 50% inhibitory concentration, IC50=6.54 μM and had a low cytotoxic effect (IC50=17.98 μM) on RAW 264.7 cells. Subsequent computational docking study revealed that this compound binds to the active site of inducible nitric oxide synthase with favorable interactions. Conclusion: our results provided promising β-carboline leads for further optimization and development with therapeutic potential as new chemopreventive and chemotherapy agents.

Cancer is responsible for millions of deaths worldwide and continues to remain one of the leading causes of death each year. According to the World Health Organization, there were an estimated 14.1 million new cancer cases and 8.2 million cancer deaths in 2012, compared to 12.7 million and 7.6 million, respectively, in 2008 (1). It is projected that the number of new cancer cases and cancer deaths worldwide will rise to 21 and 13 million, respectively, by 2030. In the United States, there were an estimated 1.7 million new cancer cases and over half million cancer deaths in 2016 (2). Because of serious side-effects, drug resistance, and relapse associated with cancer chemotherapy, there is a significant unmet medical need for developing new anticancer agents with improved efficacy and low cytotoxicity against healthy normal cells in order to minimize undesirable systemic toxicity.

On the other hand, to circumvent this toxicity issue, cancer chemoprevention represents an alternative and attractive approach to prevent the initiation, promotion, or progression of cancer using chemopreventive agents (3, 4). Mechanistically, the protective effects of chemopreventive agents result from a combination of diverse proposed mechanisms involving antioxidant, anti-inflammatory, immunomodulatory action, apoptosis induction, and chemical intervention against carcinogens, etc. (5). In recent years, accumulating evidence supports the notion that inducible nitric oxide synthase (iNOS) is associated with the onset and development of various human diseases, including cancer (6, 7); furthermore, the resulting signal molecule NO can also play a role in drug resistance and cancer metastasis (8, 9). In general, inhibition or down-regulation of carcinogen-metabolizing enzymes, which are capable of modifying potential carcinogens and generating carcinogenic substances, such as aromatase and iNOS, can impede carcinogenesis (10, 11). Specifically, aromatase acts in the late-stage conversion from reproductive hormone androgens to estrogens in peripheral tissues, and high estrogen levels can lead to the growth and development of breast tumors. As such, aromatase inhibitors have been clinically used in breast cancer therapy in postmenopausal women (12, 13). Recent clinical studies also supported the use of aromatase inhibitors for breast cancer chemoprevention therapy in high-risk postmenopausal women (14, 15). Furthermore, cancer chemoprevention can also be achieved by induction of anticarcinogenic enzymes, such as quinone reductase 1 (QR1) (16, 17). QR1 plays an important role in cellular defense in preventing the formation of oxidative stress by catalyzing the conversion of quinones to less reactive and less toxic hydroquinones (18).

β-Carboline alkaloids are an important class of anticancer natural products and medicinal molecules (19). Among many naturally occurring β-carboline molecules, callophycin A (1 in Figure 1), isolated from the red algae Callophycus oppositifolius, was found to have antiproliferative effects on various human cancer cell lines at low micromolar concentration (20). Previously, we synthesized and evaluated a series of callophycin A derivatives to explore chemical diversity at the 2 and 3 positions (11). Among them, the callophycin analog 2 with R-stereochemistry was discovered to inhibit nitrite production (50% inhibitory concentration, IC50=2.8 μM) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Subsequent mechanistic study revealed that 2 inhibits LPS-induced nitrite production by a unique and complex mechanism (21). In this work, an expanded panel of new derivatives around the tetrahydro-β-carboline core scaffold was synthesized and evaluated for anticancer and cancer chemopreventive potential.

