Novel non-cyclooxygenase inhibitory derivatives of naproxen for colorectal cancer chemoprevention

Abstract

A structure-based medicinal chemistry strategy was applied to design new naproxen derivatives that show growth inhibitory activity against human colon tumor cells through a cyclooxygenase (COX)-independent mechanism. In vitro testing of the synthesized compounds against the human HT-29 colon tumor cell line revealed enhanced growth inhibitory activity compared to the parent naproxen with 3a showing IC₅₀ of 11.4 μM (two orders of magnitude more potent than naproxen). Selectivity of 3a was investigated against a panel of three tumor and one normal colon cell lines and showed up to six times less toxicity against normal colonocytes. Compound 3a was shown to induce dose-dependent apoptosis of HT116 colon tumor cells as evidenced by measuring the activity of caspases-3 and 7. None of the synthesized compounds showed activity against COX-1 or COX-2 isozymes, confirming a COX-independent mechanism of action. Compound 3k was found to have no ulcerogenic effect in rats as indicated by electron microscope scanning of the stomach after oral administration. A pharmacophore model was developed for elucidating structure–activity relationships and subsequent chemical optimization for this series of compounds as colorectal cancer chemopreventive drugs.

Introduction

Colorectal cancer is considered as the 4th leading cause of cancer deaths with more than 600,000 documented deaths each year. The worldwide incidence of colorectal cancer is almost one million new malignant cases each year with a survival rate under 50 %, despite chemotherapy (Beck et al., 2009). The high prevalence and mortality from colorectal cancer make the search for effective prevention and/or treatments an urgent medical concern.

The use of chemopreventive approaches to control colon cancer is an attractive strategy, especially for patients with a genetic predisposition as well as for those exposed to environmental carcinogens (Chan, 2002). The relationship between inflammation and cancer is well established that suggests the use of anti-inflammatory drugs for the prevention of colorectal cancer.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a well-known class of anti-inflammatory drugs that inhibit the cyclooxygenase (COX) enzymes which are crucial for the production of eicosanoids from arachidonic acid (Vane and Botting, 1996). Reports have shown the ability of many anti-inflammatory agents, especially the NSAIDs, to inhibit tumor growth. NSAIDs have also been reported to inhibit the migration of tumor cells as well as increase of the rate of apoptosis (Jana, 2008; de Groot et al., 2007; Chan, 2002). Moreover, several studies have revealed a substantial decrease in the mortality from colorectal cancer in association with the use of aspirin and other NSAIDs (Kune et al., 1988; Thun et al., 1991; Giovannucci et al., 1994). A recent 10 year prospective study found that daily use of NSAIDs reduces the risk of colorectal cancer in the general population by as much as 35 % (Sangha et al., 2005). This protective effect was more pronounced (up to 60 %) in high risk candidates, as well as in patients who had a first-degree relative with colorectal cancer (Sangha et al., 2005).

Unfortunately, the depletion of prostaglandins as a result of chronic NSAIDs use is associated with significant side effects, including gastrointestinal ulcers, bleeding, and intestinal perforation (Rainsford, 1999). While COX-2 selective inhibitors display reduced gastrointestinal toxicity, their long-term use is associated with increased risk of myocardial infarction (Cannon and Cannon, 2012; Trelle et al., 2011). These potentially fatal adverse effects outweigh the benefits of NSAIDs or COX-2 inhibitors for preventing colorectal cancer, which tend to require high dosages administration over a long period of time. The mechanism of action that is responsible for the chemopreventive activity of NSAIDs is widely attributed to COX-2 inhibition (Wang and DuBois, 2009; Harris, 2009; Khan et al., 2011). However, in addition to mechanisms that involve the inhibition of COX-2, other reports suggest that the basis for their tumor cell growth inhibitory activity involves a COX-2-independent mechanism (Grösch et al., 2006; Narayanan et al., 2012; Ishikawa and Herschman, 2010). It has been proposed that the anticancer activity of traditional NSAIDs is exerted, at least partially, through other mechanisms that are not based on inhibiting prostaglandin synthesis (Grösch et al., 2006; Tinsley et al., 2013).

Naproxen, (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid 1, is a powerful non-selective NSAID that is extensively used as a prescription or an over-the-counter NSAID. The activity of naproxen for the treatment and/or prevention of colorectal cancer has been widely reported (Suh et al., 2011; Elsheikh and Wallace, 2012; Steele et al., 2009; Harris et al., 2008; Lanas, 2009). However, the mechanism by which naproxen exerts its growth inhibitory activity is still a matter of controversy. It has been proposed that inhibition of COX-2 by naproxen prevents the increase in prostaglandin concentrations responsible for cellular transformation into a tumor phenotype (Tuynman et al., 2004). However, increasing number of studies argue against the significance of COX inhibition for the chemopreventive activity of the NSAIDs. Reports have demonstrated that the in vitro growth inhibitory activity of the NSAIDs cannot be reversed by the addition of prostaglandins or any of their stable analogs (Hanif et al., 1996). Moreover, sulindac sulfone (a metabolite of sulindac with no COX-1 or COX-2 activity) has shown to have in vivo chemoprevention against colorectal cancer (Richter et al., 2001; Piazza et al., 1997). These findings strongly suggest a COX-independent mechanism for the chemopreventive activity of NSAIDs (Hughes et al., 2012).

In the context of our interest in developing new anticancer agents (Radwan et al., 2012) with minimal adverse effects, naproxen derivatives were identified as a potential lead. Our strategy was to develop new naproxen derivatives with enhanced growth inhibitory activity against colorectal tumor cells, while reducing the potential for side effects involving COX inhibition.

Results and discussion

The inspection of the crystal structure for naproxen-bound COX-2 (PDB 3nt1; Fig. 1) indicates the importance of the carboxylic group of naproxen for strong hydrogen bonding at the enzyme active site. Blockade or replacement of this moiety will potentially decrease naproxen binding to the COX active site. Previous studies have demonstrated that carboxylic acid modification can lead to an increase in the anticancer activity of sulindac through a COX-independent mechanism (Whitt et al., 2012; Piazza et al., 2009). Careful selection of the replacement group or substituent should result in the introduction of new naproxen derivatives that lose their affinity toward the COX enzyme, while maintaining their growth inhibitory activity against cancer cells.

