“Design and Synthesis of Some Pyridazine Containing Compounds of Expected Anticancer Activity” Thesis Presented By Demiana Samir Mikhail Master in Pharmaceutical Sciences (Organic Chemistry) (Faculty of Pharmacy, Cairo University) (2011) Submitted for The Degree of The Doctor of Philosophy in Pharmaceutical Sciences (Pharmaceutical Organic Chemistry) Under the supervision of Prof. Dr. Salwa El-Sayed Mohamed El-Meligie Professor of Organic Chemistry, Faculty of Pharmacy, Cairo University Dean of Faculty of Pharmacy, Sadat City University Ex-Head of Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University Prof. Dr. Eman Mohamed Ahmed Professor of Organic Chemistry Faculty of Pharmacy, Cairo University

Dr. Suzan Mohamed Abuel-Maaty Dr. Sawsan Abo Bakr Zaitone Associate Professor of Organic Chemistry Associate Professor of Pharmacology & Toxicology Faculty of Pharmacy, Cairo University Faculty of Pharmacy, Suez Canal University

Faculty of Pharmacy Cairo University 2017

Abstract

This study comprises a brief literature survey on cancer, its causes and treatment as well as biological activities and chemistry of pyridazine and fused pyridazine derivatives.

The Thesis involves design of some new pyridazine and fused pyridazine derivatives aiming to possess an anticancer activity through inhibition of VEGFR.

The Schemes followed for the preparation of the designed compounds are summarized asfollows:

Scheme (1) starts with the preparation of the root compound N- Carbamimidoyl-4-[(6-chloropyridazin-3-yl)amino]benzenesulphonamide (I) followed by reaction with different aromatic amines to give N-Carbamimidoyl-4- [(6-arylaminopyridazin-3-yl)amino]benzenesulphonamides IIa-f.

Scheme (2) involves nucleophilic substitution reaction of compound I with meta and para phenylenediamine to give the relevant compounds IIIa, b. Furthermore, compound IIIa was reacted with each of acetyl chloride and 3- chloropropionyl chloride to give the corresponding pyridazine derivatives IVa, b respectively. Further, compounds IIIa, b were reacted with different alkyl isothiocyanates to give the corresponding thiourea compounds Va-d and VIa-d.

On the other hand, Scheme (3A) involves the reaction of compound I with each of 2-aminophenol, different substituted anthranilic acids and 2-aminonicotinic acid to give the corresponding pyridazino[1,6-a] VII, pyridazino[6,1-b] VIIIa,c,d and pyrido[2’, 3’,4,5]pyrimido[1,2- b]pyridazine VIIIb respectively.

Scheme (3B) includes the reaction of compound I with each of substituted acetophenone phenylhydrazones, thiosemicarbazide and substituted benzoic acid hydrazides to afford the corresponding hydrazinyl derivatives IXa-c, N- Carbamimidoyl-4-[(3-amino[1,2,4]triazolo[4,3-b]pyridazin-6-yl)amino] benzenesulphonamid (X) and 3-aryl [1,2,4]triazolo[4,3-b]pyridazine derivatives XIa-c respectively.

The structure elucidation of the newly synthesized compounds was supported by IR, 1HNMR, 13CNMR for some compounds, mass spectral data as well as elemental analysis.

Moreover, all the synthesized compounds were subjected to in vitro cytotoxic activity on two cell lines: human breast cancer cell line (MCF-7) and human colon cancer cell line (HCT-116) compared with imatinib as a reference anticancer drug. Compound VIIIb was the most potent against HCT-116 with

IC50 10.42 μM while compound VIIIa was the most potent against MCF-7 with

IC50 11.18 μM.

In addition, the antitumor activity of some synthesized compounds was tested in vivo against Ehrlich’s ascites carcinoma (EAC) solid tumor grown in mice. The antitumor activity produced by compounds IIe, IVb and Vb was greater than that produced by imatinib,

Finally, the in vitro VEGFR enzyme based inhibition assay was carried out at a single dose of 10 μM for some compounds. Percentage enzyme inhibition of IVb and Vb (89.6%, 92.0%, respectively) were more significant than that of imatinib (78.7%) against MCF-7.

