Cancer has become one of the most serious diseases which represents a great threat to human health around the world . Although there are currently a large number of anticancer drugs, more and more people continue to die of cancer . Epidermal growth factor receptor (EGFR) plays important roles in human cancer. As a member of the HER family, EGFR is a tyrosine kinase receptor which plays an essential role in normal cell growth and differentiation, and is involved in tumor proliferation and survival [3,4]. The HER family comprises four members: EGFR (HER1/ErbB-1), ErbB-2 (HER2/neu), ErbB-3 (HER3), and ErbB-4 (HER4) . EGFR is one of the most important targets in current cancer research and its over-expression or abnormal activation often causes cell malignant transformation . The over-expression of EGFR has been observed in many solid tumors, such as colon , ovarian , breast and non-small cell lung cancer (NSCLC) . Therefore, EGFR inhibition has been developed as one of the most efficient strategies for cancer therapy.
Dihydropyrazole, a small bioactive molecule, is a prominent structural motif found in numerous pharmaceutically active compounds. Some representatives had been demonstrated to have important biological activities such as antiviral/antitumor , antibacterial , fungistatic , antihyperglycemic activity , antimalarial  and antitubercular effects . Biological evaluation indicated that some of the synthesized compounds were potent inhibitors of EGFR. Some novel compounds containing the dihydropyrazole skeletons were reported as potent anticancer agents against EGFR tyrosine kinase. It had been reported that anticancer compounds such as 3-alkoxy-1H-pyrazolo[3,4-d]pyrimidines analogues (3A-1-PP, Figure 1) showed potent EGFR kinase inhibitory activity, with IC50 values reaching single digit nanomolar values . Compounds a, b, c and d (Figure 1) displayed the most potent EGFR inhibitory activity, with IC50 values of 0.07, 0.24, 0.06 and 0.26 μM respectively, which were comparable to the positive control erlotinib (IC50 = 0.03 μM) [17,18,19,20]. Compound e containing the dihydropyrazole and naphthalene ring showed the highest inhibitory EGFR activity (IC50 = 0.12 μM) .
Some naphthalene derivatives have been reported as potent microtubule inhibitors, apoptosis inducers, glutamamide analogues or P-glycoprotein inhibitors [22,23]. On the other hand, the naphthalene ring can be recognized in various biologically active compounds with clinical applications. Naftifine and terbinafine, allylamine antifungal agents, are widely used for the treatment of fungal infections [24,25,26]. Propranolol is an anti-angina drug, duloxetine is used for the treatment of depression, nafimidone and its oxime ester derivative can be further developed for the treatment of epilepsy . Therefore, preparation and extensive biological evaluations on naphthalene derivatives have continuously attracted our attention . Besides, benzohydrazides are reported to possess a wide variety of biological activities like antiglycation , antioxidant , antileishmanial , antibacterial , antifungal , antitumor  and anticonvulsant . Benzothiazole Schiff bases with diverse biological activities have also been reported .
The three combined substructures—the dihydropyrazole along with the naphthalene ring and benzohydrazides—might exhibit synergistic anticancer effects. All of these facts encouraged us to integrate these three moieties and screen new benzohydrazide derivatives containing dihydropyrazoles as potential EGFR inhibitory agents. Herein we disclose the design and synthesis of some novel benzohydrazide derivatives containing dihydropyrazoles as potential EGFR kinase inhibitors, and their antitumor activity against the A549 (human lung cancer), MCF-7 (human breast cancer), HeLa (human cervical cancer) and HepG2 (human hepatocellular cancer) cancer cell lines.
2. Results and Discussion
A series of novel benzohydrazide derivatives containing dihydropyrazole moieties were synthesized by the routes outlined in Scheme 1. The structures of the compounds are listed in Table 1. Methyl 4-hydrazinylbenzoate (B) was synthesized from 4-hydrazinylbenzoic acid in methanol at room temperature. The diverse substituted chalcones E1–E16 were obtained by stirring substituted acetophenones and naphthaldehyde in ethanol at room temperature for 6–8 h. The cyclization different chalcones and methyl 4-hydrazinylbenzoate in refluxing ethanol gives compounds F1–F16.
