OF NEW THIOPHENE , PYRAZOLE , ISOXAZOLE DERIVATIVES AS ANTITUMOR , c-Met , TYROSINE KINASE AND Pim-1 KINASE INHIBITORS

The reaction of cyclohexan-1,3-dione (1) with ethyl orthoformate (2) in acetic acid gave the 2(ethoxymethylene)cyclohexane-1,3-dione (3). The latter compound was used for further heterocyclization reactions to give thiophene, pyrazole and pyran derivatives. The cytotoxicity of the newly synthesized compound against the six cancer cell lines NUGC, DLDI, HA22T, HEPG2, HONE1 and MCF showed that compounds 5, 10c, 10d, 13b, 14a, 18b, 18d, 18e and 20b were the most potent compounds. On the other hand, the toxicity of these compounds against shrimp larvae indicated that compounds 7a, 10c, 13b, 14a, 18b and 18d were non toxic against the tested organisms. Inhibition of the most potent compounds towards the tyrosine kinases c-kit, FIT-3, Vascular Endothelial Growth Factor Receptor (VEGFR)-2, Estimated Glomerular Filtration Rate (EGFR) and Platelet-Derived Growth Factor Receptor (PDGFR) revealed that compounds 5, 10c, 10d, 13b, 18b, 18d, 18e and 20b were of the highest inhibitory effect. The Pim-1 kinase test revealed that compounds 10d, 18b and 20b were of the highest inhibitory effect. In addition, the c-Met enzymatic activities showed that compounds 10c, 10d, 18b, 18e, 19 and 20b showed higher potencies against c-Met kinase than the reference foretinib. On the other hand, compounds 7a, 7b, 10d, 13a, 13b, 14a, 14b, 16a, 16b, 17, 18a-f, 19 and 20 showed higher inhibition towards PC3 cell line than the reference SGI-1776. Compounds 10c and 18b were of common potencies and their molecular docking was described.


INTRODUCTION
Major progress in cancer chemotherapy requires new drugs to eradicate the entire neoplastic diseases in human being.Finding a novel structure leads that may be of use in designing new, potent, selective, and less toxic anticancer agents remains a major challenge for medicinal chemistry researchers [1][2][3][4].Random screening of natural products and synthetics has been the source of new leads in approaches to drug discovery [5][6][7].In our ongoing search for the new potential antitumor agents, we therefore aimed to develop a series of new synthetic leads which are more accessible and more amenable to optimization through analog synthesis.
Many planar heteroaromatic derivatives have shown anti-proliferative activity in vitro and some of them are important anticancer drugs [8][9][10].Some of these types of compounds have the indazole nucleus which is a structural component of a large number of biologically active natural and unnatural compounds.Recently the high anti-proliferative activity of nitrogen containing heterocycles in two human breast cancer cell lines (MDAMB 231 and MCF-7) was reported and the authors were able to show that the tubulin is the primary target of these agents inhibiting its polymerization [11][12][13].In the previous state of the art literature, the heterocycles containing sulfur and nitrogen possess a broad range of pharmaceutical activities.The family of sulfur heterocycles includes highly stable aromatic compounds that display physicochemical properties with relevance in the design of new materials, especially those relating to cancer treatments [14].Among the sulfur heterocycles, benzo [b]thiophene is a unique heterocyclic core that has been picturized as an important pharmacophore of some bioactive molecules and therefore fascinated much interest [15,16].Thiophene containing compounds are well known to exhibit various biological activities such as BACE1 inhibitors [17] and anti-breast cancer [18].Conversely, pyrazole derivatives have been reported in the literature to demonstrate anti-tumor [19] and kinase inhibitions [20].Motivated by these results and in continuation of our previous work aimed at the synthesis of a new heterocyclic systems for anti-tumor evaluations [21][22][23] we report here the modification of cyclohexan-1,3-dione into a variety of novel condensed pyrazole, thiophene and isoxazole derivatives incorporating the cyclohexane moiety.It is relied on combination of different pharmacophores of bioactive compounds [24][25][26][27] to obtain new compounds with potent activity, increased selectivity and reduced adverse effects.Following this strategy and in a continuation of our previous study, we synthesized heterocyclic derivatives in most of the work using hydrazine and/or cyano derivatives [28][29][30] with cyclohexane-1,3dione aiming to synthesis heterocyclic scaffolds in order cumulate all the expected activities of the individual rings onto one structure.The anti-tumor evaluations followed by tyrosine kinase, Pim-1 kinases and c-Met inhibitions were measured for the newly synthesized target molecules.Additionally the confirmation of the structure of the newly synthesized compounds using different spectroscopic tools was described.

