Eco-friendly one-pot synthesis of some new pyrazolo[1,2-b]phthalazinediones with antiproliferative efficacy on human hepatic cancer cell lines

ABSTRACT This work focuses the light on some remarkable achievements in clean and efficient green experimental synthesis, characterization and evaluation of the pharmaceutical and biochemical importance of new series of pyrazolo[1,2-b]phthalazinediones which synthesized through one-pot three-component condensation reaction of the appropriate of 1,2,3-triazolyl-pyrazole-carbaldehydes with active methylene compounds (as malononitriles or ethyl cyanoacetate) and 6-nitrophthalhydrazide using the grinding method in the presence of sodium hydroxide under solvent-free condition at room temperature, in very excellent yields. All the newly synthesized compounds were characterized by physical and chemical tools (FT-IR, 1H NMR and mass spectrometry). In addition, all the new synthesized derivatives were screened for their anticancer activity against hepatic cancer cell lines to evaluate their pharmaceutical importance. GRAPHICAL ABSTRACT

Moreover, referring to the statement "The best solvent is no solvent" (35) and the growing demand for an efficient and clean methodology represent a challenge of great interest in heterocyclic synthetic chemistry. The exclusion of solvents in chemical reactions has become one of the main criteria in green chemistry (36,37). Moreover, the grinding technique is of great interest in synthetic organic chemistry as it is carried out under ecofriendly conditions and in the absence of solvent (38)(39)(40)(41). Also, the grinding process is done at room temperature and the reaction time range is from 2 to 5 minutes. So, it contributes to the development of a green strategy for the synthesis of organic derivatives in high yield with simple, fewer waste products, efficient, environmentally and economically benign compared to classical methods.

Results and discussion
Owing to the biochemical and pharmaceutical significance of 1,2,3-triazole and pyrazole rings, our growing interest in this part of the work is to incorporate between 1,2,3-triazole and pyrazole rings. Toda et al. (49) report that some of the exothermic reactions could be accomplished in excellent yield through grinding solids together (or liquid\solid) by using the mortar pestle technique which is known as grindstone chemistry. Reactions begin with grinding, with transfer of very small amounts of energy via friction (see Figure 1). Based on this simple technique, we synthesized a series of hydrazones containing 1,2,3-triazole moiety and utilizing these hydrazones in preparation of 1,2,3-triazolyl-pyrazole-carbaldhydes. Thus, condensation of acetyl triazole derivatives 1a-e (50) with phenyl hydrazine in the presence of one drop of glacial acetic acid by the grinding method at room temperature afforded the respective triazolylhydrazones 2a-e in excellent yields (Scheme 1). The chemical structure of 2a-e was established based on elemental data and also on spectral data (IR, 1 HNMR, mass). For example, the 1 HNMR spectrum of compound 2a displayed one signal at δ = 11.21 ppm attributed to the NH proton, in addition to the expected signals of the two methyl and aromatic protons. The mass spectral data of all products 2 exhibited in each case a molecular ion peak at the correct molecular weight for the respective compound (see Experimental).
When those hydrazones 2a-e were submitted to Villsmeier reaction conditions, they cyclized to afford the 1,2,3-triazolyl-pyrazole-carbaldhyde derivatives 3a-e through intramolecular cyclization reaction (Scheme 1). The structures of 3a-e were deduced from the microanalytical and spectral data. A respective example, the IR spectrum of 3d exhibited strong absorption band at v = 1715 cm −1 attributed to the carbonyl of the CHO group. Moreover, the 1 HNMR spectrum of 3d showed the absence of a signal at δ = 11.21 ppm for the (NH) group and instead there appeared two singlet signals for the two protons of pyrazole-H5 and the CHO group at δ = 9.27 and 10.52 ppm, respectively, in addition to the expected signals due to the protons of CH 3 and aryl groups (see experimental section). The mass spectra of 3d exhibited a molecular ion peak at m/z = 407 which is consistent with the assigned chemical structure.
Despite the pharmacological and synthetic importance of pyrazolo [1,2-b]phthalazinedione, several multicomponent strategies have emerged for synthesis of this ring system by cyclo-condensation of phthalhydrazide, aldehydes and active methylene compounds like ethyl cyanoacetate or malononitrile using Et 3 N as a catalyst. However, nowadays, the usage of a solid basic ecofriendly catalyst has been found to be a comfortable synthetic platform rather than the usage of volatile toxic organic bases. So, herein we used NaOH as the ecofriendly basic catalyst.
The structures of 6a-j were established via elemental analysis and spectral (IR, 1 H NMR, Mass) data. The IR spectra of compounds 6e, taken as a representative example of the products 6a-e, revealed in each case four bands at υ = 1676, 1680, 2222 and (3287, 3393) cm −1 which are assigned to the 2-carbonyl, nitrile and -NH 2 groups. Also, the 1 HNMR spectra showed in addition to the expected signal assigned for CH 3 group, pyrazole-H5 and the aromatic protons, two signals at δ = 5.12 and 6.23 ppm assigned for the pyrazole-H1 and NH 2 protons.
Otherwise, IR spectrum of 6i, taken as a representative example of the derivatives 6f-j, showed a strong biforked absorption band at v = 3423, 3387 cm −1 for NH 2 , strong stretching absorption band at v = 1720 cm −1 for ester C=O and two stretching absorption bands at v = 1665, 1687 cm −1 attributed to the two carbonyl groups of dione. Its 1 H NMR spectrum showed a triplet signal at 1.29 ppm due to three protons of CH 3 CH 2 , singlet signal at 2.52 ppm for three protons of CH 3 , quartet signal at 4.34 ppm owing to two protons of CH 2 CH 3 , singlet signal at 5.3 ppm for two protons of NH 2 , singlet signal at 6.34 ppm for pyrazole-H1, singlet signal at 9.34 ppm attributed to pyrazole-H5 and multiplet signal at 7.15-7.92 ppm due to aromatic hydrogen. In addition, mass spectral data of all new derivatives showed correct molecular ion peaks (see Experimental).
The structure of 6 was further confirmed by an alternative synthetic method. Thus, refluxing of compound 3a with malononitriles in ethanol leads to the formation of 7a. Compound 7a was then reacted with 6-nitro-2,3-dihydrophthalazinedione in ethanol in the presence of sodium hydroxide to give a compound identical in all respects (IR, mp and mixed mp.) to 6a (Scheme 2; Figure 2).