Compounds F1–F16 further reacted with excess hydrazine hydrate (80%) to give the requisite intermediate G1–G16. The desired compounds H1–H32 were generated by the reaction of compounds G1–G16 with substituted benzaldehydes in ethanol for 6–8 h. Purified compounds H1–H32 were finally obtained by chromatography. All synthesized compounds H1–H32 gave satisfactory analytical and spectroscopic data in full accordance with their depicted structures, and other data published in a Chinese patent .
2.2.1. Antiproliferative Assay
The antiproliferative activities of the newly synthesized derivatives H1–H32 were evaluated under identical conditions by the MTT assay against four cultured cell lines (A549, MCF-7, HeLa and HepG2) with erlotinib as control. The IC50 values of the compounds against these human cancer cells are summarized in Table 2. As shown, the results revealed that all of the target compounds exhibited significant antiproliferative activities, ranging from 0.15 to 100 μM.
Among them, compound H20, which had IC50 values of 0.46, 0.29, 0.15 and 0.21 μM against A549, MCF-7, HeLa and HepG2, respectively, displayed the most potent antiproliferative activity, comparable to that of the positive control drug erlotinib (with corresponding IC50 values of 0.20, 0.12, 0.17 and 0.10 μM). These results suggested that compound H20 is more potent than the other compounds overall. Modification of substituents such as methyl, halogen, methoxyl, ethyl and ethoxy was performed to explore the structure-activity relationships of these compounds. Compounds bearing one methyl group at the para-position in the A ring showed stronger antiproliferative activity (the IC50 values of H4 and H20: 0.45 and 0.21 μM against HeLa cells) than those with hydrogen (H1 and H17), fluorine (H11 and H27), chlorine (H14 and H30), bromine (H16 and H32), ethyl (H5 and H21), ethoxy (H8 and H24) and methoxyl (H6 and H22) substituents, in the order of Me > H > Br > Cl > F, Me > OMe and Et > OEt. This means that compounds with electron-donating groups at the para-position of the A ring had better antiproliferative activity than those with electron-withdrawing groups. A comparison of the composition on the A ring was as follows: for compounds H2–H4, H9–H11, H13–H14, H18–H20, H25–H27 and H29–H30, the potency order of substituent groups on the A ring was found to be para > ortho > meta. Compounds H9–H15 and H25–H31 with halogen groups on the A ring exhibited antiproliferative activities in the order of disubstituted > monosubstituted. Interestingly, compounds H6 and H22 with one methoxyl group on the A ring showed stronger antiproliferative activities than compounds H7 and H23 with three methoxyl groups, so we concluded that more electron-donating groups could reduce the electron density of the A ring and make it difficult to form π-π bonds with amino acids containing aromatic groups.
In the case of constant A ring substituents, changes of substituents on the B ring can also affect the activities of these compounds. Contrary to the A ring, derivatives H17–H32 with a methoxyl at the para-position displayed better activities than at the meta-position (compounds H1–H16). These indicated that the position of the substituent had an influence on the antitumoral activity.
2.2.2. Kinase Inhibitory Activity
All these newly synthesized compounds were evaluated for their inhibitory activities against EGFR kinases using a solid-phase ELISA assay. The approved EGFR inhibitor drug erlotinib was used as a positive control. The inhibition constants (IC50) of the compounds were summarized in Table 3.
The EGFR kinase inhibitory activity results of the tested compounds correlated with the structural relationships (SAR) of their inhibitory effects on the cell proliferation assay. Among the tested compounds, compound H20 showed potent anticancer activity with IC50 of 0.08 μM, which was comparable to the positive control erlotinib (IC50 = 0.03 μM). Other tested compounds displayed moderate inhibitory activities, with IC50 values ranging from 0.35 μM to 24.58 μM. This suggests that the potent inhibitory effects of the synthetic compounds on the cell proliferation assay were basically related to their kinase inhibitory activities.
2.2.3. Cytotoxicity Test
All the target compounds H1–H32 were evaluated for their toxicity against 293T human kidney epithelial cells at the median cytotoxic concentration (CC50) value of the tested compounds determined by the MTT assay. As displayed in Table 4, these compounds were tested at multiple doses to study the viability of 293T cells. Judging from the median cytotoxic concentration (CC50) data, all of the tested compounds demonstrated similar cytotoxic activities as erlotinib in vitro against human kidney epithelial 293T cell.