RESULTS AND DISCUSSION
In the present work, we are presenting the uses of cyclohexane-1,3-dione for the synthesis of pyrazole, thiophene, isoxazole and pyran derivatives using simple reaction sequences followed by the evaluation of the newly synthesized products against cancer cell lines and some kinases inhibitions.Thus, the reaction of cyclohexane-1,3-dione (1) with ethyl orthoformate (2) in acetic acid gave the 2-(ethoxymethylene)cyclohexane-1,3-dione (3) [31].The structure of the latter product was based on its analytical and spectral data.The 1 H NMR spectrum revealed, beside the expected signals, a triplet at  1.28 ppm indicating the presence of the CH 3 , a quartet at  3.89 ppm for the CH 2 group and a singlet at  6.79 ppm due to the presence of the CH group.Moreover, the 13 C NMR spectrum showed the presence of  at 16.3 due to the presence of the OCH 2 CH 3 group,  62.8 indicating the presence of the OCH 2 CH 3 group, two signals at 112.3, 158.2 due to the C=CH group and two signals at  177.3, 179.2 indicating the presence of the two CO groups.Compound 3 with the two active centers, the C=O group and the ethoxymethino moiety showed an interesting reactivity toward hydrazine hydrate.Thus, compound 3 reacted with hydrazine hydrate to give the 4-hydrazono-4,5,6,7-tetrahydro-2H-indazole (5) which was identified on the basis of its analytical and spectral data (see Experimental).The reaction of compound 3 with either of malononitrile (6a) or ethyl cyanoacetate (6b) gave the alkylated products 7a and 7b, respectively.Compounds 7a and 7b with different active sits towards nucleophilic reagents encourage us for their reactions with two folds of either of hydrazine hydrate (4) or phenylhydrazine (8) affording the 5-hydrazono-5,6,7,8-tetrahydro-2-substitutedpyrazolo [3,4-b]quinoline derivatives 10a-d, respectively (Scheme 1).Formation of 10a-d occurred via the intermediacy of 9a-d followed by cyclization.The structures of 10a-d were established on the basis of their respective analytical and spectral data (see Experimental).Moreover, the reaction of either of compound 7a or 7b with hydroxylamine hydrochloride (11) gave the isoxazolo [3,4-b]quinolin-5(6H)-one oxime derivatives 13a and 13b, respectively through the intermediacy of 12a,b.The analytical and spectral data were consistent with their respective structures.
The reaction of either of compound 14a or 14b with cyanoacetylhydrazine (15) gave the hydrazide-hydrazone derivatives 16a and 16b, respectively (Scheme 2).The structures of compounds 16a and 16b were based on their respective analytical and spectral data.Thus, the 1 H NMR spectrum of 16a revealed the presence of a singlet at  4.42 ppm for the CH 2 group of the cyanoacetamido moiety, a singlet at  8.31 ppm (D 2 O exchangeable) indicating the presence of the NH group.In addition the 13 C NMR spectrum which showed a signal at  58.8 equivalent to the cyanoacetamido CH 2 group, the presence of the two CN groups at  116.6, 117.4 and two signals at  164.8, 172.3 equivalent to the C=O and C=N groups, respectively.Further elucidation for the structures of compounds 16a and 16b was obtained through their synthesis via another reaction route.Thus, the reaction of molar ratio cyclohexane-1,3-dione (1) and cyanoacetylhydrazine (15) gave the hydrazide-hydrazone derivative 17.The latter compound reacted with either of malononitrile (2a) or ethyl cyanoacetate (2b) and elemental sulfur to give the same thiophene derivatives 16a and 16b, respectively (m.p., mixed m.p. and finger print IR spectra).
It is well known that multi-component reactions (MCRs) can combine three or more components together in a single reaction vessel and produce final products with a minimum of synthetic time and effort [35] because they are no need to separate any reaction intermediate [36].Such MCRs often result in high atom economy and high selectivity products [37].They are also applicable to the synthesis of heterocyclic systems [38].It is quite remarkable that many top-selling pharmaceuticals contains 4-H pyran derivatives [39][40][41][42][43] this encouraged us to synthesis 4-H pyran derivatives through the multi-component reactions of compounds 14a,b.Therefore, the reaction of either of compound 14a or 14b with any of the aromatic aldehydes 7a, 7b or 7c and malononitrile (2a The two amino groups in compounds 18a-f were of different nucleophilicity.The amino group located at C-2 close to the aromatic thiophene moiety showed more nucleophilicity than the amino group at C-8 close the pyran moiety.It is noteworthy that the amino group in the 8-aminopyran moiety possesses much lower nucleophilicity compared to aliphatic amines due to its lower location at the C-2 carbon bonded to the oxygen [44] such amino group usually requires a catalytic base to initiate its nucleophilicty [45].Thus, throughout our work, the reaction of compound 18a, as a derivative of 18a-f, with ethyl cyanoacetate (2a) without the use any catalyst in dimethylformamide under reflux gave the 2-cyanoacetamido derivative 19.