1. Introduction

The work in this Thesis, involves the synthesis, anticancer activity and chemistry of pyridazine containing compounds. The following is a brief survey on the synthesis of pyridazine derivatives and their biological activities as well as cancer, its causes and treatment.

1.1. Chemistry of pyridazine ring

The are compounds derived from by the replacement of two of the ring carbon atoms by nitrogen. The three isomeric six membered ring diazines are pyridazine, and having two nitrogen atoms in 1-2, 1-3 and 1-4 relationship respectively.

N N N N N N

Pyridazine Pyrimidine Pyrazine

Pyridazine ring system does not form a part of any natural product and thus it has been less extensively investigated than other diazines. The reason for this is that pyridazine contains a potential unit in the ring and hence, they are not readily obtainable from biochemical transformation. However, pyridazines have received much more attention from the theoretical stand point since many derivatives were found to possess potential therapeutic effect.

In this Thesis, our interest was confined to pyridazine and pyridazine containing compounds. Several ways were reported to synthesize different derivatives of pyridazine. The following is a brief survey covering the methods of synthesis of this ring.

1.1.1. Synthesis of pyridazines:

The first reported pyridazine derivative 2 was obtained by Fischer in 1886 through cyclization of phenylhydrazone (1).

O

C6H5 NNHC H N 6 5 -H2O N H3C CH2CH2COOH

CH3 1 2

Pyridazine itself was first synthesized by Tauber through oxidation of benzo[c] (3) to 3,4,5,6-pyridazinetetracarboxylic acid (4) which then was decarboxylated to pyridazine (5).

COOH KMnO COOH 4 N 5%HCl N 200oC N COOH N N N COOH 3 4 5

In 1956, Cromwell and his associates, reported the synthesis of 2,6- diphenylpyridazin-3(2H)-one (8) via the reaction of β-benzoylacrylic acid (6) with to give the hydrazone derivative 7 which upon heating with acetic anhydride and sodium acetate afforded 2,6-diphenylpyridazin-3(2H)-one (8).

C6H5 O NNHC6H5 -H2O N + C6H5NHNH2 C6H5 N C6H5 CH=CHCOOH C6H5 CHCOOH O 6 7 8

In 1989, Wermuth et al. reported the synthesis of hydroxydihydro- pyridazinone 10 by the reaction of acetophenone and pyruvic acid to yield α- methyl-β-benzoyllactic acid (9) which upon cyclocondensation with hydrazine hydrate led to pyridazinone derivative 10.

O OH HO H O O N O KOH N C6H5COCH3 NH2-NH2.H2O + OH OH O C6H5 C6H5 9 10

In 1994, Moreau and his colleagues prepared pyridazinone derivative 13 via the reaction of levulinic acid (11) with benzaldehyde to produce substituted pentanoic acid 12 which when condensed with hydrazine hydrate gave pyridazinone derivative 13.

H O O C O C6H5 HCl gas NH NH .H O + C H CHO H C COOH 2 2 2 NH H3C COOH 6 5 3 H3C N CHC6H5

11 12 13

On the other hand, 3-amino-6-benzoyl-5-oxopyridazines 15 were synthesized by refluxing compounds 14 with ethanolic sodium hydroxide which accomplished their cyclization to pyridazine derivatives 15.

O O O O CN 20%NaOH CN C6H5 C H OH C6H5 N CN 2 5 N NH N NH2 R R

14 15

R = C H , 4-CH OC H , 4-ClC H , 4-CH C H 6 5 3 6 4 6 4 3 6 4

Furthermore, iminopyridazines 17 were successfully obtained by fusing the hydrazo derivatives 16 with malononitrile in the presence of ammonium acetate.

O O O C6H5

CH COONH CN C6H5 OC2H5 + CH2(CN)2 3 4 C2H5O N N NH N NH R R

16 17

R = C H , 4-CH OC H , 4-ClC H , 4-CH C H 6 5 3 6 4 6 4 3 6 4

In addition, 6-oxo-pyridazine derivatives 18 were obtained when compounds 16 were fused with ethyl cyanoacetete.

