Accepted Manuscript Synthesis and Anticancer Screening of Novel Polynuclear Heterocyclic Compounds Derived from 2, 3-Diaminophenazine Asma. M. Mahran, Sherif. Sh. Ragab, Ahmed I. Hashem, Mamdouh M. Ali, Afaf A. Nada PII:

S0223-5234(13)00794-0

DOI:

10.1016/j.ejmech.2013.12.007

Reference:

EJMECH 6598

To appear in:

European Journal of Medicinal Chemistry

Received Date: 22 August 2013 Revised Date:

7 December 2013

Accepted Date: 9 December 2013

Please cite this article as: A.M. Mahran, S.S. Ragab, A.I. Hashem, M.M. Ali, A.A. Nada, Synthesis and Anticancer Screening of Novel Polynuclear Heterocyclic Compounds Derived from 2, 3-Diaminophenazine, European Journal of Medicinal Chemistry (2014), doi: 10.1016/ j.ejmech.2013.12.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Novel series of phenazine derivatives were synthesized and evaluated as inhibitors of the proliferation of lung carcinoma (A549) and colorectal cancer (HCT116) cell lines through inhibition of human TRK activity

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Synthesis and Anticancer Screening of Novel Polynuclear Heterocyclic Compounds Derived from 2, 3-Diaminophenazine

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Asma. M. Mahran*a, Sherif. Sh. Ragab a, Ahmed I. Hashemb, Mamdouh M. Alic, Afaf A. Nadaa Photochemistry Department, National Research Center, Dokki, Giza, Egypt.

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Department of Chemistry, Faculty of Science, Ain Shams University, Egypt

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Biochemistry Department, Division of Genetic Engineering and Biotechnology,

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National Research Centre, Dokki, Giza, Egypt

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Abstract:

2,3-diaminophenazine 1 was used as a precursor for the preparation of some novel phenazine derivatives such as imidazo[4,5-b]phenazine-2-thione 2, its methylthio 3, ethyl 1-aryl-3H-[1,2,4]triazolo[2,3,a]imidazo[4,5-b]phenazines 8a-c, ethyl (2Z)-[3aminophenazin-2-yl)amino](phenylhydrazono)ethanoate 9, pyrazino[2,3-b]phenazine derivatives

10, 12, 15-17, [1,4]diazepino[2,3-b]phenazine derivatives 13, 14, 2,3-

dibenzoylamino-phenazine 18, 1H-Imidazo[4,5-b]phenazine derivatives 20, 23a-c, 24,

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25 and 4-[(E)-(3-Amino phenazin-2-yl)diazenyl] derivatives 27-29. All compounds were tested as inhibitors of the proliferation of human lung carcinoma and colorectal cancer cell lines and through inhibition of tyrosine kinases. Most of compounds exert good activity against the two cancer cell lines. Compound 2 was found to be more

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active than the standard drug Cisplatin toward both cancer cell lines.

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Keywords: Phenazine, Imidazole, Diazepine, Pyrazine, Azo, Antitumor activity

*Correspondence: E-mail: [email protected] Phone/fax: +20237492641

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1. Introduction: It was reported that some phenazine derivatives demonstrated significant antimicrobial [1], antimycobacterial [2], antifungal activity [3-5], and are used in a large scale as anticancer [6], anthelmintic [7-9], antibiotic drugs [10-11]. On the other hand,

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they have potential therapeutic antitumor application [12], act as chemo preventive agent [13] and they recorded a great ability of reduction in blood and urine glucose level

[14]. Conventional chemotherapy, although directed toward certain macromolecules or enzymes, typically does not discriminate effectively between rapidly dividing normal cells (e.g., bone marrow and gastrointestinal tract) and tumor cells, thus leading to

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several toxic side effects. Tumor responses from cytotoxic chemotherapy are usually

partial, brief, and unpredictable. In contrast, targeted therapies interfere with molecular

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targets that have a role in tumor growth or progression. These targets are usually located in tumor cells, although some like the antiangiogenic agents may target other cells such as endothelial cells [15]. Thus, targeted therapies have a high specificity toward tumor cells, providing a broader therapeutic window with less toxicity. They are also often useful in combination with cytotoxic chemotherapy or radiation to produce additive or synergistic anticancer activity because their toxicity profiles often do not overlap with traditional cytotoxic chemotherapy. Thus, targeted therapies represent a new and

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promising approach to cancer therapy, one that is already leading to beneficial clinical effects. There are multiple types of targeted therapies available, including monoclonal antibodies, inhibitors of tyrosine kinases and antisense inhibitors of growth factor

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receptors [15].

Protein kinases (PKs) are indispensable for numerous processes in the cell.

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Tyrosine kinases are important mediators of signal transduction process, leading to cell proliferation, differentiation, migration, metabolism and programmed cell death (apoptosis) [16]. These enzymes catalyze phosphorylation of different cellular substrates. Phosphorylation in turn regulates various cellular functions. Normally, their activity is stringently regulated. However, under pathological conditions PKs can be deregulated, leading to alterations in the phosphorylation and resulting in uncontrolled cell division, inhibition of apoptosis, and other abnormalities and consequently to diseases [17]. Various cancers and other diseases are known to be caused or accompanied by deregulation of the phosphorylation. Inhibition of PKs has been shown to be a promising therapeutic strategy for cancer. In view of the above mentioned 2

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findings and as continuation of our efforts [18-20] in designing new potent, selective and less toxic biologically active compounds, we would like to report here simple convenient methods for the synthesis of some phenazine incorporating different heterocycles to evaluate them as inhibitors of the proliferation of the human lung

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carcinoma A549 and colorectal cancer HCT116 cell lines through inhibition of human TRK activity.

2. Results and discussion 2.1. Chemistry

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The required starting 2,3-diaminophenazine 1 was prepared as previously reported procedure [21]. Refluxing compound 1 with carbon disulfide in pyridine

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afforded imidazo[4,5-b]phenazine-2-thione 2. The structures of the latter product were based on microanalytical and spectroscopic data. The IR spectrum showed two absorption bands at 3495, 1425 cm-1 due to NH, and C=S group respectively. The mass spectrum reveled a molecular ion peak at m/z (%) 252 (M+, 100) corresponding to C13H8N4S. Treatment of 2 with methyl iodide in the presence of sodium ethoxide in refluxing ethanol afforded 2-methylsulfanyl-imidazo[4,5-b]phenazine 3. The structure of 3 was established on the bases of its elemental analysis and spectral data. The mass

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spectrum showed a molecular ion peak at m/z (%) 266 (M+, 100) corresponding to C14H10N4S. While its 1H NMR revealed the presence of singlet signal at δ 2.47 ppm characteristic of the S-CH3 protons (c.f. Experimental, Scheme 1). The reaction of 2 with ethyl N1-phenylhydrazon-N2-chloroacetate 4a-c [22] in ethanol in the presence of

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sodium ethoxide under reflux, gave in each case after work up only isolable products 8a-c. The structures of the latter products 8a-c were confirmed on the basis of

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microanalytical and spectral data. The mass spectrum of 8a as an example showed a molecular ion peak at m/z 408 (M+) corresponding to formula C23H16N6O2. Its IR spectrum showed absorption band at 1727 characteristic of C=O (ester), while its 1H NMR revealed the presence of a triplet signal at δ 1.38-1.41 ppm and quartet signal at δ 4.50-4.55 ppm characteristic of the ethyl ester protons. To account for the formation of 8 from reaction of 1 with 4, the step reaction mechanism outlined in (Scheme 1) is suggested. The reaction involves an initial formation of thiohydrazonate ester 5 via nucleophilic substitution of chloride in 4. The formed thiohydrazonate 5 undergoes in situ Smiles rearrangement [23-26] under the reaction conditions employed, to give the thiohydrazide intermediate 6, which in turn undergoes cyclization with concurrent loss 3

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of hydrogen sulfide to give the respective 8a-c (Scheme 1) The assignment of the formation of 8a-c was further supported by alternative synthesis of 8. Thus, treatment of 2-methylthio derivative 3 with 4 in ethanol in the presence of sodium ethoxide under reflux led to the formation of products that proved to be identical in all respects (m.p.,