Structure activity relationship
It is clear from Table 1 that the cytotoxicity of the newly synthesized products against NUGC, DLDI, HA22T, HEPG2, HONE1 and MCF using foretinib as the reference showed that compounds 5, 7a, 10c, 10d, 13b, 14a, 18b, 18d, 18e and 20b were the most cytotoxic compounds among the tested compounds.The 2-(ethoxymethylene)cyclohexane-1,3-dione (3) showed very low cytotoxicity towards the six cancer cell lines, however, its reaction with hydrazine hydrate to give the 4-hydrazono-4,5,6,7-tetrahydro-2H-indazole derivative 5 which is the nitrogen rich compound with high cytotoxicity towards the six cancer cell lines.It is of great value to note that compound 5 showed higher cytotoxicity againt HEPG2 cell line than foretinib with IC 50 228 nM.It is clear that the fused pyrazole derivative 5 showed higher cytotoxicity than the 2-(ethoxymethylene)cyclohexane-1,3-dione (3).On the other hand, the reaction of compound 3 with either of malononitrile or ethyl cyanoacetate gave compounds 7a and 7b with different potencies.Thus, it is obvious that compound 7a with the dicyano moieties showed remarkable cytotoxicity higher than 7b towards the six cancer cell lines, although compound 7b showed high potency against HEPG2 cell line with IC 50 250 nM by a value that very close to that foretinib.Considering, nitrogen rich compounds, the 5,6,7,8-tetrahydro-pyrazolo [3,4b]quinoline derivatives 10a-d, it is clear that compounds 10c (Y = OH, R' = H) and 10d (Y = OH, R' = Ph) showed the highest cytotoxicity among the four compounds.The high potencies of compounds 10c and 10d were attributed to the presence of the OH group in these compounds.
Compound 10d showed a higher cytotxiciy than foretinib towards DLDI with IC 50 58 nM.In general compound 7a showed the highest cytotoxicity which is close that those of the tricyclic compounds 10c and 10d.Reaction of either of compound 7a or 7b with hydroxylamine hydrochloride gave 5,6,7,8-tetrahydroisoxazolo [3,4-b]quinoline derivatives 13a and 13b where compound 13b (Y = OH) showed higher cytotoxicity than 13a towards the three cancer cell lines NUGC, DLDI, HEPG2 with IC 50 's 66, 222, and 217 nM, respectively.It is obvious that tricyclic pyridinopyrazole derivatives 10c,d showed higher cytotoxicity toward the six cancer cell lines than the the tricyclic pyridinoisoxazole derivative 13b as the latter showed low potency against HONE1 and MCF cell lines.
Considering the thiophene derivatives 14a,b it is clear that compound 14a (R = CN) showed higher cytotoxicity against the six cancer cell lines than compound 14b.The reactivity of 14a was attributed to the presence of the electronegative CN group.For the hydrazide-hydrazone derivatives 16a and 16b it is obvious that compound 16a (R = CN) showed high cytotoxicity only towards DLDI cell line with IC 50 408 nM while compound 16b (R = COOEt) showed low potencies against the six cancer cell lines.On the other hand, both of the hydrazidehydrazone derivative 17 showed low potencies towards the six cancer cell lines.Comparison of the thiophene derivatives 14a,b, the hydrazide-hydraone derivatives 16a,b and 17 revealed that the thiophene derivative 14a with the highest cytotoxicity among the five compounds.
Considering the multi-component products the thieno[3,2-h]chromene-3-carbonitrile derivatives 18a-f, it is clear that compounds 18b (R = CN, Y = Cl) and 18c (R = CN, Y = OCH 3 ) showed the highest cytotoxicity among the six compounds.On the other hand, compound 18d (R = COOEt, Y = H) showed high cytotoxicity against NUGC cell line with IC 50 84 nM but compound 18e (R = COOEt, Y = Cl) revealed high cytotoxicity against the five cancer cell lines NUGC, DLDI, HA22T, HEPG2 and HONE1.It is obvious through compounds 18a-f that the presence of either the CN or Cl groups enhances the high potencies of compounds 18b and 18c.The thieno[3,2-h]chromen-8-yl)-2-cyanoacetamide derivative 19 exhibited low potency towards the six cancer cell lines.It is clear that the cyanoacetamido moiety reduce the potency of compound 19.It is obvious that compounds 18b,d,e showed the highest cytotoxicity among the pyran derivatives 18a-d and 19.Considering the dihydrobenzo[b]thiophen-7(4H)ylidene)thiophene-2-carbohydrazide derivatives 20a,b with the two thieno moieties showed activities towards some selective cell lines.Thus, compound 20a (R = CN) showed high potency against HONE1 and MCF cell lines while 20b (R = COOEt) showed high potency towards all the cancer cell lines except with HEPG2 that showed low potency.In addition, compound 20b showed cytotoxicity against HA22T cell line very close to that of foretinib with IC 50 56 nM.The cytotoxicity evaluation of the synthesized compounds toward the six cancer cell lines are illustrated through Figure 1.

