Increasing the binding affinity of VEGFR-2 inhibitors by extending their hydrophobic interaction with the active site: Design, synthesis and biological evaluation of 1-substituted-4-(4-methoxybenzyl)phthalazine derivatives

Accepted Manuscript

Increasing the Binding Affinity of VEGFR-2 Inhibitors by Extending their Hydrophobic

Interaction with the Active Site: Design, Synthesis and Biological Evaluation of 1-

Substituted-4-(4-methoxybenzyl)phthalazine Derivatives

Wagdy M. Eldehna, Sahar M. Abou-Seri, Ahmed M. El Kerdawy, Rezk R. Ayyad,

Abdallah M. Hamdy, Hazem A. Ghabbour, Mamdouh M. Ali, Dalal A. Abou El Ella

PII:

S0223-5234(16)30109-X

10.1016/j.ejmech.2016.02.029

EJMECH 8380

DOI:

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 24 November 2015

Revised Date: 9 February 2016

Accepted Date: 10 February 2016

Please cite this article as: W.M. Eldehna, S.M. Abou-Seri, A.M. El Kerdawy, R.R. Ayyad, A.M. Hamdy,

H.A. Ghabbour, M.M. Ali, D.A. Abou El Ella, Increasing the Binding Affinity of VEGFR-2 Inhibitors by

Extending their Hydrophobic Interaction with the Active Site: Design, Synthesis and Biological Evaluation

of 1-Substituted-4-(4-methoxybenzyl)phthalazine Derivatives, European Journal of Medicinal Chemistry

(2016), doi: 10.1016/j.ejmech.2016.02.029.

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ACCEPTED MANUSCRIPT

Graphical abstract

Increasing VEGFR-2 Inhibitors Binding Affinity by Extending their Hydrophobic

Interaction with the Active Site: Design, Synthesis and Biological Evaluation of 4-(4-

Methoxybenzyl)phthalazine Derivatives as Novel VEGFR-2 Inhibitors

Wagdy M. Eldehna*, Sahar M. Abou-Seri*, Ahmed M. El Kerdawy, Rezk R. Ayyad,

Hazem A. Ghabbour, Mamdouh M. Ali, Dalal A. Abou El Ella

Extending the anilinophthalazine derivatives 4 with a substituted phenyl moiety

through an uriedo linker increased the hydrophobic interaction of uriedo-

anilinophthalazine derivatives 7 within the hydrophobic back pocket.

ACCEPTED MANUSCRIPT

Increasing the Binding Affinity of VEGFR-2 Inhibitors by Extending their

Hydrophobic Interaction with the Active Site: Design, Synthesis and Biological

Evaluation of 1-Substituted-4-(4-methoxybenzyl)phthalazine Derivatives

Wagdy M. Eldehna^a,*, Sahar M. Abou-Seri^b,c,*, Ahmed M. El Kerdawy^b,c, Rezk

R. Ayyad^d,e, Abdallah M. Hamdy^f, Hazem A. Ghabbour^g, Mamdouh M. Ali^h,

Dalal A. Abou El Ellaⁱ

^aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian

University, Badr City, Cairo, P.O. Box 11829, Egypt.

^bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-

Aini Street, Cairo, P.O. Box 11562, Egypt.

^cMolecular Modeling Unit, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street,

Cairo, P.O. Box 11562, Egypt.

^dDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University,

Cairo, Egypt.

^eDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Delta University For

Science and Technology, Mansoura, Egypt.

^fDepartment of Analytical Chemistry, Faculty of Pharmacy, Egyptian Russian University,

Badr City, Cairo, P.O. Box 11829, Egypt.

^gDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O.

Box 2457, Riyadh 11451, Saudi Arabia.

^hBiochemistry Department, Division of Genetic Engineering and Biotechnology, National

Research Centre, Dokki 12622, Giza, Egypt.

ⁱDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University,

Cairo, Abbassia, P.O. Box 11566, Egypt.

Abstract

A series of anilinophthalazine derivatives 4a-j was initially synthesized and tested for its

VEGFR-2 inhibitory activity where it showed promising activity (IC₅₀=0.636–5.76 µM).

Molecular docking studies guidance was used to improve the binding affinity for series 4a-j

towards VEGFR-2 active site. This improvement was achieved by increasing the hydrophobic

interaction with the hydrophobic back pocket of the VEGFR-2 active site lined with the

hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019 and Ile1044.

Increasing the hydrophobic interaction was accomplished by extending the anilinophthalazine

scaffold with a substituted phenyl moiety through an uriedo linker which should give this

extension the flexibility required to accommodate itself deeply into the hydrophobic back

pocket. As planned, the designed uriedo-anilinophthalazines 7a-i showed superior binding

affinity than their anilinophthalazine parents (IC₅₀=0.083–0.473 µM). In particular,

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compounds 7g-i showed IC₅₀of 0.086, 0.083 and 0.086 µM, respectively, which are better

than that of the reference drug sorafenib (IC₅₀=0.09 µM).

Keywords: Phthalazine; VEGFR-2; hydrophobic interaction.

*Corresponding author. Department of Pharmaceutical Chemistry, Faculty of Pharmacy,

Egyptian Russian University, Cairo, Egypt. wagdy2000@gmail.com (W.M. Eldehna),

*Corresponding author. Department of Pharmaceutical Chemistry, Faculty of Pharmacy,

Cairo University, Cairo, Egypt. saharshaarawy_69@yahoo.com (S.M. Abou-Seri).

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1. Introduction

Angiogenesis, the process of new blood vessel growth from the quiescent pre-existing

vessels, is a critical and complex process in both physiological and pathophysiological

conditions [1]. The process is regulated by a balance between pro- and antiangiogenic

molecules, and is derailed in various diseases, especially cancer [2]. In 1971, Folkman [3]

proposed that tumor growth and metastasis are angiogenesis-dependent, and hence, blocking

angiogenesis could be a strategy to hinder tumor growth.

Among the various players at the molecular level involved in the different mechanisms of

vascular growth, members of the vascular endothelial growth factor (VEGF) family have a

predominant role [4]. Vascular endothelial growth factor (VEGF) represents a family of

homodimeric glycoproteins which are critical for vasculogenesis, lymphangiogenesis and

angiogenesis [5]. VEGF ligands bind in an overlapping pattern to three different, but

structurally related, VEGF-receptor tyrosine kinases (VEGFR-TK) which are VEGFR-1 (Flt-

1), VEGFR-2 (KDR) and VEGFR-3 (Flt-4) [6].