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mixed m. p. and IR) with compound 8. Evidently, the mechanism proposed for the reaction of 3 with 4 proceeds through two steps, the first step involves acylation of 3

with 4 to give amidrazone derivative intermediate 7, which in turn undergoes in situ cyclization with concurrent elimination of methanthiol to give 8a-c as end products

(Scheme 1)

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(Scheme 1)

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Treatment of equimolar quantities of 1 and ethyl N1-phenylhydrazon-N2chloroacetate 4 in ethanol/DMF mixture at reflux temperature in the presence of triethylamine afforded a new compound, namely ethyl (2Z)-[3-aminophenazin-2yl)amino]-(phenylhydrazono) ethanoate 9. The mass spectrum of 9 showed a molecular ion peak at m/z 400 (M+) corresponding to molecular formula C22H20N6O2. Its IR spectrum showed absorption bands at 3320 and 1720 cm-1 attributed to the presence of NH, CO ester, respectively. Treatment of 9 with acetic anhydride/acetic acid mixture at

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reflux afforded single product 10 or 11 as evidenced by TLC analysis. The mass spectral data and elemental analysis of this product are consistent with the structure 10 (Scheme 2). Also, the 1H NMR spectrum does not contain the characteristic signals of the ethoxy group protons. (c.f. Experimental, Scheme 2). Oxidation of 10 with hydrogen

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peroxide gave product whose mass spectrum showed a molecular ion peak at m/z 352 (M+) corresponding to molecular formula C20H12N6O. The IR spectrum showed

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absorption bands at 3411 (NH), 1680(C=O), 1650(N=N) corresponding to 2-oxo-3phenylazo-1H-pyrazino[2,3-b]phenazine 12 (Scheme 2).

(Scheme 2)

Refluxing equimolar quantities of 1 and ethyl acetoacetate in DMF gave one

isolable product as evidenced by TLC analysis of the crude product. The mass spectral data and elemental analysis indicated a molecular formula C16H14N4O. The IR spectrum showed bands at 3368, 3061 (2NH), 2977 (CH3), 2927 (CH2), and 1660 (C=O) corresponding to 6H-7-methyl[1,4]diazepino[2,3-b]phenazine-9-one 13 (Scheme 3). 4

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The same nucleus with different derivative 6H-[1,4]diazepino[2,3-b]phenazine-7,9dione 14 was also obtained by reaction of 1 with malonic acid in presence of acetic acid. Structure of 14 was confirmed on the basis of its elemental analysis and spectral data. The mass spectrum of 14, showed a molecular ion peak at m/z 278 (M+) corresponding

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to molecular formula C16H12N4O. The IR spectrum showed three absorption bands at 3418, 2922 and 1665 cm-1 attributable to NH, CH2 and C=O groups respectively (c.f. Experimental, Scheme 3).

The reaction of 2,3-diaminophenazine 1 with chlorocarbonyl compounds was

also studied. Thus, the reaction of 1 with oxalyl chloride or dichloromaleic anhydride

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under Phillips condition (i. e. 4 N HCl) [27], led to the formation of pyrazino [2,3-b]phenazine-2,3-diones 15, 16 respectively in moderate yields. The mass spectrum of 15

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showed a molecular ion peak at m/z (278.26) corresponding to molecular formula C15H10N4O2. When 15 was treated with o-phenylenediamine in DMF, it gave quinoxalino [2,3;2',3']pyrazino[2,3-b]phenazine 17 in good yield. The structure of 17 was proved from its elemental analyses and spectral data. Thus, the IR spectrum of 17 revealed the absence of carbonyl absorption and the presence of absorption band at 3442 cm-1 due to NH. Additionally, its mass spectrum showed a molecular ion peak at m/z 336 (M+). The IR spectrum of 16 showed absorption band at 3418 cm-1

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characteristic of (NH), and doublet band near 1650 cm-1 due to 2(C=O). Furthermore, its mass spectrum showed a molecular ion peak at m/z 306 (M+) which is consistent with the assigned structure 16 (c.f. Experimental, Scheme 3). Refluxing 1 with excess of benzoyl chloride in the presence of sodium

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hydroxide gave one isolable product 18 whose mass spectral data and elemental analysis indicated a molecular formula C26H19N4O2. The 1H NMR revealed singlet

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signal at δ 12.9 ppm assigned for NH and disappearance of NH2 signal at 6.68 ppm. Additionally, the IR spectrum of 18 revealed two absorption bands at 3374, 1652 cm-1 characteristic of (NH) and (C=O) respectively, corresponding to the assigned structure 2,3-dibenzoylamino-phenazine 18 (c.f. Experimental and Scheme 3).

(Scheme 3) Heating under reflux a solution of 1 with ethoxy-methylene malononitrile [28] in DMF afforded the corresponding imidazo[4,5-b]phenazine 20 via facile intramolecular cyclization of the intermediate 19 by elimination of malononitrile. The assignment of 5

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structure 20 to the isolated product was confirmed by alternate synthesis. Thus, refluxing 1 with diethyl orthoformate in the presence of acetic acid afforded a product which was proved to be identical in all respects (m.p., mixed m.p. Mass, and IR) with 20 obtained above (Scheme 4). Besides the correct values of elemental analysis of 20,

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its mass spectrum showed a molecular ion peak at m/z 220 (M+) corresponding to formula C13H8N4. The 1H NMR spectrum showed two signals at δ 8.90 and δ 11.68 ppm corresponding to CH and NH respectively (c.f. Experimental).

Refluxing 2,3-diaminophenazine 1 with aldehydes (3-chlorobenzaldehyde, 2,4dichlo-robenzaldehyde, cinnamaldehyde) in DMF in presence of acetic acid, afforded

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the 2-aryl-1H-imidazo[4,5-b]phenazine derivatives 23a-c respectively. The structures of

23a-c were confirmed by their correct microanalyses and compatible spectroscopic data (mass, IR, 1H NMR) (see Experimental). The mass spectra of 23a,b, showed a

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molecular ion peak at m/z 331 (M++2), 367 (M++2) respectively. The IR spectra of compounds 23a-c revealed in each case the presence of characteristic NH band in the region 3404-3423 cm-1 and lack of any significant absorption bands at 3450 and 3150 cm-1 characteristic of stretching vibrations of the two amino groups. Their 1H NMR spectra showed, in addition to the aromatic protons, a signal near δ 13.4, 13.08 (s, NH) respectively, and the disappearance of signal at δ 6.24 ppm characteristic of (NH2).

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Compound 23c showed in addition to the signals of aromatic protons, the presence of signal at δ 7.00-7.43 assigned to -CH=CH- protons. The reaction of 1 with equimolar quantities of benzoic acid or butyric acid respectively was carried out in dimethylfomamide in the presence of phosphorus

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oxychloride / acetic acid (Scheme 4) afforded in each case one isolable product as evidenced by TLC analysis of the crude product. The structures of the latter products

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24, 25 were established from microanalyses, mass, IR and 1H NMR. For example, the mass spectrum of 25 showed a molecular ion peak at m/z 262 (M+) corresponding to formula C16H14N4. The 1H NMR spectrum showed beside the signals of aromatic protons, the presence of methyl protons signal at δ 1.09-1.13 ppm and signals of CH2CH2- at δ 1.19-1.22 and δ 2.47-2.58 ppm respectively (c.f. Experimental and Scheme 4).

(Scheme 4)

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Finally 2,3-diamiophenazine 1 was diazotized with hydrochloric acid and sodium nitrite at 0-5oC, to afforded the corresponding 2-aminophenazinediazonium salt 26. The latter was coupled with appropriate methylene compounds namely (3-methyl 1-phenyl-5-pyrazolone, 3-phenyl-5-isoxazolone and 5,5-dimethyl-cyclohexane-1,3-

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dione) in sodium acetate solution to give in each case one isolable product as evidenced by TLC of the crude products. The unreported new products were expected to present

in the azo form 27-29 or hydrazo 30-32. The structure elucidation of these coupling products was based on their spectral and analytical data. For example, in the mass spectra; all compounds gave the molecular ion peaks at the expected m/z values (c.f.