Toxicity
Bioactive compounds are often toxic to shrimp larvae.Thus, in order to monitor these chemicals in vivo lethality to shrimp larvae (Artemiasalina), Brine-Shrimp Lethality Assay [46] was used.
Results were analyzed with LC 50 program to determine LC 50 values and 95% confidence intervals [47].Results are given in Table 2 the following ten compounds 5, 7a, 10c, 10d, 13b, 14a, 18b, 18d, 18e and 20b which exhibited optimal cytotoxic effect against cancer cell lines.The shrimp lethality assay was considered as a useful tool for preliminary assessment of toxicity, and it has been used for the detection of fungal toxins, plant extract toxicity, heavy metals, cyanobacteria toxins, pesticides, and cytotoxicity testing of dental materials [48] natural and synthetic organic compounds [49].It has also been shown that A. salina toxicity test results have a correlation with rodent and human acute oral toxicity data.Generally, a good correlation was obtained between A. salina toxicity test and the rodent data.Likewise, the predictive screening potential of the aquatic invertebrate tests for acute oral toxicity in man, including A. salina toxicity test, was slightly better than the rat test for test compounds.In order to prevent the toxicity results from possible false effects originated from solubility of compounds and dimethyl-sulfoxide DMSO's possible toxicity effect, solutions of the test compounds were prepared in the suggested dimethyl-sulfoxide (DMSO) volume ranges.It is clear from Table 2 that compounds 7a, 10c, 13b, 14a, 18b and 18d were non toxic against the tested organisms.While compound 10d was very toxic and that compounds 5, 18e and 20a were harmful towards the tested organisms.

Compound
No.

HTRF kinase assay
We studied the activity of the synthesized compounds towards PC-3 prostate cancer cell line, results are demonstrated through Table 3.The c-Met kinase activity of all compounds was evaluated using homogeneous time-resolved fluorescence (HTRF) assay as previously reported [50,51].Briefly, 20 µg/mL poly (Glu, Tyr) 4:1 (Sigma) was pre-coated as a substrate in 384well plates.Then 50 mL of 10 mM ATP (Invitrogen) solution diluted in kinase reaction buffer (50 mM HEPES, Ph 7.0, 1 M DTT, 1 M MgCl 2 , 1 M MnCl 2 , and 0.1% NaN 3 ) was added to each well.Various concentrations of compounds diluted in 10 mL of 1% DMSO (v/v) were used as the negative control.The kinase reaction was initiated by the addition of purified tyrosine kinase proteins diluted in 39 mL of kinase reaction buffer solution.The incubation time for the reactions was 30 min at 25 o C and stopped by the addition of 5 mL of Streptavidin-XL665 and 5 L Tk Antibody Cryptate working solution to all of wells.The plates were read using Envision (PerkinElmer) at 320 and 615 nM.The inhibition rate (%) was calculated using the following equation: % inhibition = 100 -[(Activity of enzyme with tested compounds -Min)/(Max -Min)] x 100 (Max: the observed enzyme activity measured in the presence of enzyme, substrates, and cofactors; Min: the observed enzyme activity in the presence of substrates, cofactors and in the absence of enzyme).IC 50 values were calculated from the inhibition curves.