Materials and Methods

Reagents and materials. Solvents and reagents were purchased from Sigma-Aldrich, St. Louis, MO, USA or Fisher Scientific, Waltham, MA, USA and were used without further purification. Reactions were monitored either by thin-layer chromatography or by employing a Shimadzu LC-20A series high-performance liquid chromatography (HPLC) system. Compounds were purified by flash column chromatography on silica gel using a Biotage Isolera One system (Biotage AB, Uppsala, Sweden). 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III HD-400 spectrometer (Bruker Corp, Billerica, MA, USA) (400 and 100 MHz, respectively). Mass spectroscopy (MS) data were obtained using an Agilent 6120 single quad MS (Agilent Technologies, Santa Clara, CA, USA) via direct injection. The purity of compounds was determined by analytical HPLC (Shimadzu LC-20A series) (Shimadzu Scientific Instruments, Inc. Columbia, MD, USA) using a Gemini, 3 μm, C18, 110 Å column (50 mm × 4.6 mm, Phenomenex, Torrance, CA, USA) and flow rate of 1 ml/min. Gradient conditions: solvent A (0.1% trifluoroacetic acid in water) and solvent B (acetonitrile): 0-2.0 min 100% A, 2.0-7.0 min 0-100% B (linear gradient), 7.0-8.0 min 100% B, UV detection at 254 nm and 220 nm.

Design rationale. From our previous study, the S-isomer of callophycin A showed aromatase-inhibitory activity with an IC50 value of 10.5 μM (11). To further expand existing structure-activity relationship (SAR), an extended series of compounds with chemically diversified alkyl substituents at the N2 position was designed and synthesized (Figure 2). In order to evaluate the importance of the free carboxylic acid or its ester functionality at the 3 position of the tetrahydro-β-carboline scaffold, a set of β-carboline derivatives with carboxylic acid and its ester moieties removed were synthesized (Figure 3). In our previous work, carboline urea and carbamate derivatives at the 2 position exhibited low micromolar activity for the induction of QR1 and nitrite inhibition (11). On the basis of existing SAR, a series of new 2-substituted urea derivatives was designed and synthesized to further expand the SAR. In addition, to explore potential new scaffolds, four tetracyclic intramolecular cyclized hydantoin derivatives were also synthesized for biological evaluation (Figure 4). 3-Substituted amide derivatives and the N9-methylated analog were also designed and synthesized (Figure 5).

Synthesis of callophycin A analogs 5-6 is shown in Figure 2. Esterification of chiral carboxylic acid isomer 3a (S) or 3b (R) with corresponding alcohol in the presence of SOCl2 yielded 4a-d, which were then subjected to reaction with various alkyl bromides in acetonitrile at reflux in the presence of N,N-diisopropylethylamine (DIPEA), affording the N-alkylated derivatives 5a-l in 62-82% yields. Finally, 5a-l were hydrolyzed to give free carboxylic acids 6a-j in the presence of NaOH in MeOH/H2O at room temperature in 81-93% yields.

Representative procedure for the preparation of compounds 4a-d. Thionyl chloride (2.1 ml, 24.1 mmol) was added dropwise to a solution of 3a (1 g, 4.6 mmol) in methanol (50 ml) at 0°C. The reaction was stirred at room temperature and the completion of the reaction was monitored by HPLC. Excess methanol and thionyl chloride were removed by evaporation. The residue was dissolved in dichloromethane and washed successively with saturated Na2CO3 and NaCl solutions. The dried dichloromethane layer was evaporated and purified by flash column chromatography on silica gel to provide 4a.

General procedure for the preparation of compounds 5a-l. Substituted bromide (0.12 mmol) and DIPEA (0.15 mmol) were added to the solution of 4 (0.1 mmol) in acetonitrile (10 ml). The reaction was heated under reflux for 6 h. After the completion of the reaction (monitored by HPLC), solvent was removed by evaporation. The residue was dissolved in cold EtOAc (10 ml) and the mixture was then filtered to remove the precipitate. The filtrate was concentrated in vacuo to yield a light-yellow crude residue, which was further purified by flash column chromatography on silica gel to give products 5a-l.