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Experimental). The IR spectra of the isolated products revealed the presence of

absorption bands at 3482, 3414, 3412 cm- 1 respectively attributable to NH2 group, and

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absorption bands near 2931, 2919, 2958 cm-1 characteristic of CH cyclic, in addition to the characteristic bands of carbonyl group at 1631, 1685, 1611 respectively. Additionally, the 1H NMR spectra revealed the presence of a singlet signal due to the proton of C-H at δ (4.9, 5.22, 5.29) ppm respectively (c.f. Experimental). On the basis of these findings, the isolated products from the studied reaction were assigned structures 27-29 (azo tautomers) rather than the hydrazo tautomers 30-32 (Scheme 5).

2.2 Antitumor Activity

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(Scheme 5)

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Protein kinases (PKs) are indispensable for numerous processes in the cell. These enzymes catalyze phosphorylation of different cellular substrates. Phosphrylation in turn regulates various cellular functions. Normally, their activity is stringently

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regulated. However, under pathological conditions PKs can be deregulated, leading to alterations in the phosphorylation and resulting in uncontrolled cell division, inhibition of apoptosis, and other abnormalities and consequently to diseases [17]. Various cancers and other diseases are known to be caused or accompanied by deregulation of the phosphorylation. Inhibition of PKs has been shown to be a promising therapeutic strategy. Many PK inhibitors (PKIs) have been produced and tested in clinic by now. These molecules have a low molecular weight and most of them bind to protein kinases competing with ATP for the ATP-binding site [29]. The discovery of newer inhibitors has provided an opportunity of many researchers in the future. 7

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In this work we designed phenazine incorporating different heterocyclic derivatives in an attempt to use them to inhibit the proliferation of the human lung carcinoma (A549) and colorectal cancer (HCT116) cell lines through inhibition of

2.2.1 Antiproliferative activity

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human TRK activity.

As shown in table 1, the anticancer activity of the newly synthetic compounds

was tested using SRB assay as described by Skehan et al. [30] in A549 and HCT116 cell lines. For comparison, cisplatin was also tested. The results revealed that most of

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the tested compounds exert their activity against both cell lines.

In case of lung cancer cell line A549 and colorectal cancer cell line HCT116

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although compounds 24, 23c, 9, 10, 15, 13, 14 and 17 showed no activity in the two cell lines, nearly 5 compounds (1, 2, 3, 25 and 28) were mostly antiproliferative agents as potent as the standard drug, cisplatin. Compound 2 was more potent than the cisplatin in both A549 and HCT116. Other compounds (20, 23a, 23b, 16, 18, 8a, 8b, 8c, 27 and 29) showed moderate to little activity to inhibit proliferation of the two cancer cell lines.

2.2.2 Protein kinase inhibition

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The results showed that most of the tested compounds showed potent inhibition of TRK in human lung carcinoma A549 cell line and colorectal cancer HCT116 cell line as compared to the inhibition for the untreated cells as listed in (Table 1) and the results of inhibition were in consistent with the anticancer activity. In case of A549, nearly 5

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compounds (1, 2, 3, 25 and 28) were found to be potent and selective similar to the positive drug, cisplatin (87%) against human TRK with percentage of inhibition values

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were 80, 86, 85, 70 and 72% respectively as compared with control untreated cells. In case of HCT116, the same compounds (1, 2, 3, 25 and 28) were found to be potent and selective similar to the positive drug, cisplatin (85%) against human TRK with percentage of inhibition values were 72, 85, 84, 64 and 68% respectively as compared with control untreated cells. From the foregoing result it is clear that compound 2 inhibited the human TRK activity equal or more potent to cisplatin in both cell lines. These results were consistent with cell cytotoxicity activity.

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3. Conclusions

In this work we designed and synthesized new series of phenazine derivatives in an attempt to use them to inhibit the proliferation of the human lung carcinoma (A549)

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and colorectal cancer (HCT116) cell lines through inhibition of human TRK activity. The result revealed that most of the new compounds showed good activity against the two cancer cell lines and also good inhibition of human tyrosine kinase. Compound 2 was more potent than the standard drug cisplatin in both A549 and HCT116 and

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inhibited the human TRK activity equal or more potent to cisplatin in both cell lines. It was found that the results of inhibition of human tyrosine kinase were consistent with

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cell cytotoxicity activity. 4. Experimental section 4.1. Chemistry

Melting points were determined on Electrothermal Engineering LTD apparatus and are uncorrected. Infrared spectra were measured on KBr water technique on a Jasco,

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FT/IR 6100, Japan. 1HNMR and 13C- NMR spectra were obtained using a JEOL, ECA, (500 MHz) with TMS as internal standard at National Research Centre. Mass spectra were performed using 70 Kratos on Schimadzu model GC-MSQP 1000 EX equipment at Cairo University. The biological activity was performed at Biochemistry Department,

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Division of Genetic Engineering and Biotechnology, National Research Centre, Dokki, Giza, Egypt. Compound 1 was prepared according to a reported method [21].

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4.1.1 Representative procedures for synthesis of 2 and 3 Imidazo[4,5-b]phenazine-2-thione 2: 2,3-Diamiophenazine 1 (5 mmol, 1.05 g) was added to a mixture of 85 ml of pyridine and 20 ml of carbon disulfide and the solution was refluxed for 3 hours. The cold solution was acidified to PH 1 with HCl and cooled. The crude thione derivative was collected, washed with water and acetone and dried to yield 2.79 g (42%). The material was further purified by precipitation from warm (50 ml) dilute ammonia with acetic acid PH6. 9

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Compound 2 formed yellow crystals from ethanol /dioxane, yield: (2.58 g, 70 %); m.p. 350-352°C. IR (KBr, υ, cm-1): 3495 (NH), 1597 (C=N), 1425 (C=S). MS m/z (%): 254 (M++2, 1.01), 253 (M++1, 6.38), 252 (M+, 100), 225 (1), 193 (4), 180 (1), 140 (2), 126 (8) 102 (5), 77 (3). 1H NMR (DMSO-d6, δ ppm): 7.15 (s, 2H, Ar-H), 7.72-7.84 (d, 2H,

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Ar-H), 8.13-8.20 (d, 2H, Ar-H), 13.03 (s, 2 H, 2NH). Anal. Calcd. for C13H8N4S (252.29): C, 61.89; H, 3.20; N, 22.21; S, 12.71. Found: C, 61.79; H, 3.15; N, 22.11 ;S, 12.70 %. 2-Methylsulfanyl-imidazo[4,5-b]phenazine 3:

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To a stirred ethanolic sodium ethoxide solution prepared by dissolving (0.115 g, 5

mmol) of sodium metal in 30 ml ethanol, 2 (1.26 g, 5 mmol) was added. The resulting

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solution was treated portionwise with methyl iodide (5 mmol, 0.7 ml). After the addition was complete, the reaction mixture was refluxed for 10 hours. The solid precipitated was filtered off, washed with water, air dried and finally recrystallized from ethanol/ dioxane mixture to give the respective 2-methylsulfanyl-imidazo[4,5b]phenazine 3.

Yellow powder from ethanol / dioxane mixture, yield: (0.66 g, 63 %); m.p. 222-224 °C. IR (KBr, υ, cm-1): 3385 (NH), 2942 (CH), 1462 (C=N). MS m/z (%): 266 (1.00), 240

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(1.15), 211 (1.48), 156 (2.68), 98 (28.17), 78 (47.01). 1H NMR (DMSO-d6, δ ppm): 2.47 (s, 3H, S-CH3), 7.8-7.92 (m, 6H, Ar-H), 8.13(s, 1H, NH). Anal. Calcd. for C14H10N4S (266.31): C, 63.14; H, 3.78; N, 21.04; S,12.04. Found: C, 63.19; H, 3.75; N,

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21.01; S, 12.06 %.

