Synthesis and Antiproliferative Activity of 1-(4-(1H-Indol-3-Yl)-6-(4- Methoxyphenyl)Pyrimidin-2-yl)Hydrazine and Its Pyrazolo Pyrimidine Derivatives

The target compounds 1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)hydrazine (5) was synthesized by reacting 6-(1H-Indol-3-yl)-4-(4-methoxyphenyl)pyrimidine-2(1H)-thione 4 with hydrazine hydrate. Compound 5 was used as a precursor for the synthesis of new pyrazolo pyrimidine derivatives 6-9. Moreover, the 5-amino-1H-pyrazole4-carbonitrile derivative 6 was then converted into another set of novel compounds 10-14. On the other hand a series of transformations were carried out using the newly synthesized 1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl) pyrimidin2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (11) to afford the pyrazolo pyrimidine derivatives 15-18. Antiproliferatve activities for some of the newly synthesized compounds were evaluated.

Chemically, chalcones are characterized by their easy synthesis by Claisen-Schmidt condensation in good yields and the presence of the α,β unsaturated carbonyl system linked to two aromatic rings which encourage the chalcones cyclization through Michael addition to give interesting heterocyclic compounds which exhibit antitumor [6,7], antimitotic [8], antimutagenic [9] antibacterial [10], antiviral [11], antiinflammatory [12], antiulcerative [13] and hepatoprotective [14]. In the present work we have been synthesized a series of novel heterocyclic compounds derived from a previous synthesized chalcone compound [1]. The new compounds were screened for their antiproliferative activities.

Experimental General
Melting points were measured on a digital Electrothermal 9100 apparatus (Kleinfeld, Gehrden, Germany) and are uncorrected. FTIR spectra (KBr) were obtained on a Nicolet 205 spectrophotometer (Nicolet, Madison, WI, USA). The 1 H NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer (Varian inc., Palo Alto, CA, USA). Chemical shifts are expressed in δ values. The mass spectra were recorded on a Shimadzu GCMS-QP-1000EX mass spectrometer (Shimadzu, Kyoto, Japan) at 70 eV. Elemental analysis were carried out at the Micro-analytical Center of Cairo University. All reactions were routinely followed by TLC.

Synthesis of compound (18):
• Method A: To a solution of compound 15 (1.67 g, 0.005 mol) in dioxane (30 ml), hydrazine hydrate (2 ml) was added and the mixture was heated at reflux for 4 h. The reaction mixture was cooled and poured into water and the separated solid was filtered off, dried and recrystallized from dioxane. •

Results and Discussion
Through several years of research and cross referencing other related journals on the preparation of chalcones and their derivatives, chalcone compound 3 and thiopyrimidinone 4 were prepared as reported in literature [1].
The hydrazino-pyrimidine derivative 5 was synthesized by reacting the thiopyrimidinone 4 with hydrazine hydrate in refluxing alcohol, the structure of compound 5 was confirmed by IR, 1 H NMR, MS spectra and elemental analysis, where its IR revealed the absorption bands at ν max =3212 for the NH 2 and 3184, 3109 cm -1 for the two NH groups, 1 H NMR spectrum gave the signals at δ=8.93-8.95 as a broad singlet for NH 2 , hydrazine NH and at 11.65 ppm., for the indole NH respectively. MS spectrum substantiated it's exact molecular weight (cf. Scheme 1 and Experimental section).
The hydrazino-pyrimidine derivative 5 was used as an assorted precursor for the synthesis of some biologically active heterocycles, where the amino pyrazole carbonitrile compound 6 was synthesized by refluxing a 1:1 mixture of compound 5 in ethanol with ethoxymethylene malononitrile for 4 h. The structure of compound 6 was confirmed by IR, MS, 1 H NMR and elemental analysis, where its IR spectrum gave the absorption bands at ν max =3312 for NH 2 , 3175 for NH Indole and 2226 cm -1 for the CN group respectively, it's 1 H NMR substantiated an NH 2 singlet at 8.86 and a singlet at δ=11.66 ppm for NH indole.
The pyrazolo pyrimidine compound 7 was synthesized by heating an alcoholic solution of compound 5 (10.0 mmol.) with acetylacetone (10.0 mmol.) at reflux temperature for 5 h. The FTIR spectrum of compound 7 disclosed the absorption bands at 3169 for NH indole, 1635 and 1575 cm -1 for C=N groups respectively. 1 H NMR revealed NH indole singlet at δ=10.41, singlet pyrimidine H at δ=8.95 ppm and two singlets for the two CH 3 protons respectively.
The pyrimidine pyrazolone compound 8 was obtained by refluxing a solution of compound 5 and ethyl acetoacetate in acetic acid for 6 h. The structure of compound 8 was deduced from its analytical and spectral data, which were in full agreement with the proposed structure (cf. Experimental section).
Furthermore, the hydrazino-pyrimidine derivative 5 was reacted with ethyl acetoacetate in excess to afford compound 9. IR spectrum of compound 9 revealed the absorption bands at ν max =3367 and 1715 cm -1 characteristic for NH indole and C=O groups respectively, 1 H NMR exhibited the NH indole singlet at δ=10.82, two singlets at δ=1.23 and 2.43 for 2 CH 3 protons and a singlet at δ=5.6 ppm for pyranone H respectively (Scheme 2).  produce compound 10. The structure of compound 10 was confirmed by its spectral and analytical data.
The pyrazolo pyrimidinone derivative 11 was obtained on one hand by refluxing compound 6 in excess formic acid and on the other hand by heating compound 12 under reflux condition in triethyl orthoformate. The IR spectrum of compound 11 substantiated the absorption bands of indole NH, pyrimidinone NH and C=O respectively at their prospective values. 1 H NMR, MS and elemental analysis gave the confirmatory data for compound 11 (cf. Scheme 3 and Experimental section).
Treatment of compound 6 with cold concentrated sulphuric acid portion wise and sterilizing on an ice bath for about an hour. afforded the pyrazolo carboxamide derivative 12. IR spectrum of compound 12 substantiated absorption bands at 3398 for pyrazole NH 2 , 3267 for amide NH 2 , 3114 for indole NH and 1640 for the amide C=O, it's 1 H NMR displayed three singlets at δ 8.48, 9.14 and 11.42 ppm for (D 2 O exchangeable) pyrazole NH 2 , amide NH 2 and indole NH respectively.
Continuing a series of synthesis the pyrazolo carboxamide derivative 12 was transformed into the pyrazolo triazinone 13 by the addition of sodium nitrite solution to a suspended solution of 12 in concentrated hydrochloric acid at 0-5°C with stirring at room temperature.