Inhibition of selected compounds against tyrosine kinases
The most potent compounds toward the cancer cell lines 5, 7a, 10c, 10d, 13b, 14a, 18b, 18d, 18e, 20a and 20b were further investigated towards the five tyrosine kinases c-kit, FIT-3, VEGFR-2, EGFR and PDGFR and the data were expressed through Table 4.It is clear that compounds 5, 10c, 10d, 13b, 18b, 18d, 18e and 20b showed the highest inhibitory effect while compounds 7a, 14a and 20a showed the least inhibition.It is of great value to note that compounds 10d, 18d and 20b exhibit the maximum inhibitory effect among the eleven tested compounds.Specifically, the 5-hydrazono Although they are frequently implicated in acute myeloid leukemia (AML) [52] Pim kinases are over expressed in many other types of hematological malignancies and solid tumors.Specifically, over expression has been identified in bladder [53], prostate [54], and head and neck cancers [55], chronic lymphocytic leukemia [56], multiple myeloma [57] and other B cell malignancies [58].Over expression of Pim kinases was often associated with poor prognosis in each of these cancers.This relation between the Pim-1 kinase and cancer encouraged us to study the Pim-1 kinase of some compounds.Thus, the five compounds 10d, 13b, 18b, 18d and 20b were selected for testing of their inhibition for the Pim-1 kinase (Table 5) due to their antiproliferation activities against the cancer cell lines and their high activities against c-Met kinase.
The  It is important to note that throughout the biology sections that we used two different references, foretinib was used for the cytotoxicity of compounds and c-Met enzymatic inhibition while SGI-1776 was used for Pim-1 inhibition [59].
In summary, 10c and 18b were the most potent compounds beside their non toxicity against shrimp larvae and high inhibition against c-Met kinase.The in vivo activity estimate of compounds 10c and 18b represent an integration of different factors and to find differences between in vitro and in vivo responses elucidated by these compounds is hardly surprising.For that reason molecular modeling of compounds 10c and 18b was carried out and the results showed that these two compounds were close to foretinib concerning the - interaction and the binding energy score.

Docking results of compounds
Compounds 10c and 18b were selected for molecular modeling as these two compounds the most potent compounds beside their against c-Met kinase.For each docked compound, only one pose was selected based on number of binding interactions, superposition with the original ligand, bonds were measured.The docking results obtained from the docking study Table 6.Figures 2-4 showed docking of the reference foretinib, compounds respectively.

CONCLUSION
Throughout this work the cyclohexane-1,3-dione was used for the synthesis of thiophene, pyrazole and pyran derivatives.The anti-tumor evaluation of the newly synthesized compounds against the six cancer cell NUGC, DLDI, HA22T, HEPG2, HONE1 and MCF showed that compounds 10c and 18b were the most potent compounds beside their non toxicity against shrimp larvae and high inhibition against c-Met kinase.The Pim-1 kinase test indicated that compounds 10d, 18b and 20b were the most inhibitory compounds.It can be suggested that the inclusion of aromatic rings in the structure of the compound improves the cytotoxic activity of this class of compounds in addition, the presence of halogens, hydroxyl or cyano groups directly connected to this core also has a large contribution to the increase in this activity.