Fig. 1.

2D representation of naproxen at the COX-2 enzyme pocket (PDB code: 3nt1) showing crucial hydrogen bonding between naproxen caroboxylic acid and residues at the enzyme pocket. Hydrogen bonds are shown as green arrows, hydrophobic amino acids are shown in green, and polar amino acids are shown in purple (Color figure online)

Derivatives with amino acid side chain substituents were chosen for the development of the new derivatives for several important reasons. Firstly, amino acids are normal dietary constituents and generally are non-toxic in moderate doses as compared to other substitutions. Secondly, amino acids can afford a wide range of physicochemical properties for the synthesized molecules. Thirdly, being a nutritional substance, the use of amino acids permit more specific targeting for enzymes involved in the terminal phase of digestion. Accordingly, a novel series of naproxen amides of amino acid esters were designed and evaluated for COX and tumor cell growth inhibitory activity.

Docking of the designed derivatives into COX-2 enzyme

The designed compounds (3a–m, Table 1) were docked into COX-2 isozyme in order to insure the lack of potential binding at the enzyme active site. AutoDock 4.2 software (Morris et al., 2009) was used to perform automated docking of the designed compounds inside the active site of COX-2 isozyme (PDB code: 3nt1) after ligand omission (Ibrahim et al., 2012). A validation for the docking method was performed by docking the native co-crystallized naproxen ligand into its binding site of COX-2 and the results deemed satisfactory (RMSD value of 0.17 Å between the docked and co-crystalized structures and an estimated binding free energy (ΔG_bind) of −12.6 kcal/mol). All docked compounds showed significantly lower ΔG_bind compared to naproxen in the range of −8.3 to −6.7 kcal/mol (Supporting Information, Table S1). Moreover, some designed compounds either failed to be placed inside the pocket or showed steric clashes with amino acid residues at the enzyme active site (Fig. 2). This modeling study provided encouraging evidence of diminished COX inhibitory activity for the designed compounds.

Table 1.

Evaluation of the synthesized compounds 3a–m^a against the colorectal tumor cell line HT-29

Compound	R	R1	IC50 (μM)c
Naproxen (1)	–	–	1,150
3a	H	Et	14.6
3b	H	Et	123
3c	Me	Me	>250
3db	Me	Me	>250
3eb	Me	Et	175
3fb	Et	Me	209
3g	–CH(Me)2	Et	29.4
3h	–CH(Me)2	Me	102
3i	–CH2–CH(Me)2	Me	34.5
3j	–CH2–CH(Me)2	Me	74.1
3k	–CH2–COOMe	Me	120
3l	–(CH2)2–SMe	Et	49.3
3m	–CH2–Ph	Et	33.6

^an = 1 in case of 3b and n = 0 for the remaining derivatives

^bDiastereomers

^cTumor cell growth inhibitory activity was measured after 72 h of drug treatment of HT-29 colon tumor cells using the Cell Titer Glo ATP assay

Fig. 2.

Superimposition of 3a (green, ball and stick) and naproxen (yellow, ball and stick) both docked at the active site of COX-2 enzyme. Steric clashes were observed between 3a side chain and residues at the enzyme pocket are shown in red contour (Color figure online)

Chemistry

Thirteen derivatives naproxen derivatives (3a–m) were synthesized through the reaction of 1 with an appropriate amino acid ester (2) under mild conditions using mixed carboxylic–carbonic anhydrides as intermediates (Scheme 1). The carbon dioxide evolution provided a driving force for the major pathway to allow a high yield of the desired product. Analysis of the ¹HNMR data for the obtained compounds revealed retain of the stereochemical properties of the starting materials, while derivatives 3e and 3f were obtained as a 1:1 diasteriomeric mixture (Supporting Information).

Scheme 1.

Synthesis of the designed naproxen amino acid derivatives (3a–m). Reagents and Conditions: (i) Ethyl chloroformate, TEA, DCM, 0 °C, 30 min; (ii) amino acid ester, DCM, RT, 24 h

Tumor cell growth inhibitory activity for the synthesized derivatives 3a–m

Evaluation of the synthesized compounds (3a–m) against the human HT-29 colon tumor cell line revealed a general enhancement in growth inhibition activity compared to naproxen (Table 1). Derivatives 3a, 3g, 3i, 3j, 3l, and 3m were among the most active compounds showing up to two orders of magnitude increase in the potency compared to naproxen.

The encouraging activity obtained against the HT-29 cell line prompted further evaluation of the most active derivative (3a) against a panel of three human colon tumor cell lines (HT-29, SW480, and HCT116), in addition to the normal human colonocyte cell line, NCM460 (Fig. 3). Results revealed promising activity for 3a with IC₅₀ ranging between 11.9 and 17.6 μM against the tumor cell lines (Fig. 3a). The obtained activity is significantly higher than that of naproxen and the COX-2 selective inhibitor celecoxib (Fig. 3b, c). In terms of selectivity, 3a has shown a good selectivity against the tumor cells showing up to six times less activity against normal colonocytes (IC₅₀ of 58.5 μM). Tumor inhibition selectivity was not observed for either naproxen or celecoxib pointing out a different mechanisms of tumor inhibition exerted by 3a.

Fig. 3.

Dose-dependent growth inhibitory activity of, a naproxen derivative 3a, b naproxen, and c celecoxib on human colon tumor cell lines, HT-29, SW480, and HCT116 and the normal human colonocyte cell line, NCM460 after 72 h of treatment. Growth inhibitory activity was determined by measuring viable cell number using the Cell Titer Glo ATP assay

Effect of compound 3a on apoptosis induction

In order to further investigate the cytotoxic mechanism of 3a, its ability to induce apoptosis was tested through the measurement of two early stage apoptosis biomarkers; caspases-3 and 7. The results shown in Fig. 4 reveal a dose-dependent induction of apoptosis upon treatment of HCT116 cells by 3a. In addition, a twofold increase of caspase-3 and caspase-7 activity was achieved when treated with 25 μM of 3a which correlates well with its IC₅₀ values against tumor cell lines. These results proposed that the observed anti-tumor activity for 3a is associated with induction of apoptosis, while further investigation is undergoing in order to determine the exact molecular target.