General procedure for the preparation of compounds 6a-j. A solution of an appropriate ester 5a-l (0.1 mmol) and NaOH (0.5 mmol) in MeOH/H2O (10:1, 5 ml) was stirred at room temperature until the completion of the reaction (monitored by HPLC). Most MeOH was removed in vacuo and the residue was carefully neutralized with glacial acetic acid, whereupon a yellow precipitate was formed, the product was collected by filtration, washed with cold water, and dried.

General procedure for the preparation of compounds 8a-h. As shown in Figure 3, the commercially available secondary amine 7a or 7b was reacted with an appropriate alkyl bromide/chloride in acetonitrile under reflux in the presence of DIPEA, affording 8a-h in 77-94% yields. Substituted bromide or chloride (0.12 mmol) and DIPEA (0.15 mmol) were added to the solution of 7a or 7b (0.1 mmol) in acetonitrile (10 ml). This reaction was heated under reflux for 2 h. After the completion of the reaction (monitored by HPLC), the solvent was removed by evaporation. The residue was dissolved in cold EtOAc (10 ml) and the mixture was then filtered to remove the precipitate. The filtrate was concentrated in vacuo to yield a light-yellow crude residue, which was further purified by flash column chromatography on silica gel to give products 8a-h.

Figure 1.
Figure 1.

Callophycin A (1) and its synthetic analog 2.

Figure 2.
Figure 2.

Synthesis of callophycin A analogs 5-6. Reagents and conditions: a: SOCl2, R1OH, 0°C to rt; b: R2Br, DIPEA, CH3CN, reflux; c: NaOH, MeOH/H2O (10:1).

Figure 3.
Figure 3.

Synthesis of 8a-h. Reagents and conditions: a: R2-Br or R2-Cl, N,N-diisopropylethylamine (DIPEA), CH3CN, reflux.

General procedure for the preparation of compounds 9a-f and 10a-c. The urea derivatives 9a-f and 10a-c were synthesized from 4b-d or 7a and corresponding isocyanates in 66-84% yields in dichloromethane at room temperature (Figure 4). Subsequently, under the basic reaction condition, 9a-d were transformed into their corresponding intramolecular cyclized hydantoin products 11a-d in 58-68% yields.

Figure 4.
Figure 4.

Synthesis of β-carboline derivatives 9-11. Reagents and conditions: a: R2-NCO, CH2Cl2, rt; b: NaOH, THF:H2O (1:1), rt.

Figure 5.
Figure 5.

Synthesis of β-carboline amide derivatives 12a-g and N9-methylated 13. Reagents and conditions: a: R1R2NH, EDC, HOBt, CH2Cl2, 0°C to rt., overnight; b: MeI, NaH, DMF, 0°C to rt., 12 h.

The reaction of 4b-d or 7a (0.5 mmol) and an appropriate isocyanate in dichloromethane (5 ml) was stirred at room temperature overnight. After the completion of the reaction (monitored by HPLC), the reaction mixture was concentrated. The residue was purified by flash column chromatography on silica gel to give products 9a-f and 10a-c.

General procedure for the preparation of compounds 11a-d. A solution of an appropriate methyl ester 9a-d (0.1 mmol) and NaOH (1 mmol) in THF/H2O (1:1, 5 ml) was stirred at room temperature until the completion of the reaction (monitored by HPLC). THF was removed in vacuo and the residue was dissolved in EtOAc (10 ml) and washed with H2O. The organic phase was collected, dried, and concentrated. The residue was purified by flash column chromatography on silica gel to give products 11a-d.

Finally, given the promising activity of 6e and 6j in our preliminary screen, the free carboxylic acid group at the 3 position of 6e and 6j was converted to the amide functionality in 12a-g by reacting with different amines using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole monohydrate (HOBt) in dichloromethane (Figure 5). To investigate the importance of the N9 hydrogen, the N9 methylated derivative 13 was also synthesized from 5j.