VEGFR-2, a type III transmembrane tyrosine kinase receptor, is critical for angiogenesis

[7]. Hence, VEGFR-2 inhibition emerged as a prime approach for discovering new therapies

for many human angiogenesis-dependent malignancies. To date, a humanized anti-VEGF

monoclonal antibody (bevacizumab) and several small molecule VEGFR-2 kinase inhibitors

(sorafenib, regorafenib, sunitinib, vandetanib, pazopanib, axitinib and cabozantinib) have

been approved as antiangiogenic drugs [8].

Phthalazine derivatives have attracted considerable attention as antitumor agents [9-14]. In

this context, 1-anilino-4-aryl-phthalazine scaffold in particular emerged as an interesting

scaffold for designing VEGFR inhibitors. The first published anilinophthalazine derivative as

a potent VEGFR inhibitor was vatalanib (PTK787) I (Figure 1) [15]. Vatalanib I is one of the

most potent and selective VEGFR kinase inhibitors, with IC₅₀value of 43 nM for VEGFR-2

[16]. Vatalanib I showed interesting results in many clinical trials on different tumor types,

such as metastatic gastrointestinal stromal tumors (GIST), Non-small-cell lung carcinoma

(NSCLC) and metastatic pancreatic cancer [17-19]. The disclosure of vatalanib as a clinical

candidate paved the way for the design and synthesis of different anilinophthalazine

derivatives as potent inhibitors for VEGFR-2 such as IM-023911 II with IC₅₀= 190 nM

(Figure 1) [20-24].

The aforementioned facts have motivated us to design novel 1-anilino-4-aryl-phthalazine

derivatives with more potent VEGFR-2 inhibitory activity.

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Figure 1

The essential pharmacophoric features in the anilinophthalazine VEGFR-2 inhibitors

include (Figure 2): [23, 25]

a) Fused aromatic system represented by the phthalazine ring interacting as H-bond

acceptor with its N2 with Asp1046 in the conserved DFG motif and through hydrophobic

interaction with the side chains of Lys868 and Val916.

b) Substituted anilino moiety in position 1 of the phthalazine interacting as H-bond donor

with its NH with the side chain carboxylate of Glu885 of the αC helix and through

hydrophobic interaction with its substituted phenyl moiety with the hydrophobic back pocket

lined with the hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019

and Ile1044.

c) Hydrogen bond acceptor represented by a lone pair of a nitrogen (as in vatalanib I) or

oxygen atom (as in IM-023911 II) attached to position 4 of phthalazine via benzyl or phenyl

moiety, which interacts with the backbone NH of Cys919 in the hinge region of the protein.

Figure 2

In the presented work, we introduce two series of anilinophthalazine derivatives [4a-j and

7a-i] with different substitutions on the anilino moiety offering various electronic and

lipophilic environments to study their impact on the activity (Figure 1). We also replaced the

4-pyridyl moiety in the vatalanib by the relatively longer 4-methoxyphenyl moiety which

should make the oxygen lone pair more accessible for interaction with Cys919 NH group than

that of the 4-pyridyl nitrogen.

Molecular docking studies were carried out to study the interaction of the newly

synthesized compounds with VEGFR-2, their binding mode and their ability to satisfy the

pharmacophoric features required to induce the desired inhibition. They were also used further

as a guide to improve the binding between VEGFR-2 and the newly synthesized ligands.

The newly synthesized phthalazines 4a-j and 7a-i were evaluated for their potential

VEGFR-2 inhibitory and anti-proliferative activities.

2. Results and discussion

2.1. Chemistry

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The synthetic pathway employed to prepare the new phthalazine derivatives is outlined in

Schemes 1-2. Synthesis was initiated by reacting phthalic anhydride with 4-methoxy

phenylacetic acid in the presence of fused sodium acetate to afford 3-(4-

methoxybenzylidene)isobenzofuran-1(3H)-one (1) [26], which was treated with hydrazine

sulfate and sodium hydroxide to provide 4-(4-methoxybenzyl)phthalazin-1(2H)-one (2) [26].

The key intermediate 1-chloro-4-(4-methoxybenzyl)phthalazine (3) was prepared via

chlorination of 2 with refluxing phosphorous oxychloride in the presence of a catalytic

amount of N,N-dimethylaniline. The target compounds 4a-j were obtained by coupling the

chloro intermediate 3 with the appropriate aniline in a refluxing acetone (Scheme 1).

Scheme 1

IR spectra of 4a-j revealed the presence of an NH stretching band about 3300 cm^-1. Their

¹H NMR spectra showed one D₂O-exchangeable singlet signal attributed to the aniline NH

proton in the region δ 9.41-11.16 ppm, while the benzylic CH₂protons appeared as singlet

13

signal in the region δ 4.43-4.74 ppm. Furthermore, the C NMR spectra of compounds 4a-j

revealed the presence of the signal of the benzylic methylene carbon in the range δ 34.13-

37.59 ppm, while the signal due to the carbon of the methoxy group appeared around 55 ppm.

For the synthesis of ureido derivatives 7a-i, the 1-(4-aminophenyl)-3-phenylurea

intermediates 6a-i were synthesized by the reaction of 1-isocyanato-4-nitrobenzene with the

adequate aniline to afford 1-(4-nitrophenyl)-3-phenylureas 5a-i followed by hydrogenation

using Pd/C [27]. The target diarylureas 7a-i were prepared through the reaction of the key

intermediate 3 and the corresponding 1-(4-aminophenyl)-3-phenylureas 6a-i in a refluxing

acetone (Scheme 2).

Scheme 2

IR spectra of the latter products showed the absorption bands due to the (NH) and (C=O)

groups in the regions δ 3244-3340 and 1666-1714 cm^-1, respectively, whereas, their ¹H NMR

spectra revealed the presence of three D₂O-exchangeable singlet signals assigned to the two

amidic NH groups and the secondary amino NH in the regions δ 8.57-10.16, 8.63-10.89 and

9.02-10.97 ppm, respectively. Besides, the ¹³C NMR spectra of compounds 7a-i showed three

characteristic signals resonating in the range δ 158.04-158.13, 54.88-55.00 and 35.60-36.11

ppm attributable for the carbonyl, methoxy and benzylic methylene carbons, respectively.

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2.2. Activity towards VEGFR-2 kinase

2.2.1. VEGFR-2 inhibitory assay

All newly synthesized compounds were evaluated in vitro for their VEGFR-2 inhibitory

activity. Sorafenib III was included in the experiments as reference VEGFR-2 inhibitor. The

results are reported as median inhibition concentrations (IC₅₀) and summarized in Table 1.