Fig. 4.

Apoptosis induction of HCT116 cells by 3a after 48 h of treatment

Cyclooxygenase inhibitory activity and ulcerogenicity

In order to confirm the reduced COX inhibitory activity of the synthesized compounds, COX-1 and COX-2 inhibitory activity was measured using an enzymatic assay with purified isozymes. The results revealed the ability of naproxen to non-selectively inhibit COX-1 and COX-2 with IC₅₀ values of 4.2 and 3.6 μM, respectively. On the other hand, none of the synthesized naproxen derivatives (3a–m) showed significant inhibition of either isozyme up to concentrations of 100 μM (Supplementary Information, Table S2).

GI ulceration is a serious side effect associated with all NSAIDs, which limits their long-term use for cancer chemoprevention. To determine the ulcerogenic activities of synthesized naproxen derivatives, compound 3k was selected to be tested for ulcerogenicity. Naproxen was used as the positive control in this test. The ulcerogenic effect was measured by the examination of rat stomach using electron microscope scanning technique following a daily oral dose of the tested compound for 4 days. Scanning electromicrographs revealed damage of the protective mucous layer of the stomach in the case of the naproxen-treated group (Fig. 5a). On the other hand, similar toxic effects were not detected in stomach specimens of the 3k-treated group (Fig. 5b) as shown by their scanning electromicrographs which are almost identical to that of the control group (Fig. 5c). The results are consistent with the lack of COX inhibitory activity and indicate a superior GI safety profile for the synthesized compounds compared to the parent drug naproxen.

Fig. 5.

Scanning electromicrographs of rat stomach specimen following a daily oral dose for 4 days: a naproxen; b compound 3k; and c control

Generation of a pharmacophore model for the tested compounds 3a–m

A common features pharmacophore model was generated in order to elucidate structure activity relationships for the tested compounds. The “HipHop” pharmacophore module implemented in Acceylrs Discovery Studio software package (DS) was used for automated ligand-based pharmacophore generation. The tested compounds were divided according to their relative activities into highly active (3a, 3g, 3i, 3l, and 3m), moderately active (3b, 3e, 3f, 3h, 3j, and 3k), and inactive (1, 3c, and 3d) derivatives. For the optimized HipHop run, the highest weight was assigned to the highly active molecules of the training set.

HipHop generated 10 hypotheses (Hypo1-10).each composed of four features (Supporting Information). The top ranked model (Fig. 6) was considered for further analysis.

Fig. 6.

Top ranked HipHop pharmacophore model showing intra feature distances (Å). Features are color coded as follows: hydrogen bond acceptor; green and hydrophobic; cyan (Color figure online)

Results from the mapped compounds (Table 2) show a rough correlation between the derivatives’ fit values and their biological activities. The generated model was able to identify inactive compounds (1, 3c, and 3d) which were assigned low fit value scores.

Table 2.

Fit values and conformation energies for the mapped compounds into model-1

Compound	IC50 (μM)	Fit value	E (kcal/mol)a
Naproxen	1,150	2.1	9.2
3a	14.6	3.8	2.8
3b	123.4	2.4	7.6
3c	>250	2.3	6.4
3d	>250	2.1	4.1
3e	175.3	3.1	8.1
3f	209	2.9	2.3
3g	29.4	3.4	6.4
3h	102.1	2.5	1.2
3i	34.5	2.6	3.5
3j	74.1	2.7	0.9
3k	120.7	2.1	4.9
3l	49.3	3.9	5.2
3m	33.6	3.6	7.1

^aEnergy of the fitted conformer relative to its global minimum energy

Mapping of the most active compound 3a into the pharmacophore model (Fig. 7) shows the fitting of its terminal ethyl group into one of the hydrophobic features. The hydrogen bond acceptor feature was found to map to the side chain carbonyl ester moiety. While these two features are lacked in naproxen, they can be hypothesized to be critical for the improved anticancer activity of the corresponding amides.

Fig. 7.

Compound 3a (stick) mapped onto the generated pharmacophore model

The importance of the naphthalene ring could not be tested using the generated model, while a hydrophobic moiety connected to the C_α to the naphthalene was assigned to be preferential for activity. The last feature, hydrogen bond acceptor, was found to be occupied by the oxygen atom of the methoxy substituent.

In conclusion, the results revealed the critical importance of the amino acid side chain and its terminal hydrophobic ester for activity. The type of the amino acid side chain plays an important role for activity where the models suggest a hydrophobic side chain for optimal activity. Moreover, modeling results highlight the need for further investigations related to the importance of the naphthalene ring and the attached methoxy group. Current work is focusing on the replacement of the naphthalene ring by a smaller aromatic or heterocyclic ring which is attached through 3–4 carbons side chain to a hydrogen bond accepting moiety.

Experimental

Chemistry

General considerations

All the chemicals used were of commercially available reagent grade and were used without further purification. Melting Points were determined on Stuart melting point apparatus (Stuart Scientific, England) and were uncorrected. Pre-coated silica gel plates, 60G F254, obtained from Merck, Darmstadt (Germany) and were used for thin layer chromatography (TLC). Spots were visualized by using either UV-lamp at 254 nm or iodine. IR Spectra were recorded as KBr disk using Perkin Elmer FT–IR apparatus at the research center, College of Pharmacy, King Saud University, Saudi Arabia. The data are given in ύ (cm⁻¹). NMR Spectra: (A) Bruker NMR Spectrophotometer (500 MHz) at the research center, College of Pharmacy, King Saud University, Saudi Arabia, (B) Jeol NMR Spectrophotometer (400 MHz) at the research center, College of Science, King Saud University, Saudi Arabia. The chemical shifts are expressed as δ values (ppm) relative to tetramethylsilane (TMS) as internal standard. Signals are indicated by the following abbreviations: s = singlet, bs = broad singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, and m = multiplet. The J constant were given in (Hz). Mass spectra carried out using Agilent Triple Quadrupole 6410 QQQ LC/MS with ESI (Electrospray ionization) source. The purity of the synthesized final products (>98 %) was confirmed through the injection into HPLC system (Shimadzu pump LC-10AD, Japan) connected to a variable-wavelength detector (Shimadzu detector SPD-10A, Japan). An analytical reversed phase C18 column (Nucleosil 100/5 μM C18, 250 × 4.6 mm, Machery Nagel) was used which is equipped with a cartridge guard C18 column. Chromatographic separations were achieved using a mobile phase of acetonitrile and phosphate buffer (pH 4) at a flow rate of 1.8 ml/min.

