Design of New Benzo[h]chromene Derivatives: Antitumor Activities and Structure-Activity Relationships of the 2,3-Positions and Fused Rings at the 2,3-Positions

A series of novel 4H-benzo[h]chromenes 4, 6–11, 13, 14; 7H-benzo[h]chromeno[2,3-d]pyrimidines 15–18, 20, and 14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives 19a–e, 24 was prepared. The structures of the synthesized compounds were characterized on the basis of their spectral data. Some of the target compounds were examined for their antiproliferative activity against three cell lines; breast carcinoma (MCF-7), human colon carcinoma (HCT-116) and hepatocellular carcinoma (HepG-2). The cytotoxic behavior has been tested using MTT assay and the inhibitory activity was referenced to three standard anticancer drugs: vinblastine, colchicine and doxorubicin. The bioassays demonstrated that some of the new compounds exerted remarkable inhibitory effects as compared to the standard drugs on the growth of the three tested human tumor cell lines. The structure–activity relationships (SAR) study highlights that the antitumor activity of the target compounds was significantly affected by the lipophilicity of the substituent at 2- or 3- and fused rings at the 2,3-positions.


Introduction
In cancer research, multidrug resistance (MDR) is one of the major aspects that causes failure in therapeutic treatment. This phenomenon could occur either via inherited or acquired approaches, which refers to the initial resistance to a specific drug and the development of resistance after successful treatment, respectively. There have been numerous attempts to overcome these obstacles, including applying drug treatment in combination protocols. In the meantime, the development of new materials for drug design continues to be crucial in addressing this phenomenon.

Chemistry
The 4H-benzo[h]chromene derivatives 4 and 6 described in this study were prepared according to the methodology illustrated in Scheme 1. The reaction proceeded via a one-pot three component condensation of 4-methoxy-1-naphthol (1), 4-methoxybenzaldehyde (2) and malononitrile (3) or ethyl cyanoacetate (5) in an ethanolic piperidine-containing solution under microwave irradiation for 2 min at 140 • C to afford the target compounds 2-amino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo [h]chromene-3-carbonitrile (4) and ethyl 2-amino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo-[h] chromene-3-carboxylate (6), respectively. The optical activities of the target compounds 4 and 6 were measured using a Carl Zeiss polarimeter. The results indicated that compounds 4 and 6 have zero rotation (i.e., they are optically inactive) and thus are in the form of a racemic (±) mixture as illustrated in Scheme 1. target compounds were examined for their antitumor activities in comparison to the standard drugs vinblastine, colchicine and doxorubicin. The structure-activity relationships of the desired molecules highlighted the effect of the substituents at the 2,3-positions and fused rings at the 2,3-positions on the antitumor activity.
From the obtained results, it was elucidated that most of the synthesized compounds displayed excellent to modest growth inhibitory activity against the tested cancer cell lines. The investigations indicated that HepG-2 was the cell line most sensitive to the influence of the new derivatives (Table  1). Compounds 17, 18, 8a, 11 and 9 were found to be the most potent derivatives against MCF-7 cancer cells, as they were 6.8, 5.6, 5.1, 1.5, 1.3 and 19.7, 16, 1, 14.8, 4.3, 3.6 times more active than vinblastine and colchicine, respectively (Table 1), while compounds 7, 20, 8b, 15, 14 and 16 displayed good activity against the MCF-7 cancer cell as they were 2.3, 1.8, 1.7, 1.5, 1.3 and 1.0 times more active than colchicine. Besides, compounds 18, 17 and 8a were more potent and efficacious against HCT-116 cancer cells, as they were 3.3, 2.9 and 2.2 times more active than vinblastine and colchicine,