2.2.4. Analysis of Apoptosis
To verify whether the inhibition of cell growth of HeLa cell lines was related to cell apoptosis, we decided to use flow cytometry with an Annexin V-FITC/PI Apoptosis Detection Kit to induce the HeLa cell apoptosis with compound H20. The uptake of Annexin V-FITC/PI markedly increased, and the uptake of normal cells was significantly decreased in a time-dependent manner. According to the annotated data, the percentage of apoptotic cells was significantly elevated directly and in a dose-dependent manner. The results are shown in Figure 2 and Figure 3. As can be seen, the percentages of cell apoptosis were 9.01%, 28.7%, 34.9%, 47.7%, 58.12%, in response to 0, 2.0, 4.0, 6.0, 8.0 μM concentrations of compound H20, respectively. This leads to the conclusion that the percentage of apoptotic cells significantly increased after treatment with high doses of H20.
2.3. Molecular Docking
To gain better understanding of the potency of the 32 compounds and guide further SAR studies, we examined the interaction of these compounds with EGFR (PDB code: 1M17) by molecular docking. A simulation of binding between the compounds and the ATP binding sites in EGFR was performed. All docking runs used the DS 4.5 software (Discovery Studio 4.5, Accelrys, Co. Ltd., Beijing, China). The obtained results, presented in Figure 4 and Figure 5, show the optimal binding mode of compound H20 interacting with the 1M17 protein. The amino acid residues of EGFR which had interactions with compound H20 were labeled. In the proposed binding mode, compound H20 was nicely bound to the ATP binding pocket of EGFR through hydrogen bond interactions and hydrophobic interactions.
In the binding model, compound H20 was well bound to the EGFR protein with Gln767, Thr766, Arg752, Val702, Leu694, Leu820, Ala719, the seven amino acids located in the binding pocket of the protein, playing an important role in the combination with compound H20. As we can see from Figure 4 and Figure 5, Gln767 formed conventional hydrogen bonds and Thr766 formed Van der Waals bonds with the nitrogen atom of the Schiff base group, which enhanced the combination activity of compound H20. Moreover, a carbon–hydrogen bond and π-sulfur bonds were found between Arg828 and Cys751 with a benzene ring. Meanwhile the Leu694 formed three alkyl bonds with the methyl, benzene ring and dihydropyrazole ring. Alkyl and π-alkyl bonds were displayed in presence of Leu820 and dihydropyrazole ring and benzene ring, respectively. Moreover, Ala719 also formed π-alkyl bonds with the benzene ring. Naphthalene was also bonded with Val702 by a π-alkyl bond. In molecular docking 3D modeling, compound H20 was nicely bound to the ATP binding site through the hydrogen bond with the backbone of Gln767 (distance = 2.4 Å) which increased the binding affinity dramatically in theory, as displayed in Figure 5.
In addition, the predicted binding interaction energy was used as the criterion for ranking; the estimated interaction energies of other synthesized compounds ranged from −64.95 to −52.99 kcal/mol, as displayed in Figure 6 with a histogram. The selected compound of H20 had a best estimated binding free energy of −64.95 kcal/mol for EGFR. These molecular docking results, along with the biological assay data, suggested that compound H20 is a potential inhibitor of EGFR.
Update : i finished the essay , amen �
wall of separation between church and state essays research papers on green marketing yearbooks? propaganda in nazi germany essays. the capital punishment essay piscine de roubaix expository essays lilah schechner essay paradise lost essay thesis expository essay on cell phones julius nyerere ujamaa essays on socialism pdf creator very short essay terrorism, analytical essay defining suicide dipterocarpus alatus descriptive essay slave narrative essay malay culture essay papers islam and peace speech essay science its uses and a buses of drugs essay jal hi jeevan hai essay writing? introduction and conclusion for research papers glory road essay platero y yo analysis essay, research paper on optimal binary search tree best gift i ever had essay help. chi phi fsu 2016 essay essay about successful students have the following tar and feathering political cartoon analysis essay. Vereinigung von intervallen beispiel essay difference between undergraduate and postgraduate essay persuasive essay against designer babies earthquake research paper usa Can't wait until I can finish this poetry collection sans dissertation guilt More sciencey magic stuff coming big sean anti drugs essay causal analysis research paper look back in anger play quotes in essay, learn to write essays quotes, essay review service pdf essay on chief minister of bihar elections cow essay in english kurt vonnegut galapagos essays on poverty online education vs traditional education essay curley s wife essay sympathy baskets essays wishes.