General procedure for the synthesis of naproxen derivatives 3a–m

A stirred ice-cold solution of naproxen (0.806 g, 3.5 mmol) in dry dichloromethane, DCM, (30 ml) was treated with triethylamine, TEA, (0.353 g, 3.5 mmol). Ethylchloroformate (0.390 g, 3.5 mmol) was then added dropwise while stirring and the stirring was continued for further 30 min. Solution of appropriate amino acid ester (3.5 mmol) in dry DCM (10 ml) was then added gradually while stirring. The resulting mixture was further stirred at room temperature for 24 h, and the reaction was monitored by TLC using DCM:MeOH 95:5 v/v as a developing system. The solvent was removed under reduced pressure. The obtained mixture was purified by column chromatography using DCM:MeOH 95:5 v/v as a solvent. Pre-coated glass plates of silica gel 60G F254, 20 × 20 cm, 0.25 mm thick, Merck, were used for preparative chromatography when necessary.

Ethyl 2-(2-(6-methoxynaphthalen-2-yl)propanamido)acetate (3a)

Compound 3a was synthesized from the reaction of naproxen and ethyl glycinate. White powder (yield: 63 %); mp 84–86 °C. IR (KBr) ν_max/cm⁻¹ 3290 (NH), 1743 (C=O, ester), 1655 (C=O, amide); ¹H NMR, Jeol 400 MHz, (DMSO-d₆) δ 1.13 (t, 3H, J = 6.6 Hz, –CH₃ of ester), 1.41 (d, 3H, J = 7.32 Hz, –CH₃ of naproxen), 3. 79–3.82 (m, 3H, –CH of naproxen & –CH₂ of glycine), 3. 86 (s, 3H, –OCH₃ of naproxen), 4.04 (q, 2H, J = 7.36 Hz, –CH₂ of ester), 7.13–7.15 (m, 1H, ArH), 7.27 (d, 1H, J = 2.2 Hz, ArH), 7.44 (d, 1H, J = 8.04 Hz, ArH), 7.73–7.79 (m, 3H, ArH), 8.42 (t, 1H, NH, J = 5.88 Hz, D₂O-exchangeable); ¹³C NMR δ 14.56 (–CH₃ of ester), 19.07 (–CH₃ of naproxen), 41.0 (–CH₂ of glycine), 45.0 (–CH of naproxen), 55.7 (–OCH₃ of naproxen), 61.0 (–CH₂ of ester), 106.22, 119.11, 125,95, 127.09, 128.91, 129.64, 133.71, 137.57, 157.8, 170.40 (–C=O amide), 174.51 (–C=O ester); MS m/z (%) 315.6 (M⁺).

Ethyl 3-(2-(6-methoxynaphthalen-2-yl)propanamido)propanoate (3b)

Compound 3b was synthesized from the reaction of naproxen and β-alanine ethyl ester. White crystals(yield: 59 %); mp 83–84 °C. IR (KBr) ν_max/cm⁻¹ 3310 (NH), 1728 (C=O, ester), 1648 (C=O, amide); ¹H NMR, Jeol 400 MHz, (DMSO-d₆) δ 1.09 (t, 3H, J = 6. 72 Hz, –CH₃ of ester), 1.41 (d, 3H, J = 7.32 Hz, –CH₃ of naproxen), 2.39–2.42 (m, 2H, –CH₂–CO of β-alanine), 3. 23–3.28 (m, 2H, –CH₂–NH of β-alanine), 3.69 (q, 1H, J = 7.12 Hz, –CH of naproxen), 3.86 (s, 3H, –OCH₃ of naproxen), 3.97 (q, 2H, J = 7.32 Hz, –CH₂ of ester), 7.13 (dd, J = 9.16, 2.44 Hz, 1H, ArH), 7.26 (d, 1H, J = 1.84 Hz, ArH), 7.41 (d, 1H, J = 7.96 Hz, ArH), 7.69–7.78 (m, 3H, ArH), 8.08 (t, 1H, NH, J = 5.52 Hz, D₂O-exchangeable); ¹³C NMR δ 14.54 (–CH₃ of ester), 19.03 (–CH₃ of naproxen), 34.30 (–CH₂–CO of β-alanine), 35.42 (–CH₂–NH of β-alanine), 45.44 (–CH of naproxen), 55.69 (–OCH₃ of naproxen), 61.41 (–CH₂ of ester), 106.21, 119.10, 125.78, 126.96, 127.11, 128.92, 129.63, 133.66, 137.89, 157.52, 171.82 (–C=O amide), 174.01 (–C=O ester); MS m/z (%) 329.6 (M⁺).

(S)-Methyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)propanoate (3c)

Compound 3c was synthesized from the reaction of naproxen and L-alanine methyl ester. White powder (yield: 64 %); mp 98–99 °C. IR (KBr) ν_max/cm⁻¹ 3367 (NH), 1725 (C=O, ester), 1664 (C=O, amide); ¹H NMR, Bruker 500 MHz, (DMSO-d₆) δ 1.29 (d, 3H, J = 7.0 Hz, –CH₃ of alanine), 1.4 (d, 3H, J = 7.0 Hz, –CH₃ of naproxen), 3.53 (s, 3H, –OCH₃ of ester), 3.81 (q, 1H, J = 7.0 Hz, –CH of naproxen), 3.87 (s, 3H, –OCH₃ of naproxen), 4.3 (q, 1H, J = 7.0 Hz, –CH of alanine), 7.14–7.16 (m, 1H, ArH), 7.28 (s, 1H, ArH), 7.45 (d, 1H, J = 7.5 Hz, ArH), 7.71–7.79 (m, 3H, ArH), 8.45 (d, 1H, NH, J = 6.5 Hz, D₂O-exchangeable); ¹³C NMR δ 17.52 (–CH₃ of alanine), 19.15 (–CH₃ of naproxen), 44.59 (–CH of naproxen), 48.17 (–CH of alanine) 52.24 (–CH₃ of ester), 55.7 (–OCH₃ of naproxen), 106.24, 119.07, 125.86, 127.01, 127.15, 128.91, 129.64, 133.65, 137.61, 157.53, 173.58 (–C=O amide), 173.83 (–C=O ester); MS m/z (%) 315.4 (M⁺).