As can be seen in Table 1, the novel phthalazine derivatives displayed moderate to potent

VEGFR-2 inhibitory activity. Generally, series 7a-i showed superior activity in comparison to

series 4a-j (IC₅₀= 0.083 - 0.473 µM vs. 0.636 - 5.76 µM, respectively). Among the tested

phthalazine derivatives, compounds 7g-i were found to be the most potent members with

excellent VEGFR-2 inhibitory activity (IC₅₀= 0.086, 0.083 and 0.086 µM, respectively)

compared to sorafenib III (IC₅₀= 0.090 µM). Compounds 4h, 4j and 7a-f exhibited high

potency at the sub-micromolar level with IC₅₀ranged from 0.114 to 0.754 µM. On the other

hand, compounds 4a-g and 4i were the least potent analogs with IC₅₀range of 1.34 - 5.76 µM.

Table 1

2.2.2. Molecular docking study and structure activity relationship SAR

We started this work by the synthesis of a series of anilinophthalazine derivatives with

various substitutions on the anilino moiety 4a-j to investigate the impact of this variable on

activity. This series showed promising activity in the VEGFR-2 kinase inhibition assay with

IC₅₀ranged between 0.636 and 5.76 µM. At this stage molecular docking study was carried

out to investigate their plausible binding pattern and their interaction with the key amino acids

in the active site of the VEGFR-2. Information gained from the docking result analysis for

compounds 4a-j was used as a guide to perform some educated manipulations in their

chemical structure to improve the series activity.

Several crystal structures are available in the protein data bank for VEGFR-2 [28], for this

work we chose (PDB ID: 4ASD) [29] which has VEGFR-2 in the inactive conformation

(DFG-out) co-crystallized with sorafenib as inhibitor.

First, the molecular docking protocol was validated by re-docking of the sorafenib ligand in

the vicinity of the VEGFR-2 receptor active site. The re-docking validation step reproduced

the experimental binding mode of the co-crystallized ligand quite efficiently indicating the

suitability of the used setup for the intended docking study as demonstrated by the small

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RMSD of 0.470Å between the docked pose and the co-crystallized ligand (energy score (S) =

−15.19 kcal/mol) and by the ability of the docking pose to reproduce all the key interactions

accomplished by the co-crystallized ligand with the key amino acids in the active site

(Glu885, Cys919 and Asp1046) (Figure 3)

Figure 3

The ability of the synthesized compounds 4a-j to interact with the key amino acids in the

active site rationalizes their good activity as indicated by their docking pattern and docking

score compared to that of sorafenib (Figure 4 and 5, Table 2). As can be seen in Figure 4,

compound 4g (as a representative for series 4a-j) interacts by its phthalazine ring N2 through

H-bond with Asp1046 of the conserved DFG motif exposing its benzene ring to the

hydrophobic side chains of Lys868 and Val916. As H-bond donor, it interacts with its anilino

NH with the side chain carboxylate of Glu885 of the αC helix fitting its substituted phenyl

moiety into the hydrophobic back pocket lined with the hydrophobic side chains of Ile888,

Leu889, Ile892, Val898, Val899, Leu1019 and Ile1044. As planned the oxygen lone pair of

the 4-methoxyphenyl moiety interacts as H-bond acceptor with the Cys919 NH group (2D

diagrams and 3D representations for the rest of the series can be found in the supporting

information).

Figure 4

As indicated by the experimental assay (Tables 1) and supported by the docking energy

score (Tables 2), the derivative with an unsubstituted anilino moiety 4a has the lowest activity

5.76±0.58 ꢀM and docking energy score −14.74 kcal/mol. Addition of a hydrophobic

substituent on the anilino moiety (4b,4d-4i) increases the binding affinity as indicated by their

lower IC₅₀(0.754±0.07 - 3.97±0.43 µM) and better docking energy score (−15.06 - −16.32

kcal/mol) than the unsubstituted derivative 4a which could be attributed to their increased

hydrophobic interaction with the hydrophobic back pocket (left side of the Figure 4b). The

superior activity of compound 4j (IC₅₀= 0.636±0.06 µM) which is supported by its high

docking score (−15.94 kcal/mol) can be attributed to the exposure of its hydrophilic

sulphonamido moiety to the bulk solvent and its involvement in H-bonding with the imidazole

ring of His1026 in the catalytic loop (Figure 5).

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Table 2

Figure 5

Based on the docking results of the anilinophthalazine derivatives 4a-j, another series 7a-i

was designed in an attempt to increase the binding affinity to VEGFR-2 active site through

increasing the hydrophobic interaction with the hydrophobic back pocket. In this new series

7a-i, series 4a-j was extended with a second substituted phenyl group on the anilino moiety

through an uriedo linker (Figure 1) giving this second phenyl group the flexibility required to

accommodate itself into the hydrophobic back pocket. The selection of the uriedo linker is due

to the reported anti-tumor activity of a vast array of urea containing compounds [30].

As planned, series 7a-i showed better docking energy scores (S) compared to series 4a-j

(−17.85 to −20.25 kcal/mol vs. −14.74 to −16.32 kcal/mol, respectively) (Table 2) due to

extension of the hydrophobic interaction with the hydrophobic back pocket. This hypothesis is

augmented by the fact that increasing the hydrophobicity of the substituent(s) on the pendant

phenyl ring increases the docking energy score (Table 2). In agreement with the docking

energy scores (S), the expected superiority of series 7a-i was experimentally proven by their

lower IC₅₀values in comparison to series 4a-j (Table 1). Figure 6 shows the proposed binding

mode of series 7a-i as represented by compound 7h docking pose in the VEGFR-2 active site

indicating that series 7a-i does not only fulfill the key interactions required for VEGFR-2

inhibition with the key amino acids in the active site (Glu885, Cys919 and Asp1046) as series

4a-j does (figure 4) but also extending with their phenyl ureido moiety into the hydrophobic

back pocket achieving additional hydrophobic interaction which is responsible for their higher

docking score and consequently higher VEGFR-2 inhibitory activity than that of series 4a-j

(table 1 and table 2)

Figure 6

2.3. In vitro anti-proliferative activity.

All the newly synthesized compounds were selected by the National Cancer Institute

(NCI) Developmental Therapeutic Program (www.dtp.nci.nih.gov) to be screened for their

anticancer activity in vitro. The anticancer assays were performed in accordance with the

protocol of the Drug Evaluation Branch, NCI, Bethesda [31-33].