4.1.2 General procedure for synthesis of 3-ethoxycarbonyl-1-aryl-1H-[1,2,4]triazolo[3',4':2,3]imidazo[4,5-b]phenazine 8a-c:

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To a stirred ethanolic sodium ethoxide solution (prepared by dissolving 0.059 g of sodium metal in 20 ml ethanol) and absolute ethanol (10 ml), 2 (0.003 mol, 0.756 g) was added. To the resulting solution, ethyl chloro (arylhydrazono) Ethanoat 4a-c (0.003 mol) was added portionwise. After the addition was complete, the reaction mixture was refluxed until the odour of hydrogen sulfide evolving ceased (odour of rotten eggs which make blackness to lead acetate paper). The solid precipitated was filtered off, washed with water, air dried and finally recrystallized from ethanol/dioxane mixture to give the respective triazolo[3',4':2,3]-imidazo-[4,5-b]phenazine derivatives 8a-c in 6978% yield. 10

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3-Ethoxycarbonyl-1-phenyl-1H-[1,2,4]triazolo[3',4':2,3]imidazo[4,5-b]-phenazine 8a: Brown crystals from ethanol / dioxane, yield: (0.58 g, 77 %); m.p. 265-267°C. IR (KBr) IR (KBr, υ, cm-1): 3100 (CH aromatic), 2924 (CH aliphatic), 1726 (C=O ester), 1649 (C=N), 1594 (C=N). MS m/z (%): 408 (M+, 9.26), 407 (M+-1, 2.71), 381 (11.32),

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335 (5.60), 252 (15.81), 210 (2.06), 166 (4.85), 135 (8.33), 91 (27.39), 77 (54.17), 29

(100). 1H NMR (DMSO-d6, δ ppm):1.38-1.41 (t, 3H, -CH2CH3), 4.54-4.55 (q, 2H, CH2CH3), 7.40-7.42 (m, 5H, Ar-H), 8.07-8.08 (m, 6H, Ar-H). Anal. Calcd. for C23H16N6O2 (408.41): C, 67.64; H, 3.95; N, 20.58. Found: C, 67.59; H, 3.75; N,

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20.61%.

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3-Ethoxycarbonyl-1-(4-methylphenyl)-1H-[1,2,4]triazolo[3',4':2,3]-imidazo[4,5-b]phenazine 8b: Brown crystals from ethanol / dioxane, Yield: (0.51 g, 69 %); m.p. 243-245°C.IR (KBr, υ, cm-1): 3111 (CH aromatic), 2972 (CH3), 1707 (C=O ester), 1617 (C=N), 1547, (C=N), 1522 (C=C). MS m/z (%): 423 (M++1, 27.8), 422 (M+, 38.9), 325 (38.9), 211 (22.2), 153 (33.3), 91 (66.7) 77 (50), 55 (100). 1H NMR (DMSO-d6, δ ppm):1.25-1.26 (t, 3H, CH3), 2.6 (s, 3H, -CH2CH3), 4.23-4.245 (q, 2H, -CH2CH3), 7.08-7.10 (m, 4H,

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Ar-H), 7.20-7.22 (m, 6H, Ar-H). Anal. Calcd. for C24H18N6O2 (422.43): C, 68.24; H, 4.29; N, 19.89. Found: C, 68.19; H, 4.15; N, 19.96%.

3-Ethoxycarbonyl-1-(4-chlorophenyl)-1H-[1,2,4]triazolo[3',4':2,3]-imidazo[4,5-b]phenazine 8c :

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Brown crystals from ethanol / dioxane, Yield: (0.55 g, 73 %); m.p. 249-251°C. IR (KBr, υ, cm-1): 3115 (CH aromatic), 2920 (CH aliphatic), 1708 (C=O ester), 1601 (C=N),

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1556 (C=N), 1512. MS m/z (%): 442 (M+, 11.6), 441 (M+-1, 20.9), 369 (20.9), 252 (86), 168 (30.2), 129 (44.2), 77 (16.3), 60 (100). 1H NMR(DMSO-d6, δ ppm): 1.24-1.26 (t, 3H, -CH2CH3), 4.22-4.25 (q, 2H, -CH2CH3), 7.33 (s, 2H, Ar-H), 7.64-8.41 (m, 8H, ArH). Anal. Calcd. for C23H15ClN6O2 (442.85): C, 62.38%; H, 3.41%; N, 18.98%. Found: C, 62.19; H, 3.49%; N, 18.81%. 4.1.3 Procedures for Synthesis of 9, 10, 12 Ethyl (2Z)-[3-aminophenazin-2-yl)amino](phenylhydrazono)ethanoate 9: To a mixture of equimolar quantities of the 2,3-diaminophenazine 1 and ethyl chloro(phenylhydrazono)ethanoat 4a (10 mmol) in absolute ethanol / DMF mixture (20:20), 11

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was added triethylamine (1.4 ml, 10 mmol). The mixture was refluxed for 7 hours and after the reaction was complete, the reaction mixture was poured onto cold water. The precipitated solid was filtered off and recrystallized from DMF to give 9 (2.0 g, 63 %) black crystals; m.p.255-257°C. IR (KBr, υ, cm-1): 3400 (NH2), 3069 (CH), 2881 (CH

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aliphatic), 1686 (C=O ester), 1601 (C=N), 1580 (C=N). MS m/z (%): 400 (M+, 31), 387 (56), 313 (51), 262 (52), 212 (66), 168 (22.50), 118 (88), 77 (100).1H NMR(DMSO-d6,

δ ppm): 1.22 (t, 3H, -CH2CH3), 4.22-4.25 (q, 2H, -CH2CH3), 6.60 (s, 2H, NH2), 7.027.59 (m, 11H, Ar-H), 11.4 (s, 2H, 2NH). Anal. Calcd. for C22H20N6O2 (400.43): C,

SC

65.99 ; H, 5.03; N, 20.99. Found: C, 65.81; H, 5.08; N, 20.83 %.

2-Oxo-3-phenylhydrazono-1,4-dihydropyrazino[2,3-b]phenazine 10 :

M AN U

Compound 9 (2 mmol, 0.8 g) was refluxed for 7 hours in a mixture of acetic anhydride and acetic acid (4:1).The solution was evaporated under vacuum till near dryness. The remained residue was washed several times with water and recrystallized from dimethylformamide to give (0.6 g, 75 %) black crystals; m.p. 310-311°C. IR (KBr, υ, cm-1): 3442 (NH), 3256 (NH), 3010 (CH aromatic), 1684 (C=O), 1644 (2C=N), 1597 (C=N). MS m/z (%): 354 (M+, 0.87), 351 (0.76), 228 (20.37), 227 (100), 210 (2.57), 129 (2.83), 77 (0.93). 1H NMR (DMSO-d6,δ ppm): 7.10-7.83 (m, 11H, Ar-H), 10.93 (s,

TE D

1H, NH), 11.69 (s, 1H, 1NH), 13.01 (s, 1H, 1NH). Anal. Calcd. for C20H14N6O, (354.36): C, 67.79%; H, 3.98; N, 23.72. Found: C, 67.59; H, 3.89; N, 23.81%.

EP

2-Oxo-3-phenylazo-1H-pyrazino[2,3-b]phenazine 12: To a solution of 10 (2 mmol, 0.72 g) in ethanol-DMF was added hydrogen peroxide (3 ml) and the resulting mixture was stirred overnight. The solution was evaporated under

AC C

vacuum. The remaining residue was washed several times with water and recrystallized from dimethylformamide to give (0.54 g, 69 %) black powder; m.p. 296-298°C. IR (KBr, υ, cm-1): 3411 (NH), 1680 (C=O), 1650 (N=N), 1604 (C=N), 1597 (2C=N). MS m/z (%): 352 (M+, 16.04), 346 (30.22), 260 (23.13), 238 (100), 229 (23.88), 191 (20.90), 148 (20.90), 122 (19.40), 73 (42.91) 1H NMR (DMSO-d6, δ ppm): 7.10-7.13 (m, 5H, Ar-H), 7.54-7.59 (d, 2H, Ar-H), 7.74-7.77 (d, 2H, Ar-H) 7.80-7.83 (s, 2H, ArH), 11.49 (s, 1H, 1NH). Anal. Calcd. for C20H12N6O, (352.34): C, 68.18 ; H, 3.43 ; N, 23.85 . Found: C, 68.19 ; H, 3.39; N, 23.71 %.