General procedure for the preparation of compounds 12a-g. At 0°C, to a solution of an appropriate amine (0.345 mmol) in dry CH2Cl2 (5 ml), HOBt (0.69 mmol) and EDC (0.69 mmol) were added and stirred for 30 min. Then compound 6e or 6j (0.23 mmol) was added and the reaction was allowed to return to room temperature and stirred overnight. The reaction mixture was washed by water and extracted with CH2Cl2. The organic layer was washed with saturated aqueous NaHCO3 solution, brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residue was purified by flash column chromatography on silica gel to give products 12a-g in 59-73% yields.

Procedure for the preparation of compound 13. To a stirred solution of 5j (100 mg, 0.22 mmol) in dry dimethylformamide (DMF) (25 ml), NaH (11 mg, 60% suspension in mineral oil, 0.26 mmol) was added in portions under N2 atmosphere at 0°C. The reaction mixture was then warmed to room temperature and stirred for 30 min. After cooling to 0°C again, MeI (17 μl, 0.27 mmol) was added dropwise. The reaction mixture was stirred at room temperature overnight, quenched with water and the aqueous layer was extracted with ether. The combined organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography to give compound 13 in 79% yield.

Determination of QR1 activity in cell culture. QR1 activity was determined using Hepa 1c1c7 murine hepatoma cells as previously described (22). Briefly, cells were incubated in a 96-well plate with test compounds at a maximum concentration of 50 μM for 48 h prior to permeabilization with digitonin. Enzyme activity was then determined as a function of the NADPH-dependent menadiol-mediated reduction of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue formazan. Production was measured by absorption at 595 nm. Total protein, quantified using crystal violet staining, was assessed in parallel. The induction ratio (IR) of QR activity represents the specific enzyme activity of agent-treated cells compared with a dimethyl sulfoxide (DMSO)-treated control. The concentration required for double activity (CD) was determined through a dose–response assay for active substances (IR >2). Data presented are the result of three independent experiments run in duplicate. 4’-Bromoflavone (CD=0.01 μM) was used as a positive control.

Figure 6.
Figure 6.

Interaction of 12f in the active site of inducible nitric oxide synthase (PDB ID: 2Y37).

Aromatase assay. Aromatase activity was assessed by measuring the fluorescent intensity from the final product of a substrate, dibenzylfluorescein (DBF). The test substance was preincubated with a NADPH-regenerating system followed by further incubation with aromatase enzyme and DBF mixture for 30 min. Fluorescence was measured at 485 nm (excitation) and 530 nm (emission). Compounds with inhibition value of 50% or higher at the concentration of 50 μM were further assessed to determine IC50 values using TableCurve software 2D (version 4) (Systat Software Inc., Chicago, IL, USA) (22).

Measurement of the production of NO in LPS-stimulated RAW 264.7 murine macrophage cells (nitrite assay). The level of NO in the cultured media was estimated by measuring the level of nitrite due to the instability of NO and its subsequent conversion to nitrite. The nitrite assay was performed as previously described (23). Briefly, RAW 264.7 cells (ATCC, Manassas, VA, USA) were incubated in 96-well culture plates at 37°C, with 5% CO2 in a humidified air incubator for 24 h. Then cells were treated with serially diluted compounds for 15 min, followed by treatment with or without LPS (1 μg/ml) for an additional 20 h. After incubation, the nitrite released in the culture media was measured using Griess reagent [1:1 mixture (v/v) of 1% sulfanilamide in 5% H3PO4 and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride solution], and absorbance was measured at 540 nm. The concentration of nitrite was calculated using a standard curve created with known concentrations of sodium nitrite. To evaluate the cytotoxic effects of compounds on RAW 264.7 cells under the same experimental condition, sulforhodamine B (SRB) assays were performed (24).

SRB assay. After incubation of RAW 264.7 cells with test compounds, cells were fixed with 10% trichloroacetic acid solution for 30 min and stained with 0.4% SRB in 1% acetic acid solution for 30 min. Protein-bound SRB was dissolved in 10 mM Tris buffer (pH 10.0) and the absorbance was measured at 515 nm. The effect of compounds on cell survival was demonstrated as percentage survival in comparison with vehicle (DMSO)-treated control cells.