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The compounds were evaluated at one dose primary anticancer assay towards a panel of

approximately 60 cancer cell lines (concentration 10^-5M). The human tumor cell lines were

taken from nine different organs (blood, skin, lung, colon, brain, ovary, kidney, prostate and

breast). A 48 h drug exposure protocol was used and sulforhodamine B (SRB) protein assay

was applied to estimate the cell viability and growth [34]. The data reported as mean-graph of

the percent growth of the treated cells, and presented as percentage growth inhibition (GI%)

caused by the test compounds (Table 3).

The tested compounds displayed different levels of anticancer activity and possessed a

distinctive pattern of selectivity against the tested cancer cell lines.

Close examination of the GI% values in Table 3, revealed that compounds 4b, 4e-g, 7c,

7f, 7g and 7i are the most active analogues in this study, showing broad spectrum activity

toward numerous cell lines that belong to different tumor subpanels. In particular, compound

4g emerged as the most active member with mean inhibition = 44 %. It possesed

antiproliferative activity against 44 cell lines representing all subpanels (GI; 32-94 %) with

potent growth inhibitory effect over leukemia MOLT-4 and RPMI-8226, non-small cell lung

cancer NCI-H460, colon cancer HCT-116, renal cancer UO-31and prostate cancer PC-3 with

inhibition % 69, 94, 74, 73, 63 and 61, respectively. On the contrary, compounds 4d and 4j

have not shown any significant cytotoxic activity against any cell line. Nevertheless,

compounds 4a, 4c, 7d and 7h possessed fair and selective growth inhibitory activity against

sporadic cell lines.

Table 3

Regarding sensitivity of individual cell lines (Figure 7), leukemia RPMI-8226 was the

most susceptible cell line to the majority of compounds, especially compounds 4b, 4e, 4g, 4h,

7c and 7i displayed strong antiproliferative activity with GI %> 80. Meanwhile, compound 7i

showed potent growth inhibition against renal cancer RXF 393 and breast cancer HS 578T

(GI %= 100 and 96, respectively). Moreover, compounds 7c and 7f exerted cytotoxic effect

with GI more than 100% against certain cell lines. Compound 7c was proved to be lethal to

renal cancer RXF 393 and breast cancer HS 578T cell lines (GI %= 110 and 105,

respectively), while 7f exerted lethal activity over non-small cell lung cancer NCI-H460 with

GI %=110.

A control experiment was carried out to rule out that the compounds’ low cytotoxic

activity is due to the binding interaction between the hydrophobic tested compounds and the

serum albumin used in the experiment. [For further details, see Supporting Information]

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3. Conclusion

The newly synthesized anilinophthalazine derivatives 4a-j showed promising activity in

the VEGFR-2 kinase inhibition assay with IC₅₀ranged between 0.636 and 5.76 µM.

Molecular docking studies guided their binding affinity improvement through increasing the

hydrophobic interaction of the synthesized inhibitors with the hydrophobic back pocket of the

VEGFR-2 active site. Extending the anilinophthalazine scaffold with a substituted phenyl

moiety through an uriedo linker was the used strategy to increase the hydrophobic interaction

with the hydrophobic back pocket. The designed uriedo-anilinophthalazine derivatives 7a-i

showed superior binding affinity than their anilinophthalazine parents in the VEGFR-2 kinase

inhibition assay with IC₅₀ranged between 0.083 and 0.473 µM. In particular, compounds 7g-i

showed IC₅₀values of 0.086, 0.083 and 0.086 µM, respectively, which are better than that of

the reference drug sorafenib (IC₅₀= 0.09 µM). Our ultimate conclusion is that VEGFR-2

inhibitors with high binding affinity could be designed by extending the hydrophobic

interaction between the inhibitor and the hydrophobic back pocket of the VEGFR-2 active

site.

4. Experimental

4.1. Chemistry

Melting points were measured with a Stuart melting point apparatus and were uncorrected.

Infrared spectra were recorded as potassium bromide discs on Schimadzu FT-IR 8400S

spectrophotometer and expressed in wave number (cm^-1). The NMR spectra were recorded by

Varian Gemini-300BB 300 MHz FT-NMR spectrometers (Varian Inc., Palo Alto, CA). ¹H and

¹³C spectra were run at 300 and 75 MHz, respectively, in deuterated dimethylsulphoxide

(DMSO-d₆). Chemical shifts (δ_H) are reported relative to TMS as internal standard. All

coupling constant (J) values are given in hertz. Mass spectra were measured on a GCMS-

QP1000 EX spectrometer at 70 e.V. Elemental analyses was carried out at the Regional

Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt Analytical thin

layer chromatography (TLC) on silica gel plates containing UV indicator was employed

routinely to follow the course of reactions and to check the purity of products. All reagents

and solvents were purified and dried by standard techniques.

4.1.1. (Z)-3-(4-Methoxybenzylidene)isobenzofuran-1(3H)-one (1)

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Compound 1 was prepared according to reported procedure [26] (m.p. 147-149 °C).

4.1.2. 4-(4-Methoxybenzyl)phthalazin-1(2H)-one (2)

Compound 2 was prepared according to reported procedure [26] (m.p. 193-195 °C).

4.1.3. 1-Chloro-4-(4-methoxybenzyl)phthalazine (3)

4-(4-Methoxybenzyl)phthalazin-1(2H)-one 2 (2.66 g, 0.01 mol) was added portion-wise to

a cooled stirred mixture of phosphorus oxychloride (15 mL) and N,N-dimethylaniline (0.5

mL). After refluxing for 10 h, the reaction mixture was cooled then poured onto ice/water and

alkalinized with 2 N NaOH. The aqueous solution was extracted three times with ethyl

acetate. The combined organic extracts were dried over anhydrous Na₂SO₄then filtered, and

the solvent was removed under reduced pressure. The obtained solid was crystallized from

1

ethanol to give 3 (1.7 g, 60%) as beige solid; m.p. 187-189 °C; IR (KBr, ν cm^-1): H NMR

(DMSO-d₆)

δ ppm: 3.68 (s, 3H, OCH₃), 4.63 (s, 2H, CH₂), 6.81 (d, 2H, H-3 and H-5 of 4-

OCH₃-C₆H₄, J= 8.7 Hz), 7.23 (d, 2H, H-2 and H-6 of 4-OCH₃-C₆H₄, J= 9.0 Hz), 8.05 – 8.12

(m, 2H, H-6 and H-7 phthalazine), 8.25 – 8.29 (m, 1H, H-5 phthalazine), 8.34 – 8.37 (m, 1H,

H-8 phthalazine).

