Synthesis and cytotoxic activity of some novel benzocoumarin derivatives under solvent free conditions

ABSTRACT In the present study, a rapid, less expensive, clean and environmental friendly route to synthesis new pyrazoles, pyrazolopyridazines and condensed pyrimidines was developed via grinding of 2-(3-(dimethylamino)acryloyl)-3H-benzo[f]chromen-3-one (1) with different reagents. All the new compounds were characterized and established using elemental analysis and spectral data. Eight compounds were selected for in vitro antiproliferative against different human cancer cell lines entitled melanoma, cancers of the lung, leukemia, breast, brain, colon, prostate, ovary and kidney by the USA NCI. GRAPHICAL ABSTRACT


Introduction
The organic solvents are volatile and harmful, causing risks to peoples who inhale them as well as the environment. Thus, development of less hazardous synthetic methods for organic reactions is one of our objectives in current research. The grinding method is one of green chemistry techniques as it is carried out in the absence of solvent (1)(2)(3)(4). Moreover, the solvent-free reactions have numerous advantages: reduced pollution, low costs, high yields and purities of products (5,6). Otherwise, many compounds which contain coumarin nucleus exhibit antimicrobial (7), analgesic (8), ulcerogenic (9), anticoagulant (10), antiviral (11) and antimalarial (12), anti-inflammatory (13,14) anti-leishmanial (15) as well as antioxidant (16,17) activities. The antitumor activity of coumarins received a considerable interest owing to their cytotoxic activity against various types of cancer cells, including gastric cancer, liver cancer, colon cancer, breast cancer, prostate cancer, Periodontal Ligament, fibroblast, nasopharyngeal carcinoma, and normal fibroblast cell lines (18)(19)(20)(21)(22). In addition, coumarin derivatives can inhibit growth in human cancer cell lines (23) such as renal (ACHN), lung (A549, H727), leukemia (HL-60), breast (MCF7) and renal cell carcinoma (24). Coumarin and its derivatives are important components among the molecules in the drug industry. Warfarin, acenocoumarol and phenprocoumon are a derivatives of coumarin used as anticoagulant drugs. These are vitamin K antagonist which inhibits the coagulation via blocking of the coagulation factors [I, VII, IX and X] (25)(26)(27). Moreover, warfarin reduced metastases from intestinal cancer to a wide extent (28) and is also used beside the surgical treatment of cancer masses (29) (Figure 1).
In addition, scopoletin (30,31) and esculatin (32) were found in nature and they have antiproliferative, antiarthritic, antioxidant, and anti-inflammatory activities. Ensaculin (KA-672) is a drug belong to coumarin family and acts on a number of receptor systems, being both a weak NMDA antagonist and a 5HT 1A agonist (33, 34) ( Figure 1). Furthermore, combining coumarins with different bioactive molecules like: pyrazole, pyrazolo[1,5 a]pyrimidine (35), have recently been reported to exhibit significant anticancer activities. In continuation of our previous studies on solvent free condition (36,37), we report here in a facial, eco-friendly, high yielding approach to synthesis new coumarin derivatives containing different bioactive moieties by grinding and evaluate their anticancer activity.

Results and discussion
Due to the pharmaceutical importance of coumarins, pyrazoles and pyrazolopyridazines, we are interested in this part of the work to integrate these biologically active molecules. Thus, grinding of 2-(3-(dimethylamino)acryloyl)-3H-benzo[f ]chromen-3-one (1) with the appropriate of hydrazonoyl halides 2a-e in moist sodium carbonate afforded pyrazoles 4a-e, respectively. In grindstone technique, the grinding process is performed at room temperature and the reaction occurs due to heat generated by frictions between substrate and reagent. Products 4a-e were assumed to formed through 1,3-dipolar cycloaddition reaction of the nitrilimines 2 ′ ′ ′ ′ ′ (synthesized in situ from reaction of 2a-e with triethylamine) to the activated double bond in compound 1 affording cyclo adduct 3a-e which undergoes loss of dimethylamine molecule furnishing the final product (rout A). Another route (B), producing 7 does not operate based on spectral data. In 1 H NMR spectrum of 4a as an example, the ester group was resonated as triplet and quartet signals at δ 1.28 and 4.31 ppm, respectively and methyl protons was observed at δ 2.42 ppm. The H-5 of pyrazole and H-4 of coumarin were appeared as two singlet signals at δ 8.45 and 9.21 ppm, respectively. Other signals owing to aromatic protons was observed at δ 7.27-8.39 ppm (Scheme 1). 13 C NMR showed signals at 13.5 ppm due to -CH 3 of ester group and at 24.2 ppm for -CH 3 of p-tolyl, 63.5 ppm for -OCH 2 of ester beside that signals appeared at 104.6-152.5 ppm for Ar-C. Finally, three signals related to 3 C=O at δ 159.3, 160.5, 165.2 ppm. The mass spectrum of all products 4a-e exhibited a molecular ion peak for the respective compound (see Experimental).
Formation of products 4a-e rather than product 7 were further confirmed chemically by grinding pyrazoles 4a-e with hydrazine hydrate affording pyrazolo [3,4-d ]pyridazines 5a-e in a good yield (Scheme 1). Based on the above results, we exclude pyrazoles 7 and indicated that the isolable products are pyrazoles 4a-e. Structure 5a-e is well supported by spectral data as well as elemental analyses. IR spectrum of 5a lacked the absorption band of ester group and revealed the presence of broad absorption band at 3425 cm −1 corresponding to OH group. Whereas, 1 H NMR spectrum showed singlet signal at δ 2.46 ppm attributed to the protons of the methyl group in addition to two singlet signals at δ 8.97 and 9.88 ppm attributed to H-4 proton of the coumarin ring and hydroxyl group, respectively. Aromatic protons appeared in the region of δ 7.02-8.81 ppm.
However, the activity of enaminone 1 towards some selected nitrogen nucleophiles was investigated. Thus, grinding of enaminone 1 with N-nucleophiles such as hydrazine hydrate and thiocarbohydrazide afforded pyrazoles 9 and 10, respectively. The structure of these products was substantiated by spectral data and elemental analyses. IR spectrum of 10 showed a characteristic band at v 3424, 3304 cm −1 related to -NH and -NH 2 groups, 1689 for carbonyl group, and C=N and C=S groups was shown at 1621 and 1265 cm −1 , respectively. The 1 H NMR spectrum for 10 as an example, displayed signals at δ 7.32-9.18 ppm assigned to nine aromatic protons, and two singlet signals at δ 10.76, 11.12 ppm corresponding for -NH and -NH 2 groups which were exchangeable by D 2 O. Compound 9 was assumed to be formed via nucleophilic addition of hydrazine hydrate to β-carbon atom of enaminone 1 followed by elimination of dimethylamine yielded intermediate 8 which undergoes cyclocondensation affording 9 as outlined in Scheme 2.
Moreover, our present study was extended to investigate the chemical behavior of enaminone 1 towards some sulphadrugs. Thus, condensation of enaminone 1 with sulphadrugs, namely: [sulphadiazine, sulphademidine, sulphadimethoxazine or sulphisoxazole] 16a-d by grinding with few drops of acetic acid afforded benzene sulphonamide derivatives 17a-d, respectively in a good yield (Scheme 4). Chemical structure of compounds 17a-d assigned on the basis of the elemental analysis and spectra. IR spectrum of 17b showed a broad band at 3410 cm −1 corresponds to 2NH groups and at 1724 cm −1 , 1665 cm −1 due to 2 C=O functional groups. 1 H NMR spectrum revealed singlet signal at δ 2.27 ppm attributed to two methyl groups, beside that of aromatic protons at δ 6.60-9.25 ppm and two singlet signals at δ 10.60 and 11.28 ppm assignable to two NH protons exchangeable by D 2 O.