Structure of compound 13 was approved by IR, 1 H NMR, MS measurements and elemental analysis (cf. Scheme 3 and Experimental section).
Refluxing a solution of compound 12 in dimethylformamide with 20% potassium hydroxide solution and carbon disulfide (5 ml) afforded the thioxopyrazolo pyrimidinone derivative 14, which structure was deduced from its analytical and spectral data, (cf. Experimental section).
The pyrimidinone derivative compound 11 was heated in phosphorus oxychloride for 10 h. to give compound 15 which in turn was transformed by reaction with thiourea into the pyrimidin-4-thiol compound 16.
Compound 16 in ethanol and 10% sodium hydroxide solution was treated with methyl iodide then the reaction mixture was subjected to reflux affording the methylthiopyrazolo pyrimidine derivative 17.
The data were reported as mean-graph of the percent of growth of the treated cells, and presented as percentage growth inhibition (GI%) caused by the tested compounds (
With respect to broad spectrum antitumor activity, close examination of the data presented in Table 1 revealed that compounds 11, 14, and 18 are the most active derivatives showing effectiveness towards numerous cell lines belong to different tumor subpanels. Consequently, compounds 11, 14, and 18 were selected and tested against a panel of 60 different tumor cell lines at a 5 -log dose range [1][2][3][4][5].
These response parameters GI 50 , TGI, and LC 50 were calculated for each cell line, using the known drug 5 -Fluorouracil (5 -FU) as a positive control.  (Table 3).
Comparing the antitumor activities of compounds 11, 14 and 18 with those of Gefitinib and Erlotinib showed that compounds 11, 14 and 18 (Table 4) possess activities almost equal to or higher than those of Gefitinib and Erlotinib against most cell lines except non-small lung (EKVX and NCI -H522), melanoma (SK -MEL -28), ovarian cancer (IGROVI and SK -OV -3) renal cancer (ACHN and TK-10) and breast cancer (MDA-MB468) on the other hand compounds 8 and 12 showed selective activities toward CNS, renal and breast cancer cell lines, compound 9 gave selective activities toward leukemia cell lines, where compound 7 displayed moderate antitumor activities.
Compound 12 revealed selective activities towards SR leukemia cell lines with GI values of 34% whereas compound 18 disclosed weak activity against k-562 and PRMI-8226 cell lines with GI values of 23% and 17%.
Compound 7 showed moderate activities against M14 cell line, whilst compounds 9 and 12 possessed activity against UACC-62 cell line with GI values of 34% and 47%, Melanoma SK-MEL-2, SK-MEL-28 UACC-257 cell lines were found to be sensitive to compounds 12 and 18 in as shown in Table 1, at the same time compound 18 was active towards LOX IMVI and MALME-3M cell lines GI, 29% and 67%, and compound 12 has slight activity against SK-MEL-5 cell line with GI value 26%.

Conclusion
The authors herein endeavored to design and synthesize new pyrazolopyrimidine derivatives and screen some of these compounds for their anti-proliferative activity.