12

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4.1.4 Procedures for synthesis of [1,4]diazepino[2,3-b]phenazines 13, 14 6H-7-Methyl[1,4]diazepino[2,3-b]phenazine-9-one 13: A mixture of 2,3-diaminophenazine 1 (4 mmol, 0.84 g), ethyl acetoacetate (4 mmol, 0.52 ml) in dimethyl-formamide was refluxed for 12 hours. The reaction mixture was

RI PT

poured onto cold water. The separated solid was then collected by filtration, dried and recrystallized from ethanol / dioxane to give (0.58 g, 69 %) black powder, m.p. 226-

228°C. 3368 (NH), IR (KBr, υ, cm-1): 3061 (NH), 2977 (CH3), 2927 (CH2), 1660 (C=O), 1598 (3C=N) . MS m/z (%): 278 (M+, 19), 277 (34), 219 (14), 180 (5), 1

129

SC

(11), 101 (9), 97 (11), 77 (100). H NMR(DMSO-d6, δ ppm): 1.22 (s, 3H, CH3), 2.44 (s, 2H, CH2), 3.22 (s, 1H, CH), 7.02-7.59 (m, 6H, Ar-H), 11.4 (s, 1H, NH), 12.74 (s, 1H, 69.01; H, 4.95; N, 20.02 %.

M AN U

NH). Anal. Calcd. for C16H14N4O (278.30):C, 69.05; H, 5.07; N, 20.13. Found: C,

6H-[1,4]Diazepino[2,3-b]phenazine-7,9-dione 14:

A mixture of 2,3-diaminophenazine 1 (4 mmol, 0.84), malonic acid (4 mmol, 0.41 g) in acetic acid (12.5 ml) was refluxed for 8 hours. The reaction mixture was poured onto crushed ice, the separated solid was then collected by filtration, washed by water, dried

TE D

and recrystallized from ethanol / dioxane to give brown powder (1.2 g, 53 %); m.p. 246-248°C. IR (KBr, υ, cm-1): 3418 (NH) 3202 (NH), 2922 (CH2), 1665 (2C=O), 1598 (2C=N) cm-1. MS m/z (%): 279 (M++1, 17), 278 (M+, 24), 252 (100), 236 (67), 220 (44), 196 (25), 182 (26), 126 (23), 103 (29), 77 (34), 64 (75), 51 (28). 1H NMR (DMSO-

EP

d6, δ ppm): 3.32 (s, 2H, CH2), 7.70-8.02 (m, 6H, Ar-H), 10.98 (s, 2H, 2NH). Anal. Calcd. for C15H10N4O2 (278.26): C, 64.74; H, 3.62; N, 20.13. Found: C, 64.59; H, 3.49;

AC C

N, 20.06%.

4.1.5 Procedures for synthesis of pyrazino[2,3-b]phenazines 15-17 Pyrazino[2,3-b]phenazine-2,3-dione 15: To a mixture of equimolar quantities of the 2,3-diaminophenazine 1 (4 mmol, 0.84) and oxalyl chloride (4 mmol, 0.50 g) in DMF, hydrochloric acid (7 ml, 4 N) was added. The mixture was refluxed for 10 hours. The reaction mixture was poured onto cold water, the precipitated solid was filtered off and recrystallized from ethanol-dioxane mixture to give (0.52 g, 61 %) dark brown crystals; m.p. 245-247°C. IR (KBr, υ, cm-1): 3418 (2NH), 1652 (2C=O), 1529 (2C=N). MS m/z (%): 264 (M+, 1.32), 219 (3), 182 (3), 141 13

ACCEPTED MANUSCRIPT

(4), 112 (7), 95 (8), 77 (72), 60, (100). 1H NMR (DMSO-d6, δ ppm): 7.13-7.26 (t, 2 H, Ar-H), 7.61-7.70 (d, 2H, Ar-H), 7.93 (s, 2H, Ar-H), 8.13 (s, 2H, 2NH). Anal. Calcd. for C14H8N4O2 (264.22): C, 64.12 ; H, 2.31 ; N, 21.37. Found: C, 64.03; H, 2.34; N, 21.13

7,11-Dihydrofuropyrazino[2,3-b]phenazine-8,10-dione 16:

RI PT

%.

To a mixture of equimolar quantities of the 2,3-diaminophenazine 1 (4 mmol, 0.84) and dichloromaleic anhydride (4 mmol, 0.50 g) in DMF, hydrochloric acid (7 ml, 4 N) was

added. The mixture was refluxed for 10 hours. The reaction mixture was poured onto

SC

cold water, the precipitated solid was filtered off and recrystallized from ethanoldioxane to give (0.52 g, 61 %) brown crystals; m.p. 243-245°C. IR (KBr, υ, cm-1):

M AN U

3418 (2NH), 1652 (2C=O), 1598 (2C=N). MS m/z (%): 307 (M++1, 17.19), 306 (M+, 4.58), 279 (27.79), 252 (23.50), 208 (13.75), 174 (21.78), 125 (20.63), 94 (100), 76 (2.58). 1H NMR (DMSO-d6, δ ppm): 7.13-7.26 (t, 2 H, Ar-H), 7.61-7.70 (d, 2H, Ar-H), 7.62 (s, 2H, Ar-H), 13.38 (s, 2H, 2NH). Anal. Calcd. for C16H8N4O3 (304.25): C, 63.16; H, 2.65; N, 18.40. Found: C, 63.09; H, 2.59; N, 18.43 %.

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Quinoxalino[2,3;2',3']pyrazino[2,3-b]phenazine 17:

An equimolar mixture of 16 (1.5 mmol, 0.4 g), o-phenylenediamine (1.5 mmol, 0.4 g) and few drops of acetic acid was refluxed in toluene. Water was collected via DeanStark apparatus during a period of 12 hours. The solution was evaporated under vacuum

EP

and the residue was washed many times with water and recrystallized from dimethylformamide to give (0.6 g, 75 %) black powder; m.p. 301-303°C. IR (KBr, υ, cm-1): 3442 (broad, NH), 3256 (NH), 1644 (2C=N), 1597 (2C=N). MS m/z (%): 337

AC C

(M++1, 1.04), 336 (M+, 1.77), 334 (1.38), 326 (10.94), 297(20.77), 234 (923), 210 (100), 129 (10.97), 118 (80.61), 91(36.38), 77 (21.85). 1H NMR (DMSO-d6, δ ppm):7.06-7.13 (t, 4 H, Ar-H), 7.20-7.29 (d, 4H, Ar-H), 7.93 (s, 2H, Ar-H), 10.95 (s, 2H, 2NH). Anal. Calcd. for C20H12N6 (336.275): C, 71.42; H, 3.60; N, 24.99. Found: C, 71.29; H, 3.48; N, 24.88%. 4.1.6 Procedure for s Synthesis of 2,3-Dibenzoylaminophenazine 18: A mixture of 2,3-diaminophenazine 1 (10 mmol, 2.1 g), aqueous sodium hydroxide (10 %, 25 ml) and benzoyl chloride (30 mmol, 4.2 ml) was stirred at room temperature for 5 14

ACCEPTED MANUSCRIPT

hours. The separated solid was then washed with water (20 ml) and dried to give (1.9 g, 90 %) yellow crystals; m.p. 110-111°C. IR (KBr, υ, cm-1): 3374 (2NH), 1652 (2C=O), 1598 (2C=N) . MS m/z (%): 418 (M+, 0.04), 299 (0.04), 226 (0.55), 198 (2.49), 155 (10), 123 (29), 105 (100), 95 (13), 77 (16). 1H

NMR(DMSO-d6, δ ppm):7.44-7.46 (m,

RI PT

10H, Ar-H), 7.47-7.58 (t, 2H, Ar-H), 7.90-7.92 (m, 4H, Ar-H), 12.90 (s, 2H, 2NH);13C NMR (CDCl3): δ = 104.73, 126.02, 128.35, 128.61, 129.47, 129.62, 130.17, 130.35,

133.74, 133.96, 139, 141.45 (aromatic carbons); 172.87 (C-amide). Anal. Calcd. for C26H18N4O2 (418.44): C, 74.63; H, 4.34; N, 13.39. Found: C, 74.49; H, 4.49; N,

SC

13.51%.

4.1.7 Procedure for s Synthesis of 1H-Imidazo[4,5-b]phenazine 20: Method A [31] : A mixture of 2,3-diamino-phenazine 1 (4 mmol, 0.84 g) and ethoxymethylene

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malononitrile (4 mmol, 0.48 g) in dimethyl-formamide was refluxed for 12 hours. The reaction mixture was poured onto crushed ice. The separated solid was then collected by filtration, washed with water, dried and recrystallized from ethanol / dioxane to give 21 as yellow powder.