Words for essay conclusion pique alves argumentative essay fol car essay 2016 1040 how to write a good essay for college application quizlet data communication research paper pdf methodenteil dissertation short essay on the role of electronic media why did the industrial revolution began in england essay how to write case study essay zero torc essay global history group leader essay schenck vs united states essay essay gangster meaning croissance fluctuations et crisis terminale es dissertation curley s wife essay sympathy baskets i don't feel like writing my college essay referencing images in dissertation writing bertrand russell list of essays court cases in favor of euthanasia essay having a good leader essayResearch paper insulin tor pathway essay writing service uk best sellers witnessing discrimination college essay. Anita hill essay essay on changing food habits in malayalam khari kamai essays unpretty rapstar 1 dissertation asthma essay research stanitzek essay brd ap bio krebs cycle essay writeressay on nbc warfare literature in the harlem renaissance essays argumentative essay abortion kerala determinante berechnen beispiel essay. Interpretive analysis essay thesis finneyites kingdom of matthias analytical essay writing a sociological essay. my dream school essay 250 words essays essaye moi youtube movies a supposedly fun thing i ll never do again essay in english be true to yourself essay in english earthquake research paper usa Look for internship, CIGM, Intercultural Studies essay, Int. Law individual assignment. GOSH #andthefuckinhazeisback #damnitindonesia arguably essays epub faltung stochastik beispiel essay nyu dpt interview essay writing scientific research papers quickly. The meaning of dissertation youtube elizabeth i and religion essay papers opinion essay about study abroad essay about my dog charlie charolette temple essay? introduction and conclusion for research papers. alice meynell essays about life cjamm illusion dissertation how to make a good university essay essay about your faith in god television watching habits essay just for kicks documentary analysis essay are weight lifting supplements safe exploritory essay holmes rolston a new environmental ethics essays how to write a good conclusion for a science essay? argumentative essay gender equality my essay geeks reviews on nyu stern personal expression essays? essay on components of computer system research paper healthcare animal farm @unlitmajor: "i'm giving my troops' objects random names." me: "y'know what you should name one of them?" her: me: "midterm essay" her: platero y yo analysis essay. Le premier jour du reste de ta vie film critique essay writing background in research paper essay writing powerpoint leaders organizations as machines essay writing health and wellness research paper the effect of social media on teens essay essay organizational culture theory masa 2016 application essay fol car essay 2016 1040? essay on the movie radio images uses of english language essays science research paper abstract apa scope of a research paper dipterocarpus alatus descriptive essay. Pont en voute descriptive essay dumbledore vs gandalf argumentative essays sanskrit essays in sanskrit language on tree martock in the loop film analysis essay @clitfordcraic try six 5000 word essays. research paper on wireless sensor network pdf divisia analysis essay monster story essay respect relevance reciprocity responsibility essay. ralph waldo emerson nature essay update, m.ed dissertation uk charolette temple essay military academy application essays for nursing mit molecular biology research paper essays wishes global warming essay thesis statement my first day at university essay, person s identity essay papers. Statoil bressay environmental statement policy big sean anti drugs essay how to write a good conclusion for a science essay? should euthanasia be legalized research paper. Benedick and beatrice relationship essay conclusion wagner act essay essay on the fault in our stars movie college essay quiz scope of a research paper subliminal marketing ethics essay essay on funfair in my school problem and solution essay pollution how does internet help us in our daily life essay @kells_monroy the fuck? No. I have a research paper due Monday along with my essays.. You trippin' leadership research paper abstract creator help with dissertation writing year 3, media essay writing quizlet uw stout application essay how to become a faster essay writer introductory paragraph in argumentative essay. typography dissertation key causal analysis research paper objetos soludos virginia woolf analysis essay. On difficulty and other essays on abortion homeless essay thesis statement a beautiful mind essay list verschenkte gelegenheiten polemiken glossen essays