Cell cultures
Cell cultures were obtained from the European Collection of cell Cultures (ECACC, Salisbury, UK) and human gastric cancer (NUGC and HR), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and normal fibroblast cells (WI38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt).They grow as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 M glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL), at 37 o C in a humidified atmosphere containing 5% CO 2 .
Exponentially growing cells were obtained by plating 1.5 x 10 5 cells/mL for the seven human cancer cell lines including cells derived from 0.75 x 10 4 cells/mL followed by 24 h of incubation.The effect of the vehicle solvent DMSO on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.The heterocyclic compounds, prepared in this study, were evaluated according to standard protocols for their in vitro cytotoxicity against seven human cancer cell lines including cells derived from human gastric cancer (NUGC and HR), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and normal fibroblast cells (WI38).All of IC 50 values are listed in Table 1 using foretinib as the reference compound.Some heterocyclic compounds was observed with significant cytotoxicity against most of the cancer cell lines tested (IC 50 = 10-1000 nM).Normal fibroblasts cells (WI38) were affected to a much lesser extent (IC 50 > 10,000 nM).

Procedure of HTRF kinase
Briefly, 20 g/mL poly (Glu, Tyr) 4:1 (Sigma) was used as a substrate in 384-well plates.Then 50 L of 10 mM ATP (Invitrogen) solution diluted in kinase reaction buffer (50 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), Ph 7.0, 1 M dithiothreitol (DTT), 1 M MgCl 2 , 1 M MnCl 2 , and 0.1% NaN 3 ) was added to each well.Various concentrations of the tested compounds diluted in 10 L of 1% DMSO (v/v) were used as the negative control.The kinase reaction was started by the addition of purified tyrosine kinase proteins diluted in 39 L of kinase reaction buffer solution.The incubation time for the reactions was 30 min at 25 o C and ceased by the addition of 5 L of Streptavidin-XL665 and 5 L Tk Antibody Cryptate working solution to all of wells.The plates were read using Envision (PerkinElmer) at 320 and 615 nM.The inhibition rate (%) was calculated using the mathematical equation: % inhibition = 100 -[(Activity of enzyme with tested compounds -Min)/(Max -Min)] x 100 (Max: the observed enzyme activity measured in the presence of enzyme, substrates, and cofactors; Min: the observed enzyme activity in the presence of substrates, cofactors and in the absence of enzyme).

General
All melting points were determined on an Electrothermal digital melting point apparatus and are uncorrected.IR spectra (KBr discs) were recorded on a FTIR plus 460 or Pye Unicam SP-1000 spectrophotometer (Pye Unicam, UK, Cambridge). 1 H NMR and 13 C NMR spectra were recorded with Varian Gemini-200 (200 MHz, Varian UK) and JEOL AS 500 MHz (JEOL, Japan) instruments in DMSO-d 6 as solvent, using Tetraethylsilane (TMS) as internal standard chemical shifts are expressed as δ ppm.The mass spectra were recorded with Hewlett Packard 5988 A GC/MS system (Hewlett Packard, Agilent, USA) and GCMS-QP 1000Ex Shimadzu (EI, 70 eV) (Shimadzu, Japan) instruments.Analytical data were obtained from on Vario EL III Elemental CHNS analyzer.Compound 3 was previously reported, although its characterization and spectral data were not presented [31].

General procedure for the synthesis of the cyclohexylidene)propanenitrile derivatives 7a,b
To a solution of compound 3 (1.68 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) either of malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.07, 0.01 mol) was added.The reaction mixture, in each case, was heated under reflux for 4 h then left to cool and the formed solid product was collected by filtration.

b]quinoline derivatives 13a,b
To a solution either of compound 7a (1.88 g, 0.01 mol) or 7b (2.35 g, 0.01 mol) in 1,4-dioxane (40 mL) containing sodium acetate (1.0 g) hydroxylamine hydrochloride (0.69 g, 0.01 mol) was added.The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture and the formed solid product was collected by filtration.

Figure 1 .
Figure 1.Structures of the most potent compounds.

a
Ten organisms (A.salina) tested for each concentration.

Table 1 .
Cytotoxicity of novel compounds against a variety of cancer cell lines a [IC50 b (nM)].

Table 1 .
The IC 50 values are the average of at least three independent experiments.As illustrated in Table1, all the tested compounds displayed potent c-Met enzymatic activity with IC 50 values ranging from 0.29 to 12.25 nM, compared with foretinib (IC 50 = 1.16 nM).

Table 6 .
Docking study data showing amino acid interactions and the hydrogren bond lengths of target compounds and foretinib on c-Met kinase enzyme.