Method B:

A mixture of 2,3-diaminophenazine 1 (4 mmol, 0.84 g) and diethyl

TE D

orthoformate (4 mmol, 0.6 ml) in acetic acid was refluxed for 7 hours. The reaction mixture was cooled then the separated solid was collected by filtration, washed with water, dried and recrystallized from ethanol / dioxane to give (0.58 g, 69 %) yellow powder; m.p. 307-309°C. IR (KBr, υ, cm-1): 3418 (NH), 1652 (C=N), 1598 (2C=N) .

EP

MS m/z (%): 221 (M++1, 22.18), 220 (M+, 24.12), 210 (20.23), 178 (29.57), 153 (25.29), 123 (25.29), 93 (28.40), 78 (22.18), 69 (100). 1H NMR (DMSO-d6, δ ppm):

AC C

7.44-7.46 (m, 2H, Ar-H), 7.47-7.58 (m, 2H, Ar-H), 7.90 (s, 2H, Ar-H), 8.08 (s, 1H, CH), 11.86 (s, 1H, NH). Anal. Calcd. for C13H8N4 (220.22): C, 70.90; H, 3.66; N, 25.44. Found: C, 70.76; H, 3.49; N, 25.68%. 4.1.8 General procedure for synthesis of 2-aryl-1H-imidazo[4,5-b]phenazine 23a-c: A mixture of 2,3-diaminophenazine 1 (5 mmol, 1.05 g) and (5 mmol) of the aromatic aldehyde (3-chlorobenzaldehyde, 2,4-dichloro-benzaldehyde, cinnamaldehyde) in dimethylformamide with acetic acid (20 ml) was refluxed for 10-12 hours. The reaction mixture was poured onto cold water, the separated solid was then collected by filtration, dried and recrystallized from the proper solvent. 15

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2-(3-Chlorophenyl)-1H-Imidazo[4,5-b]phenazine 23a: Prepared from 2,3-diamino-phenazine 1 and 3-chlorobenzaldehyde, recrystallized from ethanol to give (0.65 g, 77 %) black crystals; m.p. 180-182°C. IR (KBr, υ, cm-1): 3418(NH), 3112(CH aromatic), 1652 (C=N), 1567 (2C=N). MS m/z (%):333 (M++2,

RI PT

0.46), 331 (M+, 2), 280 (0.93), 228 (0.31), 184 (35.50), 126 (22.50), 103 (16.80), 91 (91.80), 78 (100), 64 (100). 1 H NMR (DMSO-d6, δ ppm): 7.89-7.92 (m, 10H, Ar-H), 13.4 (s, 1H, NH). Anal. Calcd. for C19H11ClN4 (330.77): C, 68.99; H, 3.35; N, 16.94. Found: C, 88.69; H, 3.49; N, 16.86%.

SC

2-(3,5-Dichlorophenyl)-1H-imidazo[4,5-b]phenazine 23b:

Prepared from 2,3-diamino-phenazine 1 and 2,4-dichlorobenzaldehyde, recrystallized

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from ethanol / dioxane to give (0.78 g, 74 % yield) black crystals; m.p. 275-277oC. IR (KBr, υ, cm-1): 3404 (NH), 3017 (CH aromatic) 1597 (2C=N), 1527 (C=N). MS m/z (%): 367 (M++2, 0.02), 365 (M+, 1.24) 280 (0.93), 206 (6), 236 (5), 184 (35.50), 126 (22.50), 103 (16.80), 91 (91.80), 77 (7), 64 (100). 1H NMR (DMSO-d6, δ ppm): 7.257.37 (d, 2H, Ar-H), 7.63 (m, 4H, Ar-H), 8.16-8.27 (s, 3H, Ar-H), 13.02 (s, 1H, NH). Anal. Calcd. for C19H10Cl2N4 (365.23): C, 62.48; H, 2.76; N, 15.34. Found: C, 62.39; H,

TE D

2.79; N, 15.30%.

2-(Cinnamyl)-1H-imidazo[4,5-b]phenazine 23c: Prepared from 2,3-diaminophenazine 1 and cinnamaldehyde, recrystallized from ethanol to give (0.56 g, 53 % yield) yellow crystals; m.p. 205-207 oC. IR (KBr, υ, cm): 3250 (NH), 3052 (CH aromatic), 2980 (CH aliphatic), 2866 (CH aliphatic), 1633

EP

1

(C=N), 1528 (2C=N). MS m/z (%): 323 (M++1, 12.05), 322 (M+, 9.77), 306 (14.66),

AC C

286 (11.40), 256 (14.31), 211 (9.77), 185 (12.87), 129 (21.82), 95 (23.13), 77(9.45), 69 (100). 1H NMR (DMSO-d6, δ ppm): 7.00-7.43 (d, 2H, -CH=), 7.45-7.73 (m, 5H, ArH), 7.81-7.91 (d, 2H, Ar-H), 8.14 (s, 2H, Ar-H), 8.28-8.33 (t, 2H, Ar-H), 13.08 (s, 1H, NH). Anal. Calcd. for C21H14N4 (322.36): C, 78.24; H, 4.38; N, 17.38. Found: C, 78.19; H, 4.49; N, 17.21%.

4.1.9 Procedure for s Synthesis of 1H-imidazo[4,5-b]phenazines 24,25: 2-(Phenyl)-1H-imidazo[4,5-b]phenazine 24: A mixture of 2,3-diaminophenazine 1 (4 mmol, 0.84 g), benzoic acid (4 mmol, 0.5 ml) in phosphorus oxychloride (25 ml) and acetic acid (12.5 ml) was heated under reflux for 16

ACCEPTED MANUSCRIPT

8 hours. The reaction mixture was poured onto crushed ice. The separated solid was then collected by filtration, washed with water and aqueous solution of potassium carbonate, dried and recrystallized from ethanol to give red powder (0.7 g, 83 %); m.p. 360-362°C (as literature m.p. [29] ) . IR (KBr, υ, cm-1): 3198 (NH), 1532 (2C=N), 1428

RI PT

(C=N). MS m/z (%): 298 (M++2, 10), 297 (M++1, 30), 296 (M+, 100), 268 (35.50), 192 (22.50), 148 (16.80), 102 (91.80), 77 (47.70), 64 (55.60), 51 (54.21). 1H NMR (DMSOd6, δ ppm): 7.62-7.91 (m, 5H, Ar-H), 8.19-8.46 (m, 6H, Ar-H), 13.4 (s, 1H, NH). Anal.

Calcd. for C19H12N4 (296.32): C, 77.01; H, 4.08; N, 18.91. Found: C, 76.89; H, 4.19; N,

SC

18.61%. 2-Propyl-1H-imidazo[4,5-b]phenazine 25:

A mixture of 2,3-diamino-phenazine 1 (4 m mol, 0.84 g), butyric acid (4 mmol, 0.38

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g) in acetic acid (12.5 ml) was heated under reflux for 12 hours. The reaction mixture was poured onto crushed ice. The separated solid was collected by filtration, washed with water, dried and recrystallized from ethanol / dioxane to give brown crystals (0.6 g, 71 %); m.p.306-308°C. IR (KBr, υ, cm-1): 3358 (NH), 1628 (C=N), 1590 (C=N). MS m/z (%): 262 (M+, 1.32), 210 (1.80), 167 (3.06), 127 (5.21), 112 (7.08), 95 (8.11), 77 (72.26), 60 (100).1H NMR (DMSO-d6, δ ppm):0.74 (t, 3H, CH3), 1.13 (m, 2H, CH2),

TE D

2.47 (t, 2H, CH2), 6.73-6.75 (d, 2H, Ar-H), 7.01-7.17(t, 2H, Ar-H), 7.58-7.91(m, 2H, Ar-H), 10.56(s, 1H, NH). Anal. Calcd. for C16H14N4 (262.30): C, 73.26; H, 5.38; N, 21.36. Found: C, 73.19; H, 5.19; N, 21.51%.

