Design, Synthesis and Biological Evaluation of Novel 5H-Chromenopyridines as Potential Anti-Cancer Agents

A novel series of 5H-chromenopyridines was identified as anticancer agents in our continuing effort to discover and develop new small molecule anti-proliferative agents. Based on our initial lead SP-6-27 compound, we designed and synthesized novel tricyclic 5H-thiochromenopyridine and 5H-chromenopyridine analogs to evaluate the impact of an additional ring, as well as conformational flexibility on cytotoxic activity against human melanoma and glioma cell lines. All of the 5H-thiochromenopyridines have been achieved in good yields (89%–93%) using a single-step, three-component cyclization without the need for purification. The 5H-chromenopyridine analog of the potent 5H-thiochromenopyride was obtained in a good yield upon purification. All newly-prepared 5H-thiochromenopyridines showed good to moderate cytotoxicity against three melanoma and two glioma cell lines (3–15 μM). However, the 5H-chromenopyridine analogue that we prepared in our laboratory lost cytotoxic activity. The moderate cytotoxic activity of 5H-thiochromenopyridines shows the promise of developing chromenopyridines as potential anticancer agents.


Introduction
On a global scale, cancer is a major public health problem [1]. At present, one quarter of all deaths in the USA are caused by cancer [2]. A wide variety of anti-cancer agents has been described for the treatment of various kinds of cancers, including glioma and melanoma. However, many of these agents show a major failure in terms of drug resistance and toxicity. Hence, there is an emerging need for the discovery and development of novel anticancer agents to overcome drug resistance and toxicity issues.
Recently, chromene analogs have emerged as potent anticancer agents, and crolibulin is currently in a phase II clinical trial for anaplastic thyroid cancer with the National Cancer Institute (NCI) [3,4] (Figure 1).

Figure 1. Chromene-based anti-cancer agents.
Our recent anticancer screening program identified SP-6-27 as one of the potent anticancer agents in vitro [5]. The 60 National Cancer Institute Developmental Therapeutic preclinical cancer cell lines screen indicated that SP-6-27 has consistent anti-proliferative activity against nine major cancer cell lines and has been selected for further in vivo testing [6]. Although there are a number of articles demonstrating bicyclic chromenes as anti-cancer agents [4,5,[7][8][9][10][11][12][13], there are only a few reports on tricyclic chromenopyridines as anti-proliferative candidates [14,15]. Thus, the aim of the present study is to determine the potential of the novel tricyclic chromenopyridines as anticancer agents. In light of our recent success with SP-6-27, it would be interesting to envisage the biological activities of two different templates of tricyclic chromenopyridines, rigid 5H-substituted-chrominopyridines (Template A; Figure 2) and relatively flexible 5H-sustituted-thiochromenopyridines (Template B; Figure 2 Figure 2. Hypothesized chromenopyridines.
We thought that additional flexibility in Template B type molecules, conferred by the inserted sulfur atom, could play a crucial role in biological activity.
Briefly, starting with SP-6-27, we were interested in determining the effect of an additional ring (Template A-type molecules; Figure 2) and a combination of both an additional ring, as well as flexibility (Template B-type candidates; Figure 2) on cytotoxic activity on human melanoma and glioma cell lines. Here, we report the optimal structural requirements of the linear tricyclic chromenopyridines for biological activity.