4.1.4. General procedure for preparation of target compounds (4a-j):

To a stirred solution of 1-chloro-4-(4-methoxybenzyl)phthalazine 3 (2.84 g, 0.01 mol) in

refluxing acetone (15 mL), a solution of the appropriate anilne (0.012 mol) in acetone was

added. The reaction mixture was heated under reflux for 3 h. The precipitate formed after

cooling was collected by filtration and washed with water, dried then recrystallized from

ethanol-dioxane mixture except 4j, recrystallized from ethanol-DMF mixture.

4.1.4.1. 4-(4-Methoxybenzyl)-N-phenylphthalazin-1-amine (4a):

Yellow crystals (yield 66%); m.p. 263-265°C; IR (KBr, ν cm^-1): 3232 (NH), 2592 (CH

1

aliphatic); H NMR (DMSO-d₆)

δ ppm: 3.68 (s, 3H, OCH₃), 4.54 (s, 2H, CH₂), 6.83 (d, 2H,

H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 7.17 (t, 1H, H-4 of NH-C₆H₅, J= 7.8 Hz), 7.27 (d,

2H, H-2 and H-6 of 4-OCH₃-C₆H₄, J= 8.1 Hz), 7.42 (t, 2H, H-3 and H-5 of NH-C₆H₅, J= 7.8

Hz), 7.81 (d, 2H, H-2 and H-6 of NH-C₆H₅, J= 7.2 Hz), 7.99 – 8.09 (m, 2H, H-6 and H-7

phthalazine), 8.25 (d, 1H, H-5 phthalazine, J= 7.8 Hz), 8.83 (d, 1H, H-8 phthalazine, J= 7.5

Hz), 10.17 (s, 1H, NH); ¹³C NMR (DMSO-d₆)

δ ppm: 36.19 (-CH₂-), 55.01 (-OCH₃), 114.00,

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120.09, 122.31, 122.48, 123.99, 126.18, 126.43, 128.79, 129.62, 133.07, 133.33, 138.80,

152.23, 152.35, 157.93; Anal. Calcd. for C₂₂H₁₉N₃O: C, 77.40; H, 5.61; N, 12.31; Found C,

77.61; H, 5.66; N, 12.53.

4.1.4.2. 4-(4-Methoxybenzyl)-N-(3-(trifluoromethyl)phenyl)phthalazin-1-amine (4b):

White crystals (yield 73%); m.p. 215-218 °C; IR (KBr, ν cm^-1): 3446 (NH), 2677 (CH

1

aliphatic); H NMR (DMSO-d₆)

δ ppm: 3.67 (s, 3H, OCH₃), 4.49 (s, 2H, CH₂), 6.80 (d, 2H,

H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.1 Hz), 7.22 (d, 2H, H-2 and H-6 of 4-OCH₃-C₆H₄, J= 8.7

Hz), 7.32 (d, 1H, H-4 of 3-CF₃-C₆H₄, J= 7.8 Hz), 7.58 (t, 1H, H-5 of 3-CF₃-C₆H₄, J= 7.8 Hz),

7.87 – 7.98 (m, 2H, H-6 and H-7 phthalazine), 8.12 (d, 1H, H-6 of 3-CF₃-C₆H₄, J= 7.5 Hz),

8.25 (d, 1H, H-5 phthalazine, J= 8.4 Hz), 8.50 (s, 1H, H-2 of 3-CF₃-C₆H₄), 8.58 (d, 1H, H-8

phthalazine, J= 7.8 Hz), 9.41 (s, 1H, NH); ¹³C NMR (DMSO-d₆)

δ ppm: 37.59 (-CH₂-), 55.02

(-OCH₃), 113.71, 113.99, 115.93, 117.80, 118.79, 122.58, 123.47, 125.25, 125.97, 126.91,

128.89, 129.32, 129.40, 129.48, 131.36, 131.96, 141.67, 151.74, 153.39, 157.70; Anal. Calcd.

for C₂₃H₁₈F₃N₃O: C, 67.48; H, 4.43; N, 10.26; Found C, 67.59; H, 4.47 ; N, 10.52.

4.1.4.3. 4-((4-(4-Methoxybenzyl)phthalazin-1-yl)amino)benzonitrile (4c):

Yellow crystals (yield 68%); m.p. 247-249 °C; IR (KBr, ν cm^-1): 3244 (NH), 2630 (CH

1

aliphatic), 2218 (C≡N); H NMR (DMSO-d₆)

δ ppm: 3.68 (s, 3H, OCH₃), 4.64 (s, 2H, CH₂),

6.83 (d, 2H, H-3 and H-5 of 4-OCH₃-C₆H₄, J= 9.0 Hz), 7.30 (d, 2H, H-2 and H-6 of 4-OCH₃-

C₆H₄, J= 8.4 Hz), 7.83 (d, 2H, H-2 and H-6 of 4-CN-C₆H₄, J= 8.1 Hz), 8.03 – 8.16 (m, 4H, H-

3, H-5 of 4-CN-C₆H₄and H-6, H-7 phthalazine), 8.34 (d, 1H, H-5 phthalazine, J= 8.1 Hz),

8.82 (d, 1H, H-8 phthalazine, J= 7.8 Hz), 10.27 (s, 1H, NH); ¹³C NMR (DMSO-d₆)

δ ppm:

35.73 (-CH₂-), 54.85 (-OCH₃), 104.34, 113.92, 114.20, 119.25, 120.51, 120.85, 121.12,

123.79, 124.00, 126.47, 129.21, 129.61, 129.72, 132.89, 133.06, 144.18, 152.42, 153.72,

158.04; MS m/z 366 [M⁺]; Anal. Calcd. for C₂₃H₁₈N₄O: C, 75.39; H, 4.95; N, 15.29; Found C,

75.53; H, 5.01; N, 15.48.

4.1.4.4. N-(2-Chlorophenyl)-4-(4-methoxybenzyl)phthalazin-1-amine (4d):

Beige crystals (yield 59%); m.p. 155-157°C; IR (KBr, ν cm^-1): 3250 (NH), 2586 (CH

1

aliphatic); H NMR (DMSO-d₆)

δ ppm: 3.67 (s, 3H, OCH₃), 4.43 (s, 2H, CH₂), 6.80 (d, 2H,

H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 7.19 (d, 2H, H-2 and H-6 of 4-OCH₃-C₆H₄, J= 8.7

Hz), 7.25 (d, 1H, H-3 of 2-Cl-C₆H₄, J= 7.8 Hz), 7.40 (t, 1H, H-4 of 2-Cl-C₆H₄, J= 8.4 Hz),

7.54 (d, 1H, H-6 of 2-Cl-C₆H₄, J= 7.8 Hz), 7.71 – 7.73 (m, 1H, H-5 of 2-Cl-C₆H₄), 7.88 – 7.96

12

ACCEPTED MANUSCRIPT

(m, 2H, H-6 and H-7 phthalazine), 8.10 (d, 1H, H-5 phthalazine, J= 9.3 Hz), 8.44 (d, 1H, H-8

phthalazine, J= 7.8 Hz), 11.16 (s, 1H, NH, D₂O exchangable); Anal. Calcd. for C₂₂H₁₈ClN₃O:

C, 70.30; H, 4.83; N, 11.18; Found C, 70.54; H, 4.91; N, 11.36.