29:

EP

4.1.5 General procedure for synthesis of 3-amino-2-diazenylphenazine derivatives 27-

To a stirred solution of active methylene compounds (10 mmol) in ethanol (40 ml) was

AC C

added sodium acetate trihydrate (3 g, 10 mmol) and the mixture was cooled in an icebath to 0-5 oC. To the resulting cold solution, a cold solution of 2-aminophenazinediazonium chloride 26 (prepared by diazotizing 2,3-diaminophenazine 1 (10 mmol, 2.10 g) in hydrochloric acid (6 ml, 6 M) with sodium nitrite (0.7 g, 10 mmol) in water (3 ml)) was added portionwise. After all diazonium salt solution was added, the reaction mixture was stirred for two hours while cooling in ice-bath and left overnight in refrigerator. The precipitated solid was filtered off, washed with water, air dried and finally recrystallized from the proper solvent to form diazocoupling products with excellent yields. 17

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4-[(E)-(3-Aminophenazin-2-yl)diazenyl]-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3one 27: Prepared from 3-methyl-1-phenyl-5-pyrazolone, recrystallized from ethanol / dioxane to give reddish brown crystals (1.5 g, 71%); m.p. 280-282°C. IR (KBr, υ, cm-1): 3482

RI PT

(NH2), 3196(CH-Ar), 2931(CH-aliphatic), 1776(N=N), 1631 (C=O), 1530 (4C=N). MS m/z (%): 395 (M+, 11.6), 282 (14.0), 210 (76.7), 183 (53.3), 157 (25.6), 126 (34.9), 98 (30.2), 73 (46.5), 55 (100).1H NMR (DMSO-d6, δ ppm): 2.46 (s, 3H, CH3), 4.3(s,

1H,CH), 6.44 (s,2H, NH2), 7.68-7.96 (m, 11H, Ar-H). Anal.Calcd. for C22H17N7O

SC

(395.41): C, 66.88; H, 4.33; N, 24.88. Found: C, 66.98; H, 4.24; N, 24.56 % . 4-[(E)-(3-Aminophenazin-2-yl)diazenyl]-3-phenylisoxazol-5(4H)-one 28:

Prepared from 3-phenyl-5-isoxazolone, recrystallized from ethanol/ dioxane as reddish

M AN U

brown crystals (1.6 g, 76 %); m.p. 270-272°C. IR (KBr, υ, cm-1): 3414 (NH), 2919 (CH), 1893 (N=N), 1685 (C=O), 1629 (2C=N), 1532 (2C=N). MS m/z (%):384 (M+, 7.42), 340 (4.55), 281 (9.92), 213 (35.27), 210 (10.24), 182 (58.64), 155 (23.22), 116 (10.51), 91 (10.35), 78 (53.99), 45 (100).1H NMR (DMSO-d6, δ ppm): 5.69 (s, 1H, CH), 6.98(s, 2H, NH2),7.00-7.83(m, 9H, Ar-H), 8.00 (s, 2H, Ar-H)..Anal. Calcd. for

TE D

C21H16N6O2 (384.37):C, 65.62; H, 4.20; N, 21.86. Found: C, 65.49; H, 4.49; N, 21.71%. 2-[(E)-(3-Aminophenazin-2-yl)diazenyl]-5,5-dimethylcyclohexane-1,3-dione 29: Prepared from 5,5-dimethylcyclohexane-1,3-dione, recrystallized from ethanol / dioxane in reddish brown crystals (0.7 g, 33 %); m.p. 267-269°C. IR (KBr, υ, cm-1):

EP

3412 (NH), 2958 (CH), 1932 (N=N), 1611 (C=O), 1532 (2C=N), 1428 (2C=N). MS m/z (%): 361 (M+, 38.24), 360 (M+-1, 26.47), 312 (36.27), 259 (30.39), 209 (33.33), 172

AC C

(38.24), 149 (49.02), 96 (46.08), 77 (36.27), 56 (100). 1H NMR (DMSO-d6, δ ppm): 0.99 (s, 6H, 2CH3), 2.11 (s, 4H, 2CH2), 5.22 (s, 1H, CH), 7.91-8.03 (m, 6H, Ar-H), 8.22 (s, 2H, NH2) ppm. Anal. Calcd. For C20H19N5O2 (361.39): C, 66.47; H, 5.30; N, 19.38. Found: C, 66.29; H, 5.49; N, 19.21%. 4.2 Cytotoxicity

4.2.1 Cell lines and culturing Fetal bovine serum (FBS) and L-glutamine, were obtained from Gibco Invitrogen Company (Scotland, UK). Dulbecco's modified Eagle's (DMEM) medium 18

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was provided from Cambrex (New Jersey, USA). Dimethyl sulfoxide (DMSO), cisplatin, penicillin, and streptomycin were obtained from Sigma Chemical Company (Saint Louis, MO, USA). Human Tyrosine kinase (TRK) ELISA kit was purchase from

RI PT

Glory Science Co., Ltd (Del Rio, TX 78840, USA). The effect of tested compounds on the level of Human Tyrosine kinase (TRK) were determined utilizing 2 different human tumor cell lines including lung carcinoma

A549 cell line and colorectal cancer HCT116 cell line were obtained from the American Type Culture Collection (Rockville, MD, USA). The tumor cells were maintained in

SC

Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (GIBCO), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37 oC in humidified atmosphere containing 5% CO2. Cells at a concentration of 0.50 x 106

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were grown in a 25 cm2 flask in 5 ml of complete culture medium.

4.2.2 Anticancer assay

The cells in culture medium were treated with 20 µl of IC50 values of the compounds or the standard reference drug, Cisplatin dissolved in DMSO, then

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incubated for 24 h at 37 ºC, in a humidified 5% CO2 atmosphere. The cells were harvested and homogenates were prepared in saline using a tight pestle homogenizer until complete cell disruption.

To determine the level of human tyrosine kinase (TRK) in samples. A double-

EP

antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used. This assay is based on, add TRK to monoclonal antibody enzyme well which is pre-coated with human TRK monoclonal antibody, incubation; then, add TRK antibodies labeled with

AC C

biotin, and combined with Streptavidin-HRP to form immune complex; then carry out incubation and washing again to remove the uncombined enzyme. Then add Chromogen solution A, B, the color of the liquid changes into the blue, and at the effect of acid, the color finally becomes yellow. The chroma of color and the concentration of the human TRK of sample were positively correlated and the optical density was determined at 450 nm. The level of Human Tyrosine kinase (TRK) in samples was calculated (pmol/ml) as duplicate determinations from the standard curve.

19

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5. Acknowledgement We are grateful to National Research Centre, Giza, Egypt for providing the facilities and funding for this work.

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[2] S.G. Franzblaul, J. F. O'sullivan, Antimicrobial Agents and Chemotherapy, 32

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(1988) 1583-1585.

[3] Y. Ge, D. Pei, Y. Zhao, W. Li, Wang, Sh.; Xu, Y.; Current Microbiology, 54

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(2007) 277–281.

[4] H. Liu, Y. He, H. Jiang, H. Peng, X. Huang, X. Zhang, L. S. Thomashow, Y. Xu, Current Microbiology, 54 (4),) (2007) 302–306.

[5] M. Wang, H. Xu, S. Yu, Q. Feng, S. Wang, Z. Li, J. Agric. Food Chem., 58 (6) (2010) 3651–3660.

[6] J. A. Spicer, S. A. Gamage, G. W. Rewcastle, G. J. Finlay, D. J. A. Bridewell, B.C. Baguley, W.A. Denny, J. Med. Chem., 43 (7), (2000) 1350–1358.

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[7] V. Zikan, J. Sluka, CS 254604 (Cl. C07D241/46, 15 Sep 1988, Appl, 86/3,356. 08 May 1986; 2p. Chem. Abst. 111: 134194k, 1989. [8] V. Zikan, J. Sluka, J. Danek, CS 254543 (Cl. C07D241/46, 15 Sep 1988, Appl, 86/3,358. 08 May 1986 2p, Chem. Abst. 111:134195m, 1989.

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[9] V. Zikan, J. Sluka, J. Danek, CS 254605 (Cl. C07D241/46, 15 Sep 1988, Appl, 86/3,357. 08 May 1986 3p, Chem. Abst. 111:7433n, 1989

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[10] T. V. Siunova, V. V. Kochetkov, S. Z. Validov, N. E. Suzina, A. M. Boronin, Microbiology, 71 (6) (2002) 670–676, Translated from Mikrobiologiya, 71 (6),

(2002) 778–785.