(R)-Methyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)propanoate (3d)

Compound 3d was synthesized from the reaction of naproxen and D-alanine methyl ester. White powder (yield: 66 %); mp 134–135 °C. IR (KBr) ν_max/cm⁻¹ 3332 (NH), 1735 (C=O, ester), 1663 (C=O, amide); ¹H NMR, Brucker 500 MHz, (DMSO-d₆) δ 1.23 (d, 3H, J = 7.5 Hz, –CH₃ of alanine), 1.4 (d, 3H, J = 6.5 Hz, –C H₃ of naproxen), 3.64 (s, 3H, –OCH₃ of ester), 3.8 (q, 1H, J = 7. 0 Hz, –CH of naproxen), 3.87 (s, 3H, –OCH₃ of naproxen), 4. 22 (q, 1H, J = 7.0 Hz, –CH of alanine), 7.13–7.16 (m, 1H, ArH), 7.27 (s, 1H, ArH), 7.45 (m, 1H, ArH), 7.72–7.79 (m, 3H, ArH), 8.46 (d, 1H, NH, J = 7.0 Hz, D₂O-exchangeable); ¹³C NMR δ 17.42 (–CH₃ of alanine), 19.1 (–CH₃ of naproxen), 45.06 (–CH of naproxen), 48.22 (–CH of alanine), 52.38 (–CH₃ of ester), 55.7 (–OCH₃ of naproxen), 106. 22, 119.13, 125.85, 127.03, 127.13, 128.9, 129.66, 133.69, 137.62, 157.55, 173.78 (–C=O amide), 174.03 (–C=O ester); MS m/z (%) 315.5 (M⁺).

Ethyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)propanoate (3e)

Compound 3e was synthesized from the reaction of naproxen and DL-alanine ethyl ester. White powder (yield: 59 %); mp 121–123 °C; the ratio of L/D: 50/50; IR (KBr) ν_max/cm⁻¹ 3331 (NH), 1745, 1708 (C=O, ester), 1674 (C=O, amide); ¹H NMR, Bruker 500 MHz, (DMSO-d₆) δ 1. 02 (t, 1.5H, J = 7.0 Hz, –CH₃ of D-alanine ester), 1.18 (t, 1. 5H, J = 7.0 Hz, –CH₃ of L-alanine ester), 1.23 (d, 1.5H, J = 7.5 Hz, –CH₃ of D-alanine), 1.28 (d, 1.5H, J = 7.5 Hz, –CH₃ of L-alanine), 1.41 (m, 3H, –CH₃ of naproxen), 3.8 (m, 1H, –CH of naproxen), 3.87 (s, 3H, –OCH₃ of naproxen), 3.97 (q, 1H, J = 7.0 Hz, –CH₂ of D-alanine ester), 4.09 (q, 1H, J = 7.5 Hz, –CH₂ of L-alanine ester), 4.18 (q, 0.5H, J = 7.5 Hz, –CH of D-alanine), 4.28 (q, 0.5H, J = 7.0 Hz, –CH of L-alanine), 7.15 (m, 1H, ArH), 7.28 (s, 1H, ArH), 7.45 (m, 1H, ArH), 7.72–7.79 (m, 3H, ArH), 8.45 (m, 1H, NH, Hz, D₂O-exchangeable); ¹³C NMR δ 13.90 (–CH₃ of L-alanine ester), 16.92 (–CH₃ of D-alanine ester), 18.56 (–CH₃ of D-alanine), 18.69 (–CH₃ of L-alanine), 18.95 (–CH₃ of naproxen), 44.11 (–CH of naproxen), 44.36 (–CH of naproxen), 47.58 (–CH of L-alanine), 51.65 (–CH of D-alanine), 55.1 (–OCH₃ of naproxen), 57.37 (–CH₂ of D-alanine ester), 60.14 (–CH₂ of L-alanine ester), 105.63, 118.48, 125. 27, 126.43, 126.56, 128.31, 129.05, 133.6, 137.02, 156.94, 171.38 (–C=O D-amide), 173.0 (–C=O L-amide), 173.25 (–C=O D-ester), 173.77 (–C=O L-ester); MS m/z (%) 329.4 (M⁺).

Methyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)butanoate (3f)

Compound 3f was synthesized from the reaction of naproxen and methyl DL-α-aminobutyrate. White powder (yield: 70 %); mp 97–98 °C; the ratio of L/D: 50/50. IR (KBr) ν_max/cm⁻¹ 3381 (NH), 1748 (C=O, ester), 1686 (C=O, amide); ¹H NMR, Bruker 500 MHz, (DMSO-d₆) δ 0. 75 (t, 1.5H, J = 6.5 Hz, –CH₃ of D-butyrate), 0.89 (t, 1.5H, J = 6.5 Hz, –CH₃ of L-butyrate), 1.40 (d, 3H, J = 7.0 Hz, –CH₃ of naproxen), 1.55–1.57 (m, 2H, –CH₂Me of butyrate), 3.53 (s, 1.5H, –OCH₃ of D-butyrate ester), 3.65 (s, 1. 5H, –OCH₃ of L-butyrate ester), 3.86–3.87 (m, 4H, –CH and –OCH₃ of naproxen), 4.1–4.25 (m, 1H, –CH of butyrate), 7.13–7.15 (m, 1H, ArH), 7.28 (s, 1H, ArH), 7.41–7.44 (m, 1H, ArH), 7.72–7.77 (m, 3H, ArH), 8.4 (d, 1H, NH, J = 7.5 Hz, D₂O-exchangeable); ¹³C NMR δ 13.63 (–CH₃ of butyrate), 18.49 & 20.2 (–CH₃ of naproxen), 35.46 (–CH₂ of butyrate), 44.48 (–CH of naproxen), 48.55 (–CHCO of butyrate), 51.61 & 52.03 (–OCH₃ of ester), 55.11 (–OCH₃ of naproxen), 105.64, 118.52, 125.31, 126.5, 128.31, 129.06, 133.09, 136.84, 156.97, 170.46 (–C=O amide), 171.06 (–C=O amide), 173.4 (2 –C=O ester); MS m/z (%) 329.4 (M⁺).