4.1.4.5. N-(3-Chlorophenyl)-4-(4-methoxybenzyl)phthalazin-1-amine (4e):

White crystals (yield 59%); m.p. 241-243 °C; IR (KBr, ν cm^-1): 3290 (NH), 2675 (CH

1

aliphatic); H NMR (DMSO-d₆)

δ ppm: 3.68 (s, 3H, OCH₃), 4.60 (s, 2H, CH₂), 6.83 (d, 2H,

H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.1 Hz), 7.20 (d, 1H, H-4 of 3-Cl-C₆H₄, J= 8.1 Hz), 7.30 (d,

2H, H-2 and H-6 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 7.44 (t, 1H, H-5 of 3-Cl-C₆H₄, J= 8.1 Hz),

7.78 (d, 1H, H-6 of 3-Cl-C₆H₄, J= 8.1 Hz), 8.04 – 8.15 (m, 3H, H-2 of 3-Cl-C₆H₄and H-6, H-

7 phthalazine), 8.33 (d, 1H, H-5 phthalazine, J= 7.8 Hz), 8.93 (d, 1H, H-8 phthalazine, J= 8.4

Hz), 10.49 (s, 1H, NH); ¹³C NMR (DMSO-d₆)

δ ppm: 35.48 (-CH₂-), 54.85 (-OCH₃), 113.92,

114.19, 120.52, 121.71, 121.97, 123.83, 124.17, 124.36, 126.54, 129.02, 129.64, 129.75,

130.34, 133.02, 133.84, 140.29, 152.57, 152.82, 158.06; MS m/z 377 [(M+2)⁺], 375 [M⁺];

Anal. Calcd. for C₂₂H₁₈ClN₃O: C, 70.30; H, 4.83; N, 11.18; Found C, 70.52; H, 4.90; N,

11.38.

4.1.4.6. N-(4-Chlorophenyl)-4-(4-methoxybenzyl)phthalazin-1-amine (4f):

Yellow crystals (yield 72%); m.p. 233-235°C; IR (KBr, ν cm^-1): 3334 (NH), 2594 (CH

1

aliphatic); H NMR (DMSO-d₆)

δ ppm: 3.70 (s, 3H, OCH₃), 4.61 (s, 2H, CH₂), 6.85 (d, 2H,

H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 7.29 (d, 2H, H-2 and H-6 of 4-OCH₃-C₆H₄, J= 8.7

Hz), 7.52 (d, 2H, H-3 and H-5 of 4-Cl-C₆H₄, J= 8.7 Hz), 7.78 (d, 2H, H-2 and H-6 of 4-Cl-

C₆H₄, J= 8.7 Hz), 8.10 – 8.21 (m, 2H, H-6 and H-7 phthalazine), 8.38 (d, 1H, H-5 phthalazine,

J= 7.8 Hz), 8.89 (d, 1H, H-8 phthalazine, J= 8.4 Hz), 10.60 (s, 1H, NH, D₂O exchangable);

Anal. Calcd. for C₂₂H₁₈ClN₃O: C, 70.30; H, 4.83; N, 11.18; Found C, 70.51; H, 4.89; N,

11.34.

4.1.4.7. N-(4-Chloro-3-(trifluoromethyl)phenyl)-4-(4-methoxybenzyl)phthalazin-1-amine (4g):

White crystals (yield 67%); m.p. 240-242°C; IR (KBr, ν cm^-1): 3242 (NH), 2692 (CH

aliphatic);¹H NMR (DMSO-d₆)

δ ppm: 3.69 (s, 3H, OCH₃), 4.74 (s, 2H, CH₂), 6.85 (d, 2H,

H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 7.35 (d, 2H, H-2 and H-6 of 4-OCH₃-C₆H₄, J= 9.0

Hz), 7.79 (d, 1H, H-5 of 4-Cl-3-CF₃-C₆H₃, J= 8.7 Hz), 8.15 – 8.28 (m, 3H, H-6 of 4-Cl-3-

CF₃-C₆H₃and H-6, H-7 phthalazine), 8.43 (s, 1H, H-2 of 4-Cl-3-CF₃-C₆H₃), 8.48 (d, 1H, H-5

13

phthalazine, J= 8.4 Hz), 9.14 (d, 1H, H-8 phthalazine, J= 8.4 Hz), 11.15 (s, 1H, NH); C

NMR (DMSO-d₆)

δ

ppm: 34.13 (-CH₂-), 54.97 (-OCH₃), 114.14, 114.23, 120.89, 121.44,

13

ACCEPTED MANUSCRIPT

122.11, 124.51, 125.03, 126.84, 127.73, 127.88, 129.89, 132.16, 134.63, 135.62, 137.54,

153.12, 153.31, 157.94, 158.29; Anal. Calcd. For C₂₃H₁₇ClF₃N₃O: C, 62.24; H, 3.86; N, 9.47;

Found C, 62.39; H, 3.89; N, 9.63.

4.1.4.8. 4-(4-Methoxybenzyl)-N-(3-methoxyphenyl)phthalazin-1-amine (4h):

Yellow crystals (yield 60%); m.p. 157-159°C; IR (KBr, ν cm^-1): 3419 (NH), 2927 (CH

aliphatic); ¹H NMR (DMSO-d₆)

δ ppm: 3.69 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 4.62 (s, 2H,

CH₂), 6.85 – 6.88 (m, 3H, H-4 of 3-OCH₃-C₆H₄and H-3 and H-5 of 4-OCH₃-C₆H₄), 7.31 –

7.42 (m, 5H, H-2, H-5 and H-6 of 3-OCH₃-C₆H₄and H-2 and H-6 of 4-OCH₃-C₆H₄), 8.11 –

8.21 (m, 2H, H-6 and H-7 phthalazine), 8.38 (d, 1H, H-5 phthalazine, J= 8.1 Hz), 9.04 (d, 1H,

H-8 phthalazine, J= 7.8 Hz), 10.89 (s, 1H, NH); ¹³C NMR (DMSO-d₆)

δ ppm: 35.08 (-CH₂-),

54.97 (-OCH₃), 55.27 (-OCH₃), 109.76, 111.42, 114.12, 116.01, 121.09, 124.96, 126.85,

127.15, 128.50, 129.80, 129.98, 134.59, 138.17, 152.27, 152.46, 158.15, 159.88; MS m/z 371

[M⁺]; Anal. Calcd. for C₂₃H₂₁N₃O₂: C, 74.37; H, 5.70; N, 11.31; Found C, 74.55; H, 5.78; N,

11.53.