Results and Discussion
The development of a single-step, multicomponent reaction strategy to prepare a small library of compounds was of considerable interest. Anderson et al. first reported the synthesis of 5H-substitutedchromenopyridines (Template A-type; Figure 2) utilizing two-step condensation of 3H-substituted-phenol with an arylmethylidene derivative of malonitrile and malonitrile, respectively [16]. In the following year, Melekhin et al. reported a single-step, two-component preparation of 5H-substituted-chromenopyridines (Template A-type; Figure 2) via condensation of resorcinol with an arylmethylidene derivative of the malonitrile dimer [17]. To the best of our knowledge, Evdokimov et al. are the first to demonstrate a single-step, three-component cyclization technique, which utilized salicylaldehyde, thiophenol and malonitrile, to prepare 5H-substituted-thiochromenopyridines (Template B-type; Figure 2) [18]. Recently, Ghomi et al. demonstrated tin oxide nanoparticles, as well as zirconium nanoparticle as efficient catalysts to promote four-component cyclization of salicylaldehyde, thiols and two equivalent malonitriles, resulting in 5H-substituted-thiochromenopyridines in a good yield [19,20]. Here, we report an efficient, as well as fast three-component cyclization reaction, which works both under normal reflux and microwave irradiation, to prepare 5H-substituted-thiochromenopyridines, as shown in Scheme 1. The synthetic procedure for the preparation of six 5H-substituted-thiochromenopyridines (1a-1f), consisting of the C 8 -methoxy substituent, is presented in Scheme 1.
Briefly, 4-methoxy-salicylaldehyde was subjected to a three-component cyclization reaction with 2-amino-1,1,3-propenetricarbonitrile (malonitrile dimer) and the aromatic thiol of interest, employing a catalytic amount of trimethylamine in ethanol under a refluxing condition. The desired thiochromenopyridines were obtained in great yields upon simple recrystallization, ruling out the need for a tedious purification process. At this point, we made an effort to prepare a few thiochromenopyridines consisting of C 8 -dimethylamino substituents utilizing 4-dimethylamino-salisaldehyde following the same synthetic method. Scheme 2 demonstrates our effort to prepare C 8  To our surprise, 4-dimethylamino-salisaldehyde was observed as unreacted and 4-methoxy-thiophenol reacted with the malonitrile dimer to afford the undesired, highly-substituted pyridine derivative 2. At this point, we thought of employing two equivalents of malonitriles, as demonstrated by Evdokimov et al. [18], instead of the malonitrile dimer. Interestingly, the reactions worked as desired. Scheme 3 demonstrates our second and successful attempt to prepare three thiochromenopyridines (1g-1i) consisting of the C 8 -dimethylamino substituent. This finding suggests that unlike the malonitrile dimer, two equivalents of malonitrile work both for electron-deficient and electron-rich salisaldehyde. In continuation, we made an effort to conduct the three-component cyclization under microwave irradiation utilizing two equivalents of malonitrile in order to further shorten the reaction time. Our effort to achieve eight 5H-substituted-thiochromenopyridines is summarized in Scheme 4. Under controlled microwave irradiation, the reaction time is further shortened to 10 min. However, the yield is compromised to 30%-45%.
In this study, ten 5H-substituted-chromenopyridines (1a-1j), as well as three 4H-substituted chromenes (1k-1m) were designed and synthesized, as shown in Figure 3. Upon obtaining nine 5H-substituted-thiochromenopyridines (1a-1i), we tested their antiproliferative activity against three melanoma cell lines and two glioma cell lines in vitro. Table 1 represent the IC50 values of the prepared compounds against three melanoma and two glioma cell lines. Interestingly, all nine compounds are found moderately active against melanoma cell lines, and eight of them are observed as moderately active against glioma cell lines, as indicated by Table 1. Interestingly, Compound 1a, consisting of a benzyl head group, is tolerated in melanoma cell lines, but not in glioma cell lines. It is noteworthy that the C 8 -methoxy substituent offers better activity in both cell lines compared to the C 8 -dimethylamino substituent. Table 1 suggests that 1b and 1c are the two most potent compounds (IC50 ~ 3.6 μM) against melanoma. Likewise, 1e and 1f are the two most potent for glioma cell lines with IC50 values in the 3 μM range. This finding also suggests that the fluoro head group (1b, 1c) is preferentially active against the melanoma cell lines, and the dimethylamino head group (1f) is preferentially active against the glioma cell lines. At this point, we decided to prepare Template A-type chromenopyridines ( Figure 2) that do not consist of flexible carbon-sulfur-carbon bonds. We thought that this may provide insight into the role of flexibility offered by the carbon-sulfur-carbon bonds (1a-1i) for the biological activity. We chose to prepare Template A-type derivatives of 1b and 1f based on the observed activity. Scheme 5 demonstrates our effort to prepare rigid 5H-substituted-chromenopyridine following the procedure demonstrated by Melekhin et al. [17] However, only 1j, a rigid analogue of 1b, worked out in our hands, as summarized in Scheme 5. To our surprise, 1j was observed as inactive against all melanoma and glioma cell lines. Hence, this finding suggests that the flexibility offered by carbon-sulfur-carbon bonds (1a-i1) is critical for cytotoxic activity. However, we have learned from SP-6-27 that rigid chromenes are active. To the best of our knowledge, there is no report on the cytotoxicity of the 4H-substituted-thiochromenes. Hence, it would be interesting to see if the flexibility offered by the bicyclic 4H-substituted thiochromenes is tolerated.
Interestingly, none of the thiochromenes (1k-1m) were found to be active against both cell lines. Briefly, our initial findings suggest that the tricyclic thiochromenopyridines (1a-1i) have potential to be developed as novel anti-cancer agents. However, the activity is limited to the lower micromolar range. Hence, additional research is needed to optimize the activity of these compounds.