(S)-Ethyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)-3-methylbutanoate (3g)

Compound 3g was synthesized from the reaction of naproxen and L-valine ethyl ester. White powder (yield: 60 %); mp 98 °C. IR (KBr) ν_max/cm⁻¹ 3348 (NH), 1719 (C=O, ester), 1676 (C=O, amide); ¹H NMR, Jeol 400 MHz, (DMSO-d₆) δ 0.87–0.92 (m, 6H, 2 –CH₃ of valine), 1.04 (t, 3H, J = 7.32 Hz, –CH₃ of ester), 1.41 (d, 3H, J = 7.32 Hz, –CH₃ of naproxen), 2. 03–2.06 (m, 1H, –CH(Me)₂ of valine), 3.86 (s, 3H, –OCH₃ of naproxen), 3.9–4.05 (m, 3H, –OCH₂ of ester and –CH of naproxen), 4.16–4.2 (m, 1H, –CHCO of valine), 7.13–7.15 (m, 1H, ArH), 7.27 (s, 1H, ArH), 7.45–7.47 (m, 1H, ArH), 7.72–7.78 (m, 3H, ArH), 8.3 (d, 1H, NH, J = 8.56 Hz, D₂O-exchangeable); ¹³C NMR δ 14.94 (–CH₃ of ester), 18. 85 (2 –CH₃ of valine), 19.29 (–CH₃ of naproxen), 19.54 (2 –CH of naproxen), 30.51 (–CH₃ of valine), 44.71 (–CH(Me)₂ of valine), 51.79 (–CH₃ of ester), 55.69 (–OCH₃ of naproxen), 57.96 (–CHCO of valine), 60.72 (–CH₂ of ester), 106.22, 119.06, 125.83, 126.97, 127.17, 128.89, 129.62, 133. 65, 137.64, 157.52, 171.96 (–C=O amide), 174.35 (–C=O ester); MS m/z (%) 357.4 (M⁺).

(R)-Methyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)-3-methylbutanoate (3h)

Compound 3h was synthesized from the reaction of naproxen and D-valine methyl ester. White powder (yield: 58 %); mp 73–75 °C. IR (KBr) ν_max/cm⁻¹ 3381 (NH), 1745 (C=O, ester), 1655 (C=O, amide); ¹H NMR, Bruker 500 MHz, (DMSO-d₆) δ 0.71 (d, 3H, J = 6.5 Hz, –CH₃ of valine), 0.76 (d, 3H, J = 6.5 Hz, –CH₃ of valine), 1.40 (d, 3H, J = 6.5 Hz, –CH₃ of naproxen), 1.95–2.04 (m, 1H, –CH(Me)₂ of valine), 3.67 (s, 3H, –OCH₃ of ester), 3.86 (s, 3H, –OCH₃ of naproxen), 3.96 (q, 1H, J = 6.5 Hz, –CH of naproxen), 4.16 (d, 1H, J = 6.5 Hz, –CHCO of valine), 7.13–7.15 (m, 1H, ArH), 7.27 (s, 1H, ArH), 7.48–7.5 (m, 1H, ArH), 7.74–7.78 (m, 3H, ArH), 8.34 (d, 1H, NH, J = 8.0 Hz, D₂O-exchangeable); ¹³C NMR δ 16.92 (2 –CH₃ of valine), 19.15 (–CH₃ of naproxen), 44.36 (–CH of naproxen), 47.58 (–CH(Me)₂ of valine), 51.65 (–CHCO of valine), 51.79 (–CH₃ of ester), 55.1 (–OCH₃ of naproxen), 105.63, 118.49, 125.27, 126.43, 126.56, 128.32, 129.06, 133.06, 137.02, 156.94, 173.0 (–C=O amide), 173.26 (–C=O ester); MS m/z (%) 343.4 (M⁺).

(S)-Methyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)-4-methylpentanoate (3i)

Compound 3i was synthesized from the reaction of naproxen and L-leucine methyl ester. White powder (yield: 60 %); mp 96–98 °C. IR (KBr) ν_max/cm⁻¹ 3325 (NH), 1734 (C=O, ester), 1670 (C=O, amide); ¹H NMR, Jeol 400 MHz, (DMSO-d₆) δ 0.85 (d, 3H, J = 6.12 Hz, –CH₃ of leucine), 0.89 (d, 3H, J = 6.72 Hz, –CH₃ of leucine), 1.12–1.16 (m, 1H, –CH(Me)₂ of leucine), 1.39 (d, 3H, J = 6.72 Hz, –CH₃ of naproxen), 1.39–1.7 (m, 2H, –CH₂ of leucine), 3.52 (s, 3H, –OCH₃ of ester), 3.81–3. 86 (m, 4H, –CH & –OCH₃ of naproxen), 4.3–4.32 (m, 1H, –CH of leucine), 7.13–7.15 (m, 1H, ArH), 7.28 (s, 1H, ArH), 7.44 (d, 1H, J = 8.56 Hz, ArH), 7.70–7.79 (m, 3H, ArH), 8. 40 (d, 1H, NH, J = 7.8 Hz, D₂O-exchangeable); ¹³C NMR δ 19.26 (–CH₃ of naproxen), 21.86 & 23.3 (2 –CH₃ of leucine), 24.87 (–CH(Me)₂), 45.01 (–CH of naproxen), 50.86 (–CHCO of leucine), 52.22 (–CH₃ of ester), 55.69 (–OCH₃ of naproxen), 106.22, 119.07, 125.86, 127.0, 128.89, 129.63, 133.65, 137.55, 157.53, 173.49 (–C=O amide), 174.13 (–C=O ester); MS m/z (%) 357.3 (M⁺).