Pharmacological screening
In vitro cytotoxic assay The cytotoxic activity of eight compounds was determined by USA National Cancer Institute against different human cancer cell lines, using Adriamycin and 5-Florouracil as positive controls. The results are cited in Table 1

Experimental
Melting points were determined on an electrothermal apparatus and are uncorrected. IR spectra were recorded (KBr discs) on a Shimadzu FT-IR 8201 PC spectrophotometer. 1 H and 13 CNMR spectra were recorded in CDCl 3 and (CD 3 ) 2 SO solutions on a Varian Gemini 300 MHz and JNM-LA 400 FT-NMR system spectrometer and chemical shifts are expressed in ppm units using

Synthesis of pyrazoles 4a-e
A mixture of enaminone 1 (0.293 g, 1 mmol), the appropriate hydrazonoyl halides 2a-e (1 mmol each) and moist sodium carbonate (0.2 g, 2 mmol) was thoroughly ground with a pestle in an open mortar at room temperature until the mixture turned into a melt. The initial syrupy reaction mixture solidified within 3-10 min.
After completion of the reaction as monitored by TLC, the mixture was diluted with water, filtered and recrystallized from a DMF/EtOH affording 4a-e, respectively. General method for synthesis of pyrazolo [3,4d]pyridazines 5a-e A mixture of pyrazoles 4a-e (1 mmol each) and hydrazine hydrate (0.1 mL, 2 mmol) was ground with a pestle in a mortar at room temperature for 10-15 min. (monitored by TLC). The reaction mixture was poured into water and the solid product was collected by filtration washed with ethanol, dried and recrystallized from ethanol affording pyrazolo [3,4-d ]pyridazines 5a-e, respectively.

Synthesis of 2-(1H-pyrazol-3-yl)-3H-benzo[f ]chromen-3-one (9)
A mixture of enaminone 1 (0.293 g, 1 mmol) and hydrazine hydrate (0.1 mL, 2 mmol) was ground with a pestle in a mortar at room temperature. After few seconds the mixture became sticky, the grinding process was continued for 10-15 min. (monitored by TLC). The formed product was washed with ethanol, filtered and recrystallized from ethanol to give the desired product 9. Off

Reaction of enaminone 1 with sulphadruges
A mixture of enaminone 1 (0.293 g, 1 mmol), sulphadrugs 16a-d (1 mmol each), and few drops of acetic acid was ground with a pestle in a mortar at room temperature for 5-10 min. until the mixture turned into a melt, during which time a color change occurred (the change is colorless to yellow to orange). After completion of the reaction as monitored by TLC. The mixture was poured into water and the solid product was filtered, washed with ethanol, dried and recrystallized from DMF affording the desired products 17a-d, respectively.

NCI-60 screening methodology
The cytotoxicity against cancer cell lines were conducted at USA National Cancer Institute according to reported standard procedure (Sulphorhodamine B assay) (46)(47)(48).

Conclusions
In this research, benzo[ f ]chromen-3-one moiety is introduced as new class in antitumor agent against different cancer cell lines. Briefly, target compounds were prepared via reactions of 2-(3-(dimethylamino)acryloyl)-3H-benzo[ f ]chromen-3-one with hydrazonoyl halides, N-nucleophiles and sulphadrugs by grinding reactants together at room temperature. The anticancer activity was performed at USA National Cancer Institute (NCI) against different human tumor cell lines called melanoma, leukemia and cancers of the brain, lung, colon, ovary, prostate, breast and kidney. The results displayed that pyrazole 4e and pyrazolo [3,4-d ]pyridazines 5a, 5c-5e exhibited powerful anticancer activity against most cell lines.