SAR Studies
The corresponding partition coefficients (log P), which are known as an index of lipophilicity, were calculated using ACD/Labs log P ver. 14.02, are listed in Table 1. The preliminary structureactivity relationships (SAR) study focused on the effects of the replacement at 2,3-positions or 2,3positions of the fused rings on the antitumor activities of the synthesized compounds. The study includes a comparison of the cytotoxic activities of compounds 4, 6 and their analogues against the MCF-7, HCT-116 and HepG-2 cell lines. For instance, the SAR study of compound 4 and its analogues has confirmed that the more potent and efficacious activity than vinblastine and colchicine of compounds 8a, 11 and 9 against the MCF-7 cancer cells and the good activity of compounds 7 and 8b against MCF-7 cancer cells as compared to compound 10 and colchicine was attributable mainly to the presence of the -NHAc, -N=CHNH2, -N=CHOEt, -N=CHPh and -NAc2 moieties at the 2-position and some hydrophobic groups are preferred over others at this 2-position, as indicated by the increasing values of Log P shown in Table 1. Besides, blocking the 2-(-NH2) group of compound 4 with other hydrophobic moieties, for example with a 2-(-N=CHNMe2) as in compound 10, resulted in the reduction of the potency. Replacement of the 3-cyano with a 3-ester (a hydrophobic group) caused a reduction of the potency of compound 6 as compared to compound 4, while blocking the 2-(-NH2) group of compound 6 with hydrophilic or hydrophobic groups such as (-NHCHO or N=CHNMe2) reduced the potency of compound 13 and improved the potency of compound 14 against MCF-7 cancer cells as compared to colchicine. Incorporating a pyrimidine ring at the 2,3postions of compound 4 with hydrophobic groups (=NH-8, -Me-9) for compound 17 and (=NH-8, -NH2-9) for compound 18 resulted in a strong improvement of potency against MCF-7 cancer cells as compared to vinblastine and colchicine, while the presence of a hydrophilic 8-(-C=O) group for compound 15 or a hydrophobic group 8-(-NH2) for compound 16 barely reduces their potency while more reduction of potency is observed with hydrophobic groups (=NH-8, -N=CHPh-9) for compound 20 against the MCF-7 cancer cell as compared to colchicine. This behavior suggests that the antitumor activity is significantly affected by the lipophilicity as indicated by a decreasing value of log P as shown in Table 1 and hydrophobic groups are more beneficial than hydrophilic groups, as well as the 7H-benzo[h]-chromeno [2,3-d]pyrimidine nucleus is more valuable than the 4H-benzo[h]chromene nucleus.
Further investigation of the impact of the substitution pattern at the previous positions of the synthesized compounds on the antitumor activities was then conducted. Compound 8a, bearing a hydrophobic substituent (-NHAc) at the 2-position, exhibited an increase in the activity against HCT-116 cancer cells compared to vinblastine and colchicine, whereas compounds 7, 9, 8b, 11, 10 and 4 with hydrophobic substituents (-N=CHPh, -N=CHOEt, -NAc2, -N=CHNH2, -N=CHNMe2 and NH2) at the 2-position displayed a remarkable enhancement in the antitumor activity against HCT-116 cancer