(S)-Ethyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)-4-methylpentanoate (3j)

Compound 3j was synthesized from the reaction of naproxen and L-leucine ethyl ester. White powder (yield: 54 %); mp 97–99 °C. IR (KBr) ν_max/cm⁻¹ 3375 (NH), 1717 (C=O, ester), 1669 (C=O, amide); ¹H NMR, Bruker 500 MHz, (DMSO-d₆) δ 0.88 (d, 3H, J = 7.0 Hz, –CH₃ of leucine), 0.91 (d, 3H, J = 6. 5 Hz, –CH₃ of leucine), 1.02 (t, 3H, J = 6.5 Hz, –CH₃ of ester), 1.16–1.18 (m, 1H, –CH(Me)₂ of leucine), 1.40 (d, 3H, J = 6.5 Hz, –CH₃ of naproxen), 1.55–1.66 (m, 2H, –CH₂ of leucine), 3.82–3.87 (m, 4H, –CH and –OCH₃ of naproxen), 3.98 (q, 2H, J = 6.5 Hz, –CH₂ of ester), 4.29–4.32 (m, 1H, –CH of leucine), 7.14–7.16 (m, 1H, ArH), 7.28 (s, 1H, ArH), 7.44 (d, 1H, J = 8.0 Hz, ArH), 7.71–7.78 (m, 3H, ArH), 8.39 (d, 1H, NH, J = 7.5 Hz, D₂O-exchangeable); ¹³C NMR δ 13.79 (– CH₃ of ester), 18.4 (–CH₃ of naproxen), 21.0 (2 –CH₃ of leucine), 24.87 (–CH(Me)₂), 44.45 (–CH of naproxen), 53.53 (–CHCO of leucine), 55.10 (–OCH₃ of naproxen), 60.36 (–CH₂ of ester), 105.62, 118.49, 125.3, 126.44, 126.5, 128.1, 128.28, 129.04, 129. 11, 133.09, 156.94, 171.36 (–C=O amide), 173.35 (–C=O ester); MS m/z (%) 371.4 (M⁺).

(S)-Dimethyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)succinate (3k)

Compound 3k was synthesized from the reaction of naproxen and L-aspartic acid dimethyl ester. White powder (yield: 75 %); mp 110–111 °C. IR (KBr) ν_max/cm⁻¹ 3354 (NH), 1744 (C=O, ester), 1648 (C=O, amide); ¹H NMR, Bruker 500 MHz, (DMSO-d₆) δ 1.39 (d, 3H, J = 7.0 Hz, –CH₃ of naproxen), 2.69–2.78 (m, 1H, H_a of –CH₂ aspartic), 2.79–2.86 (m, 1H, H_b of –CH₂ aspartic), 3.54 (s, 3H, –OCH₃ of ester), 3.57 (s, 3H, –OCH₃ of ester), 3.79–3.80 (m, 1H, –CH of naproxen), 3.87 (s, 3H, –OCH₃ of naproxen), 4.64–4.65 (m, 1H, –CH of aspartic), 7.15 (d, 1H, J = 8.5 Hz, ArH), 7.29 (s, 1H, ArH), 7.42 (d, 1H, J = 8.5 Hz, ArH), 7.70–7.79 (m, 3H, ArH), 8.52 (d, 1H, NH, J = 7.5 Hz, D₂O-exchangeable); ¹³C NMR δ 18.52 (–CH₃ of naproxen), 35.46 (–CH₂ of aspartic), 44.47 (–CH of naproxen), 48.54 (–CHCO of aspartic), 51.61 (–CH₃ of ester), 52.03 (–CH₃ of ester), 55.10 (–OCH₃ of naproxen), 105.62, 118.53, 125.31, 126.49, 128.31, 129.06, 133.09, 136.85, 156.96, 169.46 (–C=O amide), 171.07 (–C=O ester), 173.37 (–C=O ester); MS m/z (%) 373.3 (M⁺).

(S)-Ethyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)-4-(methylthio)butanoate (3l)

Compound 3l was synthesized from the reaction of naproxen and L-methionine ethyl ester. White powder (yield: 77 %); mp 85–86 °C. IR (KBr) ν_max/cm⁻¹ 3306 (NH), 1737 (C=O, ester), 1646 (C=O, amide); ¹H NMR, Jeol 400 MHz, (DMSO-d₆) δ 1.03 (t, 3H, J = 6.72 Hz, –CH₃ of ester), 1. 40 (d, 3H, J = 7.32 Hz, –CH₃ of naproxen), 1.88–1.95 (m, 2H, –CH₂–CH₂–S– of methionine), 2.03 (s, 1H, –S–CH₃ of methionine), 2.45–2.49 (m, 2H, –CH₂–CH₂–S– of methionine), 3.81–3.86 (m, 4H, –CH and –OCH₃ of naproxen), 3.98 (q, 2H, J = 6.96 Hz, –CH₂ of ester), 4.34–4.36 (m, 1H, –CH of methionine), 7.13–7.15 (m, 1H, ArH), 7.28 (s, 1H, ArH), 7.44 (d, 1H, J = 8.56 Hz, ArH), 7.71–7.78 (m, 3H, ArH), 8.42 (d, 1H, NH, J = 7.92 Hz, D₂O-exchangeable); ¹³C NMR δ 14.43 (–CH₃ of ester), 15.14 (–SCH₃), 19.14 (–CH₃ of naproxen), 30.15 (–S–CH₂), 31.04 (–S–CH₂–CH₂), 45.08 (–CH of naproxen), 51.66 (–CHCO of methionine), 55.69 (–OCH₃ of naproxen), 60.95 (–CH₂ of ester), 106.23, 119.08, 125.87, 127.03, 127.11, 128.9, 129. 62, 133.67, 137.56, 157.53, 172.22 (–C=O amide), 174.18 (–C=O ester); MS m/z (%) 389.3 (M⁺).