General
All reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and were used without further purification. The solvents for moisture-sensitive reactions were freshly distilled, and the reactions were carried out in an argon atmosphere. Routine thin-layer chromatography (TLC) was performed on aluminum-backed Uniplates (Analtech, Newark, DE, USA). Nuclear magnetic resonance spectra were obtained on a Varian Inova-500 spectrometer ( An amount of 2-amino-1,1,3-propenetricarbonitrile (1 mmol), desired thiophenol or thionapthanol (1 mmol) and triethyl amine (0.1 mmol) were added in a solution of 4-methoxy-salisaldehyde (1 mmol) in 7 mL EtOH. The resulting mixture was allowed to reflux over 4 h, at which point the product precipitates out of the solution. The resulting precipitate was filtered and dried under vacuum. The residue was then dissolved in 3 mL DMF. The insoluble particles were filtered off. A volume of 4 mL H2O was then poured into the resulting filtrate, leading to the precipitation of the pure product out of the solution. The precipitates were filtered off and dried under vacuum, leading to the pure 5H-substitutedthiochromenopyridines (89% to 91%) as light yellowish solids.

2,4-Diamino-8-methoxy-5-(naphthalen-1-ylthio)-5H-chromeno[2,3-b]pyridine-3-carbonitrile
(1e): Compound 1e was prepared following the general procedure for the preparation of Compounds 1a-1f. An amount of 385 mg (0.90 mmol, 90%) 1e was obtained as a light yellowish solid. 1   An amount of malonitrile (2 mmol), desired thiophenol (1 mmol) and triethyl amine (0.1 mmol) were added in a solution of 4-dimethylamino-salisaldehyde (1 mmol) in 7 mL EtOH. The resulting mixture was allowed to reflux over 4 h, at which point the product precipitates out of the solution. The resulting precipitate was filtered off and dried under vacuum. The residue was then dissolved in 3 mL DMF. The insoluble particles were filtered off. A volume of 4 mL H2O was then poured into the resulting filtrate, leading to the precipitation of the pure product out of the solution. The precipitates were filtered off and dried under vacuum, leading to the pure 5H-substituted-thiochromenopyridines (1g-1i, 89%-91%) as light pinkish solids. [2,3-b]pyridine-3-carbonitrile (1g): Compound 1g was prepared following the general procedure for the preparation of Compounds 1g-1i. An amount of 388 mg (0.90 mmol, 92%) 1g was obtained as a light pinkish solid. 1   A mixture of 4-substituted-salisaldehyde (1 equivalent), malonitrile (2 equivalent) and the desired substituted-phenylthiol or naphthol (1 equivalent) was mixed in 3 mL ethanol in a microwave tube. A catalytic amount of trimethylamine (2-4 drops) was added to the solution. The reaction mixture was irradiated at 150 °C using 300 psi pressure and 300 Watts of power over 10 min in CEM Discover. The reaction mixture was cooled down to room temperature; the precipitates were filtered and dried under vacuum. The resulting residue was dissolved in 3 mL DMF. The insoluble particles were removed by filteration. A volume of 4 mL H2O was added to the DMF layer, leading to the precipitation of the desired product. The precipitates were filtered and dried under vacuum, resulting in the pure product as a yellowish or a pinkish solid in a decent yield (30%-45%), as reported in Scheme 4. The characterizations of the obtained products (1a-1i), under microwave irradiation, comply with the ones obtained under regular reflux condition. [2,3-b]pyridine-3-carbonitrile (1j): An amount of 124 mg 3-methoxy-phenol (1 mmol) was added in a solution of 240 mg (Z)-2-amino-4-(4-fluorophenyl)buta-1,3-diene-1,1,3-tricarbonitrile (3a), prepared following the literature reported procedure, in 15 mL ethanol. The volume of a catalytic amount of piperidine was added to the reaction mixture. The reaction mixture was brought to reflux overnight. The reaction mixture was cooled down, evaporated out, and the crude was purified utilizing 2% MeOH in methylene chloride, resulting in 30 mg (8.3%) product as a yellowish solid. 1  An equimolar mixture of 4-methoxy-salisaldehyde, malonitrile and the desired substituted phenylthiol were mixed in 7 mL EtOH. The reaction mixture was brought to reflux overnight under argon atmosphere. The reaction was allowed to cool down to room temperature, resulting in the precipitation of the product. The product was filtered off and recrystallized in diethyl ether, leading to the pure 4H-substitutedthiochromenes (1k-1m) in good yields.