O C H O O 6 5 CN CNCH COOC H C2H5O C6H5 OC2H5 + 2 2 5 N N N O NH R R 16 18

R = C6H5, 4-CH3OC6H4, 4-ClC6H4, 4-CH3C6H4

In 2004, tetrahydropyridazin-3-one 20 was prepared via the condensation of oxobutanoic acid derivative 19 with hydrazine hydrate.

O O

S OH S NH O NH2NH2.H2O N

O O

19 20

In 2008, Kumar et al. demonstrated the synthesis of pyridazinone derivatives 22 through Friedel-Crafts acylation of acetanilide using succinic anhydride in presence of anhydrous aluminium chloride to afford the corresponding γ-keto acids 21, which upon subsequent cyclization with various hydrazine derivatives in provided the corresponding pyridazinone derivatives 22.

NHCOCH3

NHCOCH3 NHCOCH3 O O O AlCl3 NH2NHR + C2H5OH N OH O N R O O 21 22

R = C H , 4-FC H , C H N 6 5 6 4 3 6 2

Moreover, a group of investigators reported the synthesis of pyridazinones 24 via the cyclization of compounds 23 with ethyl cyanoacetate or diethyl malonate.

R R

CNCH2COOC2H5/C2H5ONa R R + or CH2(COOC2H5)2/C2H5ONa R1 O N NNH2 N O H

23 R = H, Br, OCH3 24 1 R = CN, COOC2H5

Aim of the work

Our main objective in this Thesis was to design and synthesize new active anticancer drugs through inhibition of VEGFR.

A marked increase in VEGF levels has been observed in various types of cancer including breast cancer and colon cancer. For both types of cancer significant correlations between VEGF and the extent of tumor vascularisation, tumor stages and metastasis have been reported. Blood VEGF levels are often used to estimate the degree of tumor development.

Breast cancer can be propagated and metastasize under the influence of the elevated levels of VEGF, which has been indicated as a potent angiogenic factor that has an effect on tumor progression.

It was reported that breast cancer is an angiogenic-dependent disease. Angiogenesis plays an essential role in breast cancer development, invasion, and metastasis. Breast cancer is potentially treatable by antiangiogenic therapy. Transfection of tumor cells with inhibitors of angiogenesis decreases growth and metastasis.

Patients with elevated serum concentration of VEGF having gastrointestinal and colon carcinoma is more progressive and tumor metastasized in these patients rapidly.

Inhibition of angiogenic activity is a logical therapeutic target and may provide means of shrinking primary tumors and diminishing metastatic spread.

Review

Scheme 1: Cl NH Cl 2 i N N + N N NH HN S Cl O N NH2 H H O N NH2 S O I O NH

ii IIa Ar =

IIb Ar = CH3 NHAr F N IIc Ar = N

Cl HN N IId Ar = H N NH2 S O Cl O NH IIe Ar = IIa-f

Cl

Br

IIf Ar =

Reagents and conditions: i)(CH3)2CHOH/reflux 4h, ii) ArNH2/CH3CH2CH2CH2OH/reflux 15h.

ii

HN NHCOR Scheme 2: N HN NH2 N N HN N H N NH2 HN S O H O NH N NH2 S IVa, b Cl IIIa O O NH i iii N N HN NHCSNHR

HN N

H N N NH2 S O HN I O NH H N NH2 S O O NH Va-d NH2

IVa R=-CH3 IVb R=-CH2CH2Cl HN NHCSNHR Va R=-CH3 Vb R=-CH CH 2 3 N Vc R=-CH2CH=CH2 iii N HN Vd R=-(CH2)3CH3 VIa R=-CH3 HN N VIb R=-CH2CH3 N VIc R=-CH2CH=CH2 H VId R=-(CH ) CH N NH2 2 3 3 S O HN IIIb O NH H N NH2 S O O NH VIa-d

Reagents and conditions: i)3 or 4-(NH2)2C6H4/abs. C2H5OH/reflux 12h, ii) RCOCl/abs. C2H5OH/ reflux 9h.iii)RCSN/abs. C2H5OH/reflux10h

Scheme 3A: N

i N N

HN H N NH2 S Cl O O NH N VII N

R HN R1 H X N NH2 S O N R2 I O NH

ii N O N

HN H N NH2 S O O NH VIIIa R= -H, R1= -H, R2= -H, X= C VIIIa-d VIIIb R= -H, R1= -H, R2= -H, X= N 1 2 VIIIc R= -OCH3, R = -OCH3, R = -H, X= C VIIId R= -H, R1= -H, R2= -I, X= C

Reagents and conditions: i) 2-aminophenol/DMF/24 h, ii) substituted anthranilic acids and 2-aminonicotinic acid/CH3CH2CH2CH2OH/24 h.