Antiproliferative activities
The in vitro growth inhibition activities of the newly synthesized compounds 2a-e, 3a-e and 6a-j were examined against human hepatocellular carcinoma (HepG-2) comparing with the well-known anticancer standard (Doxorubicin) under the same condition by using colorimetric MTT assay. Data generated are used to plot a dose-response curve in which the concentration of tested compounds required to kill half of the cell population (IC 50 ) was determined. IC 50 values were calculated for each experiment separately and mean values ± SD are represented in Table 1. Each compound at each concentration was tested in triplicate in a single experiment, which was repeated three to five times. The results of the studies on antiproliferative activities of tested compounds are summarized in Table 1 and Figure 3.
The results of the studies on antiproliferative activity of tested compounds in Table 1 and Figure 1 show that compound 6f revealed the highest antiproliferative activity (IC 50 = 3.01 ± 0.21µg/mL) toward the human hepatic cancer (HepG2) cell line while it is not active against the normal cell line (BALB/3T3). Moreover, compounds (2b, 3b, 3c, 3e, 6a, 6c and 6i) indicated high activity against the HepG2 cell line but they also showed lower activity against the normal cell line BALB/3T3. On the other hand, compound 2c showed lower antiproliferative activity against HepG2 (IC 50 = 69.20 µg/mL) but it is not active against BALB/3T3 in the used range of concentration.