Molecular docking. Molecular docking was performed using a protein-ligand docking program GLIDE (XP mode) (Schrödinger, LLC, Portland, OR, USA) (25, 26). In brief, this was achieved by simulation of docking of compounds into the active binding site of iNOS protein for which the 3D structure was obtained from Protein Data Bank (https://www.rcsb.org/, PDB ID: 2Y37). Compounds were built with ChemBio3D (CambridgeSoft Corp, Waltham, MA, USA) and optimized at molecular mechanical level using Schrödinger software (Schrödinger, LLC, Portland, OR, USA). They were then docked into the binding site of iNOS protein and the binding position with the most favorable score was examined.

Results

Chemistry. All the compounds were successfully synthesized in moderate to good yields and their structures were characterized by 1H and 13C NMR and MS. The purity of all compounds was determined to be >95% (monitored at 254 and 220 nm) by reverse phase HPLC.

Biological assays. Forty-eight β-carboline derivatives (Figures 2, 3, 4 and 5) were evaluated in cancer chemopreventive and anticancer assays including the induction of QR1 and inhibition against aromatase and nitrite production. Briefly, testing for activity was initially performed at 50 μM. For compounds exhibiting >50% inhibition or with IR value >2.0 in the QR1 assay at this concentration, further testing was performed to determine an IC50 (or CD for QR1) value. The antiproliferative effects of these compounds were also tested using a SRB assay; compounds leading to <50% of cell survival at 50 μM were considered cytotoxic, and IC50 values were determined. Results are summarized in Table I.

QR1 induction. For QR1 induction, among the N-alkylation derivatives of 5, the R-isomer 5f demonstrated higher activity (IR=4.0, CD=10.9 μM) than its S-isomer 5a (IR=2.9, CD=42.4 μM). There were no major differences observed between the two chiral isomers with other N2-substituents. In contrast, most compounds derived from 6 with a free carboxylic acid group showed increased QR1 induction activity. Among the series of compounds without the carboxylic acid group (8 and derivatives), only 8e with 1-bromo-2-naphthylmethyl group at the N2 position demonstrated potent activity, with an IR value of 3.2 (CD=1.3 μM). For urea derivatives with an alkyl side chain (9 and 11), only weak QR1 inductive activity was observed. However, higher activity was observed with the cyclized hydantoin compound 11c (IR=2.2, CD=41.4 μM). In general, the introduction of an amide functionality at the 3 position instead of the carboxylic acid group led to increased QR1 induction (12a, 12e, 12f vs. 6e, 6j). In contrast, the S-isomer ethylamino-substituted amide derivative 12a (IR=2.6, CD=32.3 μM) was much more active than its corresponding R-isomer 12b (IR=1.4). Among the R-isomer amide derivatives 12b-g, isobutyl 12e (IR=2.2, CD=39.9 μM) and 4-methylpiperazinyl 12f (IR=2.4, CD=30.4 μM) showed higher activity than other substituents. In addition, the N1-methylated compound 13 (IR=3.0, CD=26.0 μM) showed higher activity relative to its free NH variant 5j.

Aromatase inhibitory activity. Among all the tested compounds, only tetracyclic derivatives 11a-c with an extended hydantoin ring system led to over 34% inhibition when tested at 50 μM, and 11a exhibited the most potent inhibition (59.9% inhibition at 50 μM).