4.1.4.9. 4-(4-Methoxybenzyl)-N-(4-methoxyphenyl)phthalazin-1-amine (4i):

Yellow crystals (yield 64%); m.p. 243-245°C; IR (KBr, ν cm^-1): 3273 (NH), 2619 (CH

aliphatic); ¹H NMR (DMSO-d₆)

δ ppm: 3.69 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 4.51 (s, 2H,

CH₂), 6.83 (d, 2H, H-3 and H-5 of 4-OCH₃-C₆H₄, J= 9.0 Hz), 7.02 (d, 2H, H-3 and H-5 of 4-

OCH₃-C₆H₄-NH, J= 9.3 Hz), 7.26 (d, 2H, H-2 and H-6 of 4-OCH₃-C₆H₄, J= 8.4 Hz), 7.62 (d,

2H, H-2 and H-6 of 4-OCH₃-C₆H₄-NH, J= 8.7 Hz), 8.01 – 8.10 (m, 2H, H-6 and H-7

phthalazine), 8.24 (d, 1H, H-5 phthalazine, J= 7.2 Hz), 8.87 (d, 1H, H-8 phthalazine, J= 7.5

Hz), 10.41 (s, 1H, NH); ¹³C NMR (DMSO-d₆)

δ ppm: 36.18 (-CH₂-), 54.94 (-OCH₃), 55.27 (-

OCH₃), 113.96, 114.37, 120.26, 124.34, 125.39, 126.32, 126.58, 129.47, 129.64, 130.29,

133.17, 133.88, 151.86, 152.10, 156.96, 157.96; Anal. Calcd. for C₂₃H₂₁N₃O₂: C, 74.37; H,

5.70; N, 11.31; Found C, 74.53; H, 5.81; N, 11.52.

4.1.4.10. 4-((4-(4-Methoxybenzyl)phthalazin-1-yl)amino)benzenesulfonamide (4j):

White crystals (yield 70%); m.p. 251-253°C; IR (KBr, ν cm^-1): 3406, 3325, 3259 (NH, NH₂),

2644 (CH aliphatic), 1327, 1147 (SO₂); ¹H NMR (DMSO-d₆)

δ ppm: 3.69 (s, 3H, OCH₃), 4.64

(s, 2H, CH₂), 6.85 (d, 2H, H-3 and H-5 of 4-OCH₃-C₆H₄, J= 9.0 Hz), 7.30 (d, 2H, H-2 and H-

6 of 4-OCH₃-C₆H₄, J= 8.4 Hz), 7.86 (d, 2H, H-2 and H-6 of 4-SO₂NH₂-C₆H₄, J= 8.7 Hz), 7.99

(d, 2H, H-3 and H-5 of 4-SO₂NH₂-C₆H₄, J= 8.4 Hz), 8.08 – 8.21 (m, 2H, H-6 and H-7

14

ACCEPTED MANUSCRIPT

phthalazine), 8.38 (d, 1H, H-5 phthalazine, J= 7.8 Hz), 8.85 (d, 1H, H-8 phthalazine, J= 7.8

Hz), 10.35 (s, 1H, NH, D₂O exchangable); Anal. Calcd. for C₂₂H₂₀N₄O₃S: C, 62.84; H, 4.79;

N, 13.32; Found C, 62.97; H, 4.86; N, 13.47.

4.1.5. substituted 1-(4-nitrophenyl)-3-phenylurea (5a-i)

Compounds 5a-i were prepared according to reported procedures [35].

4.1.6. substituted 1-(4-aminophenyl)-3-phenylurea (6a-i)

Compounds 6a-c, e, f, i [35], 6g [36] and 6h [37] are previously reported.

4.1.6.1. 1-(2-Chlorophenyl)-3-(4-aminophenyl)urea (6d):

White crystals (yield 69%); m.p. 209-211°C; IR (KBr, ν cm^-1): 3489, 3394, 3305 (NH, NH₂),

1

1631 (C=O); H NMR (DMSO-d₆)

δ ppm: 4.78 (s, 2H, NH₂, D₂O exchangable), 6.53 (d, 2H,

H-3 and H-5 of 4-NH₂-C₆H₄-NH, J= 6.9 Hz), 6.97 (t, 1H, H-4 of 2-Cl-C₆H₄, J= 7.5 Hz), 7.07

(d, 2H, H-2 and H-6 of 4-NH₂-C₆H₄-NH, J= 8.7 Hz), 7.25 (t, 1H, H-5 of 2-Cl-C₆H₄, J= 7.8

Hz), 7.39 (d, 1H, H-3 of 2-Cl-C₆H₄, J= 7.5 Hz), 8.07 (s, 1H, NH_urea, D₂O exchangable), 8.14

(d, 1H, H-6 of 2-Cl-C₆H₄, J= 8.4 Hz), 8.90 (s, 1H, NH_urea, D₂O exchangable).

4.1.7. General procedure for preparation of target compounds (7a-i):

A solution of 1-chloro-4-(4-methoxybenzyl)phthalazine 3 (2.84 g, 0.01 mol) in acetone was

added to a stirred solution of 6a-i (0.01 mol) in refluxing acetone. The reaction mixture was

refluxed for 2 h then left to cool. The precipitated solid was filtered, dried and recrystallized

from dioxan to give 7a-i.

4.1.7.1. 1-(4-((4-(4-Methoxybenzyl)phthalazin-1-yl)amino)phenyl)-3-phenylurea (7a):

Yellow crystals (yield 65%); m.p. 250-253 °C; IR (KBr, ν cm^-1): 3253 (NH), 2922 (CH

1

aliphatic), 1697 (C=O); H NMR (DMSO-d₆)

δ ppm: 3.70 (s, 3H, OCH₃), 4.52 (s, 2H, CH₂),

6.85 (d, 2H, H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 6.97 (t, 1H, H-4 of NH-C₆H₅, J= 7.5

Hz), 7.26 – 7.31 (m, 4H, H-3, H-5 of NH-C₆H₅and H-2, H-6 of 4-OCH₃-C₆H₄), 7.46 – 7.53

(m, 4H, HN-C₆H₄-NH), 7.61 (d, 2H, H-2 and H-6 of NH-C₆H₅, J= 8.4 Hz), 8.16 – 8.20 (m,

2H, H-6 and H-7 phthalazine), 8.36 (d, 1H, H-5 phthalazine, J= 9.0 Hz), 8.85 (d, 1H, H-8

phthalazine, J= 9.3 Hz), 9.12 (s, 1H, NH_urea, D₂O exchangable), 9.30 (s, 1H, NH_urea, D₂O

exchangable), 10.88 (s, 1H, NH, D₂O exchangable); Anal. Calcd. for C₂₉H₂₅N₅O₂: C, 73.25;

H, 5.30; N, 14.73; Found C, 73.41; H, 5.38 ; N, 14.91.