(S)-Ethyl 2-((S)-2-(6-methoxynaphthalen-2-yl)propanamido)-3-phenylpropanoate (3m)

Compound 3m was synthesized from the reaction of naproxen and L-phenylalanine ethyl ester. White powder (yield: 76 %); mp 93–95 °C. IR (KBr) ν_max/cm⁻¹ 3346 (NH), 1727 (C=O, ester), 1643 (C=O, amide); ¹H NMR, Jeol 400 MHz, (DMSO-d₆) δ 1.0 (t, 3H, J = 7.32 Hz, –CH₃ of ester), 1.3 (d, 3H, J = 6.72 Hz, –CH₃ of naproxen), 2.85–2.93 (m, 1H, H_a of –CH₂–Ph phenylalanine), 2.98–3.02 (m, 1H, H_b of –CH₂–Ph phenylalanine), 3.78 (q, 1H, J = 6.72 Hz, –CH of naproxen), 3.86 (s, 3H, –OCH₃ of naproxen), 3.96 (q, 2H, J = 7.32 Hz, –CH₂ of ester), 4.42–4.47 (m, 1H, –CH of phenylalanine), 7.13–7.41 (m, 8H, ArH), 7.68–7.77 (m, 3H, ArH), 8.47 (d, 1H, NH, J = 7.96 Hz, D₂O-exchangeable); ¹³C NMR δ 14. 4 (–CH₃ of ester), 19.02 (–CH₃ of naproxen), 37.19 (–CH₂–Ph), 45.04 (–CH of naproxen), 54.12 (–CHCO of phenylalanine), 55.69 (–OCH₃ of naproxen), 60.93 (–CH₂ of ester), 106.22, 119.07, 127.03, 127.1, 128.69, 128.76, 129. 63, 129.71, 137.46, 137.85, 157.53, 171.96 (–C=O amide), 173.89 (–C=O ester); MS m/z (%) 405.3 (M⁺).

Biology

Cell culture

The human HT-29, SW480, and HCT116 colon tumor cell lines were obtained from ATCC and grown under standard cell culture conditions in RPMI 1640 medium containing 10 % fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5 % CO₂. The human NCM460 normal colonocyte cell line was obtained from INCELL Corporation and grown in INCELL’s enriched M3:10 medium (Cat # M310A500); which is M3 medium plus supplements and 10 % [v/v] fetal bovine serum [FBS], and contains antibiotics. The cells were grown following the manufacture’s protocol at 37 °C in a humidified atmosphere with 5 % CO₂.

Growth assays

Tissue culture treated 96-well microtiter plates were seeded at a density of 5,000 cells/well. For growth inhibition assays, cells were incubated at 37 °C for 18–24 h, treated with the specified compound or vehicle control (0.1 % DMSO final) and incubated at 37 °C for an additional 72 h. Treatment effects on cell growth were determined using the luminescent Cell Titer Glo Assay (Promega) that measures ATP levels as an indicator of viable cell number according to the manufacturer’s specifications.

Apoptosis assay

For measuring apoptosis induction, 96-well microtiter plates were seeded at a density of 10,000 cells per well. Cells were treated with the specified compound or vehicle control for 24 h. The Luminescent Caspase-Glo 3/7 Assay (Promega) was performed together with the MultiTox-Fluor assay (Promega), according to the manufacturer’s instructions. The live cell signal from the MultiTox-Fluor assay was first done to normalize for cell number and then caspase 3/7 activity was measured.

Cyclooxygenase inhibitory assays

COX-1 and COX-2 activities were measured using purified ovine COX-1 and recombinant human COX-2 enzymes in the COX Inhibitor Screening Assay Kit from Cayman Chemical Company (Ann Arbor, MI). The assay was performed according to the manufacturer’s recommendations.

Molecular modeling studies

Pharmacophore generation

All experiments were conducted on an Intel Pentium dual-core processor workstation with 2 MB cache memory and 4 GB RAM. Hypotheses generation were applied against previously described data sets, and their functionality is available as part of Accelrys Discovery Studio 2.5 (DS) software. Molecules were edited using the DS 3D visualizer where conformational models for each compound were generated using the Poling Algorithm. The “best conformational search” option was used, specifying 250 as the maximum number of conformers. The models emphasized a conformational diversity under the constraint of 20 kcal/mol energy threshold above the estimated global minimum based on use of the CHARMm force field. The molecules associated with their conformational models were submitted to HipHop hypothesis generation. Misses, the number of molecules which do not have to map to all features in generated hypotheses; FeatureMisses, the number of maximal molecules which do not have to map to each feature in generated hypotheses and CompleteMisses; the number of molecules which do not have to map to any feature in a given hypothesis, were set as 3, 2, and 2, respectively. A “Principal” value of 2 and MaxOmit features of 0 have been assigned to all training set molecules with an inter-feature value of not less than 3.0 Å.

Molecular docking

Molecular docking of the synthesized compounds into COX-2 enzyme was carried out using the AutoDock 4.2 software package (Radwan et al., 2012). The graphical user interface AutoDockTools (ADT 1.5.4) was employed to setup the enzymes: all hydrogens were added, Gasteiger charges were calculated, and non-polar hydrogens were merged to carbon atoms. The 3D structures of ligand molecules were built, optimized (PM3) level, and saved in pdb format with the aid of Chem3D Ultra 8.0 implemented in ChemOffice Ultra 8.0 package (Chem Draw Ultra 8.0, 2004). In all docking experiments, a grid box size of 50 × 50 × 50 points in x, y, and z directions was built, the box was centered on the ligand naproxen within the binding pocket of the protein. A grid spacing of 0.375 Å and a distances-dependent function of the dielectric constant were used for the calculation of the energetic map. One hundred runs were generated by using Lamarckian genetic algorithm searches, and the most favorable free energy of binding was selected as the resultant complex structures. Molecular operating environment (MOE 2008.10) was used to optimize and visualize the produced complexes through their relaxation under MMFF94x forcefield.