SAR Studies
The corresponding partition coefficients (log P), which are known as an index of lipophilicity, were calculated using ACD/Labs log P ver. 14.02, are listed in Table 1. The preliminary structure-activity relationships (SAR) study focused on the effects of the replacement at 2,3-positions or 2,3-positions of the fused rings on the antitumor activities of the synthesized compounds. The study includes a comparison of the cytotoxic activities of compounds 4, 6 and their analogues against the MCF-7, HCT-116 and HepG-2 cell lines. For instance, the SAR study of compound 4 and its analogues has confirmed that the more potent and efficacious activity than vinblastine and colchicine of compounds 8a, 11 and 9 against the MCF-7 cancer cells and the good activity of compounds 7 and 8b against MCF-7 cancer cells as compared to compound 10 and colchicine was attributable mainly to the presence of the -NHAc, -N=CHNH 2 , -N=CHOEt, -N=CHPh and -NAc 2 moieties at the 2-position and some hydrophobic groups are preferred over others at this 2-position, as indicated by the increasing values of Log P shown in Table 1. Besides, blocking the 2-(-NH 2 ) group of compound 4 with other hydrophobic moieties, for example with a 2-(-N=CHNMe 2 ) as in compound 10, resulted in the reduction of the potency. Replacement of the 3-cyano with a 3-ester (a hydrophobic group) caused a reduction of the potency of compound 6 as compared to compound 4, while blocking the 2-(-NH 2 ) group of compound 6 with hydrophilic or hydrophobic groups such as (-NHCHO or N=CHNMe 2 ) reduced the potency of compound 13 and improved the potency of compound 14 against MCF-7 cancer cells as compared to colchicine. Incorporating a pyrimidine ring at the 2,3-postions of compound 4 with hydrophobic groups (=NH-8, -Me-9) for compound 17 and (=NH-8, -NH 2 -9) for compound 18 resulted in a strong improvement of potency against MCF-7 cancer cells as compared to vinblastine and colchicine, while the presence of a hydrophilic 8-(-C=O) group for compound 15 or a hydrophobic group 8-(-NH 2 ) for compound 16 barely reduces their potency while more reduction of potency is observed with hydrophobic groups (=NH-8, -N=CHPh-9) for compound 20 against the MCF-7 cancer cell as compared to colchicine. This behavior suggests that the antitumor activity is significantly affected by the lipophilicity as indicated by a decreasing value of log P as shown in Table 1 and hydrophobic groups are more beneficial than hydrophilic groups, as well as the 7H-benzo[h]-chromeno[2,3-d]pyrimidine nucleus is more valuable than the 4H-benzo[h]chromene nucleus.
Further investigation of the impact of the substitution pattern at the previous positions of the synthesized compounds on the antitumor activities was then conducted. Compound 8a, bearing a hydrophobic substituent (-NHAc) at the 2-position, exhibited an increase in the activity against HCT-116 cancer cells compared to vinblastine and colchicine, whereas compounds 7, 9, 8b, 11, 10 and 4 with hydrophobic substituents (-N=CHPh, -N=CHOEt, -NAc 2 , -N=CHNH 2 , -N=CHNMe 2 and NH 2 ) at the 2-position displayed a remarkable enhancement in the antitumor activity against HCT-116 cancer cells compared to colchicine. This behavior suggests that the substitution at 2-position could be tolerated and the incorporation of hydrophobic substituents is beneficial for increasing the value of Log P as shown in Table 1. Replacing the 3-cyano-with 3-ester (a hydrophobic group) resulted in a loss of the activity for compound 6 compared to compound 4. The blocking of the 2-(-NH 2 ) group in compound 6 with hydrophilic or hydrophobic groups such as 2(-NHCHO) or 2-(-N=CHNMe 2 ) caused a remarkable enhancement in the antitumor activity for compounds 13 and 14 against HCT-116 cancer cells compared to colchicine. The introduction of a pyrimidine ring at the 2,3-postions of compound 4 with a hydrophobic group 8-(=NH), 9-(-NH 2 ) for compound 18 and 8-(=NH), 9-(-Me) for compound 17 resulted in a remarkable enhancement of potency against HCT-116 cancer cells as compared to vinblastine and colchicine. In contrast, the presence of a hydrophilic 8-(-C=O) group in compound 15 resulted in a partial loss of the activity. The same behavior has been observed after incorporation of hydrophobic groups (-NH 2 -8; =NH-8, -Me-9; =NH-8, -N=CHPh-9) into compounds 16, 17 and 20, hinting that grafting a pyrimidine ring at the 2,3-postions with a lipophilic substituent (hydrophobic group) like imino/amino or imino/methyl groups is more beneficial than other lipophilic substituenta (hydrophilic or hydrophobic groups) like carbonyl, imino or benzylideneamino moieties for the activity by increasing the value of Log P as shown in Table 1.
Concerning the activity against HepG-2, compounds 7, 18, 17 and 8a,b were the most active analogs through this study with IC 50 values of 0.7 ± 0.11, 0.8 ± 0.08, 0.9 ± 0.11, 0.9 ± 0.1 and 3.9 ± 0.3, respectively, in comparison to the reference drugs vinblastine and colchicine (IC 50 = 4.6 ± 0.01 and 10.6 ± 0.04 µg/mL). Additionally, compounds 15, 11, 10 and 4 displayed good activity against HepG-2 in comparison to colchicine and the other compounds 9, 6, 14, 16, 13 and 20. These results imply that the introduction of a hydrophobic group (-N=CHPh or -NHAc) at the 2-position of the chromene nucleus and the incorporation of a pyrimidine nucleus at the 2,3-positions with a hydrophobic group (=NH-8, -NH 2 -9 or =NH-8, -Me-9) were indispensable for the activities against HepG-2 by decreasing the value of log P as shown in Table 1. In addition, compound 17 was found to be the most potent derivative against MCF-7 as compared to doxorubicin, as it was almost equipotent as doxorubicin, while compounds 18 and 17 were almost equipotent as doxorubicin against HCT-116. Besides, compounds 7 and 18 with IC 50 = 0.7 ± 0.11 and 0.8 ± 0.08 µg/mL displayed significant growth inhibitory activity against HepG-2 in comparison to doxorubicin, and compounds 17 and 8a were equipotent as doxorubicin, indicating that hydrophobic groups like benzylideneamino and acetylamino moieties at 2-postion is preferred for antitumor activity more than other hydrophobic groups with decreasing value of log P as shown in Table 1  Finally, we can deduce that the substitution pattern at the 2,3-positions or fused rings at the 2,3-positions on the synthesized 4H-chromene and pyrimidine moieties are crucial elements for the antitumor activity. The incorporation of pyrimidine rings at the 2,3-postions with groups (=NH-8, -NH 2 -9 and =NH-8, -Me-9) or (-N=CHPh and -NHAc) at the 2-postion of the chromene nucleus is favorable and greatly enriches the activity more than the other hydrophobic and hydrophilic groups tested.