Nitrite inhibition. As shown in Table I, among the N2-alkylated 3-substituted ester derivatives 5a-l, the R-isomers (5f, IC50=30.97 μM; 5j, IC50=16.63 μM) generally exhibited activity comparable to their corresponding S-isomers (5a, IC50=33.10 μM; 5e, IC50=17.69 μM) except that the R-isomer 5g (IC50=33.62 μM) was more active than its S-isomer 5b. Moreover, the bulky ethyl and isopropyl ester compounds 5k and 5l were inactive compared to their methyl ester counterparts 5e (the S-isomer, IC50=17.69 μM) and 5j (the R-isomer, IC50=16.63 μM). In contrast, in the free carboxylic acid series 6a-j, the stereoisomers of alkylation derivatives 5-6 had a stereospecific effect on nitrite inhibitory activity. For instance, the R-isomers (6h, IC50=38.43 μM; 6i, IC50=28.43 μM; 6j, IC50=22.22 μM) were more active than their corresponding S-isomers. In addition, compounds with N2 substituted 1-bromo-2-naphthylmethyl group (5e, 5j, 6e, 6j) demonstrated more potent activity, with IC50 values ranging from 16.63 to 27.13 μM, lower than all the other N2 alkyl substituents tested. Among the series of compounds 8a-h without a carboxylic acid group, 8c with N2 substituted 2-naphthoxypropyl group and 8e with 1-bromo-2-naphthylmethyl exhibited inhibitory activity with IC50 values of 31.06 and 28.91 μM, respectively. For the urea compounds (9c, 9e, 9f and 10a-c) with an alkyl side chain, none of these compounds showed notable NO inhibition activity. Interestingly, tetracyclic hydantoin derivatives 11c and 11d inhibited nitrite production, with IC50 values of 27.80 and 35.11 μM, respectively. In general, the introduction of the amide functionality at the 3 position (e.g., 12a, IC50=13.39 μM; 12b, IC50=14.49 μM; 12e, IC50=42.88 μM; and 12f, IC50=6.54 μM) led to enhanced inhibitory activity compared to 6e (IC50=27.13 μM) and 6j (IC50=22.22 μM) with a 3-substituted carboxylic acid group. It should be noted for the ethyl-substituted amide derivatives, S-isomer 12a (IC50=13.39 μM) had almost the same activity as its corresponding R-isomer 12b (IC50=14.49 μM). Overall, the R-isomer amide derivative 12f with 4-methylpiperazinyl moiety had the most potent activity (IC50=6.54 μM) among all the compounds tested.

Cytotoxic effects on RAW 264.7 cells. None of the alkylated derivatives 5-6 had any cytotoxic effect on RAW cells. Among the series of alkylated compounds of 8 without a carboxylic acid group, 8c with a N2-substituted 2-naphthoxypropyl moiety was cytotoxic toward RAW cells with an IC50 value of 39.90 μM. From the urea and tetracyclic hydantoin series (9-11), only 11c with a hexyl group exhibited a cytotoxic effect on RAW cells, with an IC50 value of 49.95 μM. Among 3-substituted amide derivatives 12a-g, only 12f (containing a 4-methylpiperazinyl group; IC50=17.98 μM) had cellular cytotoxic effect on RAW cells.

View this table:
Table I.

Biological activities (data mean value±standard deviation, μM) of compounds 5-13 evaluated in cancer-chemopreventive and anticancer assays.

Molecular docking with iNOS. In an effort to provide structural insight and gain further understanding of the potency of our tested compounds, we proceeded to examine the potential interactions of 12f with iNOS protein using the 3D structure obtained from Protein Data Bank (PDB ID: 2Y37). As shown in Figure 6, the carboline compound 12f was found to bind in the same pocket as the co-crystalized ligand. The tetrahydro-β-carboline moiety of 12f was buried in a pocket woven between TYR367, VAL346, PRO344, and iron protoporphyrin IX (HEME). One hydrogen bond was formed between NH on the carboline moiety with a carboxylic acid group on HEME. The naphthalene ring moiety was accommodated in another side pocket defined by GLU488, GLN486, and TYR485, forming hydrophobic interactions. Moreover, two additional hydrogen bonds were formed between the acylpiperazine moiety and amino acid GLN381 and ARG260.