[11] M. A. Veselova , Klein, Sh.; Bass, I. A.; Lipasova, V. A.; Metlitskaya, A. Z.; Ovadis, M. I.; L. S. Chernin, I. A. Khmel, ISSN 1022-7954, Russian Journal of Genetics, (2008) 44 (12), 1400–1408. [12] G. W. Rewcastle, W.A. Denny, B. C. Baguley, J. Med. Chem., 30 (5) (1987) 843–851. [13] M. Conda-Sheridan, L. Marler, E. Park, T. P. Kondratyuk, K. Jermihov, A. D.

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Mesecar, J. M. Pezzuto, R. N. Asolkar, W. Fenical, M. Cushman, J. Med. Chem., 53 (24) (2010) 8688–8699. [14] M. S. Abdelfattah, T. Kazufumi, M. Ishibashi, J. Nat. Prod., 73 (12) (2010) 1999–2002.

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[15] A. Arora, E.M. Scholar, J. Pharmacol. & Exp. Ther., 315(3) (2005) 971-979. [16] M. K. Paul, A. K. Mukhopadhyay. Intern. J. Med. Sci, 1(2) (2004) 101-115.

[17] I. Shchemelinin, L. Sefc, , E. Nečas, Folia Biol. (Praha), 52, (2006) 81-101 , 137-148.

[18] A. S. Shawali, A. M. Mahran, A. A. Nada, J. Heteroatom Chemistry, 18 (4)

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(2007) 393-398.

[19] A. M. Mahran, N. A. Hassan, Arch. Pharm. Res., 29 (1) (2006) 46-49.

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[20] A. M. Mahran, Egypt. J. Chem., 52 (2) (2009) 265-275.

[21] J. Manassen, S. Kalif, J. Am Chem. Soc, 88 (1966) 1943-1947. [22] G. Favrel, Bull. Soc. Chim. Fr., 31 (1904) 150.

[23] A. J. Elliott, P. D. Callaghan, M. S. Gibsonand, S. T. Nemeth, Can. J. Chem., 53 (1975) 1484-1490.

[24] J. F. Bunnett, R. E. Zahler, Chem. Rev., 49 (1951) 273-412.. [25] J. F. Bunnett, Quart. Rev. (London), 12 (1958) 12.

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[26] K. Ishii, M. Hatanaka, I. Ueda, Chem Pharm Bull, 39 (1991) 3331-3334. [27] M. A. Philips, J. Chem. Soc., (1928) 2393-2399. [28] B. Stanovnik, J. Svete, Chem. Rev., 104 (2004) 2433-2480. [29] M. A. Bogoyevitch, R. K. Barr, A. J. Ketterman, Biochim. Biophys. Acta 1754,

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(2005) 79-99.

[30] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D.Vistica, J. T.

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Warren, H. Bokesch, S. Kenney, M. R. Boyd, J. Natl. Cancer Inst., 82 (1990) 11071112.

[31] A. M. Amer, A. A. El-Bahnasawi, M. R. H. Mahran, M. Lapib, Monatshefte fur Chemie, 130 (1999)1217-122.

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Table 1. The IC50 values and percent of human TRK inhibition of the tested compounds against lung carcinoma A549 and colorectal cancer HCT116 cell linesa.

a

80% 86% 85% 11% 14% 50% 2% 1% 5% 15% 16% 12% 4% 72% 2% 87%

72% 85% 84% 4% 18% 37% 0.4% 0.5% 3% 17% 12% 11.50 2% 68% 0.6% 85%

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6.00 4.40 5.20 11.20 9.60 8.75 14.60 17.50 11.40 7.90 6.80 16% 11.90 6.80 13.90 4.75

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8.80 7.70 8.40 21.00 19.00 17.60 27.60 23.60 21.70 13.80 11.50 6.80 22.00 9.80 22.30 8.20

% of TRK inhibitionb A549 HCT116

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1 2 3 8a 8b 8c 9 10 13 14 15 16 17 18 20 23a 23b 23c 24 25 27 28 29 DMSO Cisplatin

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Compound

IC50 (µg/ml) A549 HCT116

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Data were expressed as average of three independent experiments. The percentage changes as compared with control untreated cells (DMSO treated) b

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N

NH 2

N

NH 2

H N

N

CS 2

MeI

N

H N

N

N

SMe

S

pyridine

N

2

1

EtONa

N H

3 COOEt

EtONa Cl

4 N NH Ar COOEt

N

COOEt S

N

N

N

N NH Ar

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H N

N

SMe

N NH Ar

N

N 7

5

-MeSH Ar

H N

N

N

-H 2 S NH

N

N

N N

S 6

COOEt

N

COOEt

N N

N

Ar

SC

8a-c

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Ar: a, C 6 H 5 ; b, 4-CH 3 C 6 H 4 ; c, 4-ClC 6 H 4

Scheme 1. Synthesis of compounds 8a-c

N

NH2

N

N

N H

N NHPh

N N

4

Ph

O

EP

N

COOEt Cl

Et 3N

H 2O 2

NH2 N

NH

NH

N

9

COOEt

Ac 2O/AcOH

-EtOH H N

N N

N H

-H 2O N NHPh

N

O

N

10

12

AC C

N

EtOH/DMF

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NH2

N

1

+

Scheme 2. Synthesis of compounds 9, 11, 12

23

H N N H

11

N N Ph OEt

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N

N H H N

13 N

CH2 (COOH)2

AcOH

N

NH2

N

N

Cl

Cl

N

NH2

Cl

O

NH2

O

N H

15

O

N

N

O

O

O N

DMF

N H

16 N

PhCOCl NaOH

H N

N

N

N H

N

17

H N

O Cl

O

H N

N

DMF

NH2

1

N H

14

O

O O

NH

N

Ph

Ph

NH O

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18

SC

O

CH3 O

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DMF

O

H N

N

CH3 COCH2 COOEt

Scheme 3. Synthesis of compounds 13-18

N

.. NH2

N

NH

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EtO

CN

H

CN

EP

-2EtOH

NH2

AC C

N N

NH2

ArCHO

DMF/AcOH

CN

H N

N

OEt N

21

N

-EtOH

N

N

NH2

Ar

N

H N

N

N

Ar 22

POCl 3 /AcOH

N

N H

N

1

PhCOOH

H N

20

CN

19

N

H

H

DMF

CH(OEt) 3

-CH 2 (CN) 2

N

H N

N

N

23a-c Ph

Ar = a, 3-Cl-C 6 H 4, b, 2,4-Cl 2-C 6H 3, c, CH=CH-C 6 H 5

24 CH 3 CH 2 CH 2 COOH

N

H N

N

N

CH 2 CH 2 CH 3

POCl 3/AcOH

25

Scheme 4. Synthesis of compounds 20, 23-25

24

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CH3 N

O

H

N

N

Ph

NH2

N

NH2

NaNO2 HCl

+

N

N2 Cl

N

NH2

27

-

NH N

28

N

NH2 O

N

N

NH N

N

NH2 O

N O

O

N

NH N

N

NH2 O

32

31

SC

30

N Ph

N N

N

Ph

N

AC C

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Scheme 5. Synthesis of compounds 27-29

25

N N

NH2 O

N

O

O

CH3 N

N

O

26

1

H

N

N

AcONa.3H2O O

N N Ph Ph

O

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N

NH2 O

N

Ph

CH3

N N

29

NH2 O

H

O

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• Novel heterocyclic compounds derived from 2,3-diaminophenazine were synthesized. • All compounds were characterized based on microanalytical and spectral

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data.

• The compounds were screened for their antitumor activity against A549 and HCT116.

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• Five compounds were mostly antiproliferative agents as potent as the Cisplatin.

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• Compound 2 inhibited the TRK activity equal or more potent to the

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Cisplatin.

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HNMR spectrum of 2

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HNMR spectrum of 3

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HNMR spectrum of 8b

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HNMR spectrum of 8c

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HNMR spectrum of 13

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HNMR spectrum of 14

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HNMR spectrum of 17

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HNMR spectrum of 18

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HNMR spectrum of 20

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HNMR spectrum of 23b

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HNMR spectrum of 23c

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HNMR spectrum of 24

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1

HNMR spectrum of 25

AC C

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HNMR spectrum of 27

AC C

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HNMR spectrum of 28

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HNMR spectrum of 29