Experimental
All 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 NMR spectra were recorded in (CD 3 ) 2 SO solutions on a JNM-LA 400 FT-NMR system spectrometer and chemical shifts are expressed in δ ppm units using TMS as an internal reference. Mass spectra were recorded on a GC-MS QP1000 EX Shimadzu. Elemental analyses were carried out at the microanalytical center of Cairo University. Chemistry A mixture of the appropriate acetyl triazole derivatives 1a-e (10 mmol) and phenyl hydrazine (10 mmol) was ground in a mortar at room temperature, in the presence of drops of acetic acid (2 mmol), for 10-20 minutes. The reaction mixture was poured into water and the solid product was collected by filtration followed by washing with ethanol. The crude product was then recrystallized from acetic acid to give the corresponding hydrazone derivatives 2a-e. The products 2a-e together with their physical constants are listed below.  Synthesis of pyrazoles 3a-e Phosphorus oxychloride (20 mL, 20 mmol) was added dropwise with stirring to dimethyformamide (150 mL) at 0-5°C. Then the appropriate hydrazone 2a-e (20 mmol) was added portion-wise with continuous stirring, left overnight at room temperature, poured onto ice-cold water and neutralized with ammonium hydroxide solution (5%). The formed precipitate was filtered, dried and recrystallized from acetic acid to give the corresponding pyrazoles 3a-e, respectively. The products 3a-e together with their physical constants are listed below.   General procedure for synthesis of pyrazolo [1,2b]phthalazine derivatives 6a-j A mixture of pyrazole-4-carbaldehydes 3a-e (5 mmol), malononitrile (4a) or ethylcyanoacetate (4b) (5 mmol), and 6-nitro-2,3-dihydrophthalazine-1,4-dione (5) (1.035g, 5 mmol) was taken in a mortar at room temperature. Solid sodium hydroxide (2 mmol) was added to few drops of water. The reaction mixture was ground by the pestle, under the hood, for 20-30 minutes (monitored through TLC). The reaction mixture was then poured into 2N HCl, and the solid product was collected by filtration followed by washing with water and EtOH. The crude product was recrystallized from ethanol to obtain the pure 6a-j. The products 2a-e together with their physical constants are listed below. (1) Reaction of 7a with 6-nitro-2,3-dihydrophthalazine-1,4-dione (5) Equimolar amounts of 7a (0.377 g, l mmol) and 6-nitro-2,3-dihydrophthalazine-1,4-dione (3a) (0.207g, 1mmol) in ethanol (10 mL) containing an equivalent amount of NaOH (0.058 g, 1 mmol) was refluxed for 2 h, gave product identical in all respects (m.p., mixed m.p. and IR spectra) with compound 6a.

Pharmacology
Antiproliferative activity

Compounds
All compounds were dissolved in DMSO (stock solution 10 mg/mL) and subsequently diluted in culture medium to reach the required concentrations (ranging from 100 to 0.1 µg/mL).

An antiproliferative assay in vitro
Twenty-four hours before addition of the tested compounds, the cells were plated in 96-well plates (Sarstedt, Germany) at a density of 1 × 10 4 cells per well. The assay was performed after 72 h exposure to varying concentrations of the tested compounds. The in vitro cytotoxic effect of all compounds was examined using the SRB assay.

Conclusions
A new series of pyrazolophtalazinedione derivatives have been successfully developed and characterized, in excellent yields by the grinding method under solventfree condition at ambient temperature. The newly synthesized compounds seem to be interesting for pharmaceutical studies. They revealed high potency as anticancer agents. They inhibit the growth of cancer cells but with lower cytotoxic effects on normal cells in the used range of concentrations. The encouraging promising results obtained from anticancer studies on the newly synthesized derivatives make the synthesis of new series of these compounds and studying of their pharmaceutical importance an active area for more and more investigations.

Disclosure statement
No potential conflict of interest was reported by the authors. Dr. Marwa S. El-Gendey was born in Giza. She graduated from Al-Azhar University -College of sciencechemistry department at 1996 with general score B+ with honors. She received the master degree at 2002 in organic chemistry then the PhD. at 2007 on the same field. She began her functional gradation as lecture at chemistry departmentcollege of science -Al-Azhar University at 1997 then as an assistant teacher then lecturer at the same department at 2007. She specialized in organic chemistry as she has done several studies on heterocyclic compounds with biotic effects on microorganisms and anti-tumor effects. She now works as an associate professor at Al-Taif University.