Biology
Cell Culture and Cytotoxicity Assay Melanoma Human A375, WM164 and MDA-MB-435 melanoma cells were cultured in 10% fetal bovine serum supplemented with DMEM medium with 1% antibiotics at 37 °C 5% carbon oxide. The cell viability was determined by the MTS assay as described previously [21,22]. In brief, 5000 cells were seeded into each well of 96-well plates overnight. Then, a serial dilution of the compounds dissolved in culture medium was used to treat the cells for 48 h. After that, the medium was aspirated, and MTS agent was added to detect the cell density in each well by reading the optical absorbance at 490 nm.

Glioma
Human MT330 and SJG2 glioma cells were grown in DMEM with 10% fetal bovine serum. Cells were maintained in the presence of penicillin (100 IU/mL) and streptomycin (100 mg/mL) at 37 °C with 5% CO2. Cell viability was determined using the MTT assay. Cells (1000-2000/well) were plated overnight in 96-well plates and treated with various concentrations of the drugs for 4 days. MTT reagent (10 μL/well) was added to the cells; cells were solubilized, and the optical density at 570 nm was measured.

Conclusions
We have identified a series of novel thiochromenopyridines as potential cytotoxic agents against a number of human cancer cell lines. To the best of our knowledge, this is the first report of thiochromenopyridines as potential anticancer agents. This study reveals that the addition of a third ring to SP-6-27, to give 1j, leads to no detectable activity (ND). In continuation, insertion of a sulfur atom, as in 1k, 1l, 1m, leads to no detectable activity. Finally, insertion of a sulfur atom, as well as a third ring, as in 1a-1i, provided activity, although in the 3-15 µM range. Briefly, this study suggests that the flexible carbon-sulfur-carbon bonds in 5H-substituted-thiochromenopyridines are important for cytotoxicity. We propose that the observed diminished activity of 5H-substituted-chromenopyridine (1j) is due to the absence of flexible-carbon-sulfur-carbon bonds. However, the same flexibility is responsible for the observed inactivity of 4H-substituted-thiochromens (1k-1m). To summarize, flexible carbon-sulfur-carbon bonds are required for the activity of tricyclic chromenopyridines, but not for the bicyclic chromenes. In addition, compounds with C 8 -methoxy substituents (1b, 1c, 1e and 1f) offer better activity as compared to the C 8 -dimethylamino substituents (1h, 1i and 1j). The good to moderate activity observed with the thio-tricyclic chromenopyridines established them as initial hits, and further research will be needed to develop chromenopyridines as potential leads.