R

N Scheme 3B: N C

CH3 N i N

HN H N NH2 S O O NH IXa-c

Cl N N

NH2 N ii N N N

HN HN H H N NH2 N NH2 S S O O I O NH O NH X

N N

iii N R N

IXa R= -H HN IXb R= -Cl H N NH2 IXc R= -Br S XIa R= -H O O NH XIb R= -CH3 XIc R= -Cl XIa-c

Reagents and conditions: i) substituted acetophenone phenylhydrazones/160-180 oC/ 7h, ii)thiosemicarbazide/abs. C2H5OH/20 h, iii)benzoyl hydrazine derivatives/ 35 h.

Summary

This Thesis comprises four chapters. The first is an introduction which is composed of a brief literature survey on the different methods for the synthesis of pyridazine derivatives, a brief survey on cancer, its causes and treatment as well as the biological activities of pyridazine and their cytotoxic activities. The second chapter clarifies the objectives of the work and Schemes designed for the preparation of the new required target compounds. The third chapter explains the theoretical discussion of the experimental work and discussion of the results of cytotoxic activity. The fourth chapter includes the experimental part of this thesis which contains the detailed procedure used for the synthesis of the target compounds as well as the intermediates. In addition, the physical properties of the synthesized compounds, their elemental analysis and spectral data are illustrated. Also, it demonstrates the in vitro cytotoxic activity for all the synthesized compounds on two cell lines: human breast cancer cell line (MCF-7) and human colon cancer cell line (HCT-116) compared with imatinib as a reference anticancer drug. Moreover, the antitumor activity of some synthesized compounds was tested in vivo against Ehrlich’s ascites carcinoma (EAC) solid tumor grown in mice. Finally, the in vitro VEGFR enzyme based inhibition assay was carried out at a single dose of 10 μM for some compounds.

Results and conclusion

The present study reports the synthesis and anticancer activity of some pyridazine containing compounds. The obtained results revealed that compound Vc, which showed good cytotoxic activity against colon cancer cell line (HCT-116) also, exhibited the highest inhibition in the VEGFR kinase assay (92.2%).

On the other hand, the antitumor activity produced by compounds IVb and Vc was greater than that produced by imatinib, when tested in vivo against EAC also, showed percentage enzyme inhibition (89.6%, 92.0%, respectively) on breast cancer cell line (MCF-7) more significant than that of imatinib (78.7%).

"تصوين و تشييذ بعض الوركباث التً تحتىي علً حلقت البيريذازيي و الوتىقع لها تأثير هضاد للسرطاى"

رسالة هقدهة هن الواجستير/ دهياًت سوير هيخائيل

هاجسحيرالعلوم الصيدلية )كيوياء عضوية(

جاهعة القاهرة )3122(

للحصول على درجة دكحوراة الفلسفة فى العلوم الصيدلية

)كيوياء عضوية صيدلية(

جحث إشراف األستار الذكتىرة/سلىي السيذ هحوذ الوليجً

أسحاذ الكيوياء العضوية كلية الصيدلة - جاهعة القاهرة

عويد كلية الصيدلة-جاهعة هدينة السادات

رئيس قسن الكيوياء العضوية الصيدلية كلية الصيدلة-جاهعة القاهرة )سابقا( األستار الذكتىرة/إيواى هحوذ أحوذ

أسحاذ الكيوياء العضوية كلية الصيدلة - جاهعة القاهرة

أ.د.م/سىزاى هحوذ أبى الوعاطً أ.د.م/سىسي أبى بكر زيتىى

أسحاذ هساعد الكيوياء العضوية أسحاذ هساعد بقسن األدوية و السووم

كلية الصيدلة - جاهعة القاهرة كلية الصيدلة- جاهعة قناة السويس

كليت الصيذلت

جاهعت القاهرة

7102