Discussion

A series of callophycin A analogs bearing a tetrahydro-β-carboline motif was synthesized and evaluated. This work revealed several compounds to have low micromolar activity in our assays. Specifically, 8e with N2-substituted 1-bromo-2-naphthylmethyl group demonstrated excellent activity in QR1 induction with CD of 1.3 μM. Consistent with our previous findings (11), this series of tetrahydro-β-carboline derivatives were largely inactive in the aromatase inhibition assay. Among all the tested compounds, 11a exhibited the most potent inhibition (59.9% inhibition at 50 μM) against aromatase. With regard to the nitrite assay, the R-isomer of the amide derivative 12f bearing a 4-methylpiperazinyl group exhibited the most potent inhibitory activity (IC50=6.54 μM) against NO production, with a cytotoxic effect (IC50=17.98 μM) on RAW cells. The cytotoxicity of 12f is likely due to the extra amine functionality and increased cellular membrane penetration. In general, only three compounds (8c, 11c, and 12f) developed cellular cytotoxic effects on RAW cells simultaneously with iNOS inhibition under the same experimental condition. Since the cytotoxic IC50 values of these compounds were higher than their corresponding IC50 values against NO production, it can be concluded that iNOS inhibition was not caused by a false-positive effect due to general cellular cytotoxicity.

There are several factors that can affect NO synthesis: i) the cellular level of L-arginine as a substrate of iNOS and iNOS-specific cofactors, ii) enzymatic activity of iNOS, iii) protein and mRNA expression levels of iNOS, and iv) existence of endogenous antagonists (27). Active compounds found in the current study may affect enzymatic activity of iNOS by interacting with the active site or allosteric site (e.g. interaction with the dimerization site of iNOS). It is known that NOS is active only as a homodimer (28). Thus, inhibitors of iNOS dimerization have therapeutic potential in effectively controlling iNOS activity and reducing the high levels of NO produced under pathological conditions (29). Recently, pyrimidine imidazole derivatives were reported to be versatile iNOS inhibitors by interacting with both dimer and monomer entities of the iNOS enzyme (30). On the other hand, active compounds from nitrite assay may affect enzymatic activity by directly binding to the active enzyme site (substrate-binding site). For example, L-NG-monomethyl arginine, a derivative of arginine (iNOS substrate), is known to act as a competitive inhibitor of NOS (31).

In order to predict the binding mode of 12f, found to be the most active compound in the nitrite assay, molecular docking simulation was performed. The docking study revealed that 12f interacts favorably with and binds with iNOS in the active site, including three hydrogen bonds, as well as hydrophobic interactions. There are two main domains in iNOS, the oxygenase domain and the reductase domain. Compound 12f is predicted to bind with the oxygenase domain that includes regions involved in dimerization and the L-arginine binding site. None of the residues (TYR367, VAL346, PRO344, GLU488, GLN486, and TYR485) that interact with 12f exist outside of dimerization regions (32); the inhibition may occur not by allosteric inhibition, but by active-site inhibition. Nevertheless, further experimental studies remain to be performed to determine the exact mechanism of inhibition by 12f, including directly inhibiting the enzyme or another alternative mechanism such as induction by LPS. Together, these results provide promising β-carboline leads for further optimization and development with therapeutic potential as new chemopreventive and chemotherapy agents.

Acknowledgements

This work was partly supported by the National Institutes of Health grant P20GM103466.

Footnotes

  • This article is freely accessible online.

  • Conflicts of Interest

    The Authors declare that there are no conflicts of interest in regard to this study.

  • Received May 30, 2018.
  • Revision received June 16, 2018.
  • Accepted June 19, 2018.

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Synthesis and Structure–Activity Relationships of Tetrahydro-β-carboline Derivatives as Anticancer and Cancer-chemopreventive Agents
MINGMING ZHANG, EUN-JUNG PARK, TAMARA P. KONDRATYUK, JOHN M. PEZZUTO, DIANQING SUN
Anticancer Research Aug 2018, 38 (8) 4425-4433; DOI: 10.21873/anticanres.12744
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