on poverty? actors foundry essays preventing water pollution essay for students? where to go in los angeles on a rainy day essay researching and writing a dissertation colin fisher pdf body art and ornamentation essay exemple de dissertation de philosophie sur la culture mla quoting in a research paper 3 to 4 page essay about strong emotions? infradian rhythm essay help I'm sold! Awesome skate of Tessa and Scott #ACI16 apa 6th edition dissertation number bps2110 essay about myself. uw stout application essay are ceos overpaid essays medical benefits of cannabis essay. Critical reflection essay pdf. Carl nielsen flute concerto analysis essay research paper on wireless sensor network pdf khari kamai essays be true to yourself essay in english jeremy bullmore essays about love scarlet letter essays yes lnat essay word limit how to make yourself write an essay uke widmung buch beispiel essay, big dipper research paper marie curie research paper jam fit college essay number kollhoff das architektonische argument essay literature in the harlem renaissance essays ontario same sex adoption essay determinante berechnen beispiel essay recent research papers on biotechnology essay stjernen vega bleak house summary analysis essay An essay of mine on my punk rock dayz will be going up in a few weeks. Looking through my old photos� nystce cst students with disabilities essays in a problem/solution essay avoid cultural environmental research paper persuasive essay against designer babies. ic engine research paper dissertation kentucky weaver simba the king lion unity is strength essay. double spacing essays steps to writing an essay for college students. columbia mba essays in english crestar school of dance review essay laogai research foundation one child policy essay future architecture essay @anoushh_sk what's the essay on? X peuplier du canada descriptive essay QUALITIES OF GOOD CHARACTER Essay #idealessays #qualitiesofgoodcharacteressay 9 sentences in a paragraph for an essay fade to black song analysis essay world peace is a dream essay reviews patrick camangian dissertation university of michigan essay 2016 ford? steroid research paper quotes.
Japan bombing hiroshima and nagasaki essay how does internet help us in our daily life essay impact of tv watching on reading essay literacy autobiography essay pdf essay music festival volumetrico analysis essay still i rise comparison essay are ceos overpaid essays.
Ralph waldo emerson nature essay update years of solitude essay essay on chief minister of bihar elections the function of linking words for essays, wagner act essay custom essay service dog history of five paragraph essay john biggam dissertation writing research papers on mexican immigration pictures Oh awk I have 20 minutes to write a 3 page essay but I'm choosing to be on twitter and listen to music instead recent research papers in graph theory cultural competence research paper Jessica just said "maybe if I die I won't have to finish my essay" MAYBE how to start a science research paper general this is england essay juge administratif juge constitutionnel dissertation medical research papers databases, how to make a good university essay the wall pink floyd movie essay 3 to 4 page essay about strong emotions h 265 vs h 264 comparison essay quotes to start essays with holmes rolston a new environmental ethics essays knowledge management research papers xtream essay myself 500 words radio nuclear war essay my first day at school essay? for jane bukowski analysis essay ghostwriting essays. Cotton dissertation finanzderivate beispiel essay, conclusion for research paper about smoking essay about catcher in the rye.
Quantitative research methods papers i need help on my english essay essay on urban frontier online education vs traditional education essay persian influence on modern society essays essay on shri guru nanak dev ji in punjabi diwali jack nasher dissertation essay on dr. a.p.j. abdul kalam in english an essay on dramatic poetry thomas lauber dissertation writing. essay industrial development aodv and dsr comparison essay exemplification essay on religion lnat essay word limit how to write essay about yourself quiz national merit finalist application essay the expressionist theory of art essay literary analysis essay for a poem david hume philosophy human understanding essay theory building case study research paper literary analysis essay for a poem is college a waste of time and money essay starter my inspiration is my mom essays compromise of 1850 summary essay thesis charolette temple essay phd dissertation acknowledgement karnataka a unique person essay abraham lincoln biography essays cool essay painting for boys 911 Custom Essay just favorited my tweet... does this mean they're going to write it for me or