General Information
Commercial-grade solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Melting points were measured with a Stuart Scientific (Stone, Staffordshire, UK) apparatus and are uncorrected. IR spectra were determined as KBr pellets on a FT/IR 460 plus spectrophotometer (Jasco, Tokoyo, Japan). 1 H-NMR (500 MHz) and 13 C-NMR spectra (125 MHz) were recorded using an AV 500 MHz spectrometer (Bruker, Billerica, MA, USA).
Chemical shifts (δ) are expressed in parts per million (ppm). The 1 H-NMR and 13 C-NMR spectra of the compounds are provided in the Supplementary Material. The MS were measured using a GC/MS-QP5050A spectrometer (Shimadzu, Tokoyo, Japan). The microwave synthesis was performed using a mono-mode Milestone Sr1 device (Milestone, Shelton, CT, USA) while mass spectra were determined on a Shimadzu GC/MS-QP5050A spectrometer. Elemental analyses were carried out at the Regional Centre for Mycology and Biotechnology (RCMP) at Al-Azhar University (Cairo, Egypt) and the results were within ±0.25% of the theoretical values. Analytical thin layer chromatography (TLC) on silica gel precoated F 254 (Merck, Billerica, MA, USA) plates was used to check the purity of the compounds. A mixture of imadate 9 (4.14 g, 0.01 mol) and dimethylamine (0.45 g, 0.01 mol) in methanol (30 mL), was stirred at room temperature for 1 h then left overnight to precipitate. The solid product was collected by filtration, washed with methanol and recrystallized from proper solvent to afford 10.

Cell Culture
The tumor cell lines breast adenocarcinoma (MCF-7), human colon carcinoma (HCT-116) and hepatocellular carcinoma (HepG-2) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/mL gentamycin. The cells were maintained at 37 • C in a humidified atmosphere with 5% CO 2 and were subculture two to three times a week.

Cytotoxicity Evaluation Using Viability Assay
The tumor cell lines were suspended in medium at concentration 5 × 10 4 cell/well in Corning 96-well tissue culture plates and then incubated for 24 h. The tested compounds with concentrations ranging from 0 to 50 µg/mL were then added into 96-well plates (six replicates) to achieve different conc. for each compound. Six vehicle controls with media or 0.5% DMSO were run for each 96 well plate as a control. After incubating for 24 h, the numbers of viable cells were determined by the MTT test. Briefly, the media was removed from the 96 well plates and replaced with 100 µL of fresh culture RPMI 1640 medium without phenol red then 10 µL of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of PBS) to each well including the untreated controls. The 96-well plates were then incubated at 37 • C and 5% CO 2 for 4 h. An 85-µL aliquot of the media was removed from the wells, and 50 µL of DMSO was added to each well and mixed thoroughly with the pipette and incubated at 37 • C for 10 min. Then, the optical density was measured at 590 nm with the microplate reader (Sunrise, TECAN, Inc., Morrisville, NC, USA) to determine the number of viable cells and the percentage of viability was calculated as [1 − (ODt/ODc)] × 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each conc. using GraphPad Prism software [43,44] (San Diego, CA, USA).

Conclusions
The synthesis of new compounds with potential applications as drug replacements is an area of high interest in literature in order to overcome the drug resistance issue.  5-c]pyrimidine derivatives have been synthesized, starting from 2-amino-4-(4methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile and ethyl 2-amino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carboxylate. The new molecules have been evaluated for their antitumor activities against three cancer cell lines: breast adenocarcinoma (MCF-7), human colon carcinoma (HCT-116) and hepatocellular carcinoma (HepG-2). This pharmacological study was also undertaken to evaluate the effects of the substituents and the pyrimidine rings on the antitumor activities. The obtained results have shown that most of these synthesized compounds exhibited good antitumor activities towards the tested cell lines. The SAR study along with the biological assay confirmed that the incorporation of pyrimidine rings at the 2,3-positions with 8-(=NH), 9-(-NH 2 ) and 8-(=NH), 9-(-Me) or (-N=CHPh and -NHAc) at the 2-position of the chromene nucleus enhances the activity more than other groups.