15

ACCEPTED MANUSCRIPT

4.1.7.2. 1-(4-((4-(4-Methoxybenzyl)phthalazin-1-yl)amino)phenyl)-3-(3-

(trifluoromethyl)phenyl)urea (7b):

Yellow crystals (yield 77%); m.p. 205-207 °C; IR (KBr, ν cm^-1): 3282 (NH), 2933 (CH

1

aliphatic), 1714 (C=O); H NMR (DMSO-d₆)

δ ppm: 3.70 (s, 3H, OCH₃), 4.54 (s, 2H, CH₂),

6.85 (d, 2H, H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 7.28 (d, 2H, H-2 and H-6 of 4-OCH₃-

C₆H₄, J= 9.0 Hz), 7.49 – 7.65 (m, 7H, H-4, H-5 and H-6 of 3-CF₃-C₆H₄and 4H of HN-C₆H₄-

NH), 8.01 (s, 1H, H-2 of 3-CF₃-C₆H₄), 8.12 – 8.20 (m, 2H, H-6 and H-7 phthalazine), 8.35 (d,

1H, H-5 phthalazine, J= 9.3 Hz), 8.92 (d, 1H, H-8 phthalazine, J= 9.0 Hz), 9.71 (s, 1H,

NH_urea), 9.91 (s, 1H, NH_urea), 10.94 (s, 1H, NH); ¹³C NMR (DMSO-d₆)

δ ppm: 35.60 (-CH₂-),

54.88 (-OCH₃), 113.94, 114.19, 117.85, 118.90, 121.03, 121.42, 122.37, 124.80, 124.96,

125.81, 126.95, 128.57, 129.30, 129.72, 129.84, 129.90, 134.05, 137.92, 138.56, 140.67,

152.00, 152.67, 158.13; MS m/z 543 [M⁺]; Anal. Calcd. for C₃₀H₂₄F₃N₅O₂: C, 66.29; H, 4.45;

N, 12.88; Found C, 66.38; H, 4.49 ; N, 13.04.

4.1.7.3. 1-(4-Cyanophenyl)-3-(4-((4-(4-methoxybenzyl)phthalazin-1-yl)amino)phenyl)urea

(7c):

Brown crystals (yield 65%); m.p. 241-243°C; IR (KBr, ν cm^-1): 3251 (NH), 3034 (CH

aliphatic), 2216 (C≡N), 1716 (C=O); ¹H NMR (DMSO-d₆)

δ ppm: 3.70 (s, 3H, OCH₃), 4.54

(s, 2H, CH₂), 6.85 (d, 2H, H-3 and H-5 of 4-OCH₃-C₆H₄, J= 9.0 Hz), 7.27 (d, 2H, H-2 and H-

6 of 4-OCH₃-C₆H₄, J= 8.1 Hz), 7.55 – 7.73 (m, 8H, 4H of 4-CN-C₆H₄and 4H of HN-C₆H₄-

NH), 8.07 – 8.20 (m, 2H, H-6 and H-7 phthalazine), 8.35 (d, 1H, H-5 phthalazine, J= 7.2 Hz),

8.92 (d, 1H, H-8 phthalazine, J= 8.1 Hz), 9.79 (s, 1H, NH_urea), 10.10 (s, 1H, NH_urea), 10.97 (s,

1H, NH); ¹³C NMR (DMSO-d₆)

δ ppm: 35.63 (-CH₂-), 55.00 (-OCH₃), 103.06, 114.07,

117.70, 118.99, 119.28, 121.01, 124.89, 125.82, 126.94, 127.04, 128.54, 129.79, 133.26,

134.22, 135.03, 138.29, 144.26, 152.02, 152.28, 158.13; MS m/z 500 [M⁺]; Anal. Calcd. for

C₃₀H₂₄N₆O₂: C, 71.99; H, 4.83; N, 16.79; Found C, 72.17; H, 4.89 ; N, 17.03.

4.1.7.4. 1-(2-Chlorophenyl)-3-(4-((4-(4-methoxybenzyl)phthalazin-1-yl)amino)phenyl)urea

(7d):

Yellow crystals (yield 53%); m.p. 249-251 °C; IR (KBr, ν cm^-1): 3255 (NH), 2945 (CH

1

aliphatic), 1703 (C=O); H NMR (DMSO-d₆)

δ ppm: 3.69 (s, 3H, OCH₃), 4.53 (s, 2H, CH₂),

6.84 (d, 2H, H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 7.03 (t, 1H, H-4 of 2-Cl-C₆H₄, J= 7.8

Hz), 7.27 – 7.32 (m, 3H, H-5 of 2-Cl-C₆H₄and H-2, H-6 of 4-OCH₃-C₆H₄), 7.43 (d, 1H, H-3

16

ACCEPTED MANUSCRIPT

of 2-Cl-C₆H₄, J= 8.4 Hz), 7.58 – 7.65 (m, 4H, HN-C₆H₄-NH), 8.07 – 8.17 (m, 3H, H-6 of 2-

Cl-C₆H₄and H-6, H-7 phthalazine), 8.31 (d, 1H, H-5 phthalazine, J= 8.1 Hz), 8.60 (s, 1H,

NH_urea), 8.94 (d, 1H, H-8 phthalazine, J= 8.7 Hz), 10.16 (s, 1H, NH_urea), 10.89 (s, 1H, NH);

¹³C NMR (DMSO-d₆)

δ ppm: 35.80 (-CH₂-), 55.00 (-OCH₃), 114.04, 118.80, 120.76, 121.57,

122.28, 123.28, 124.75, 125.18, 126.76, 126.81, 127.41, 128.91, 129.17, 129.73, 130.42,

133.83, 134.56, 136.00, 138.01, 151.97, 152.05, 152.32, 158.07; MS m/z 511 [(M+2)⁺] and

509 [M⁺]; Anal. Calcd. for C₂₉H₂₄ClN₅O₂: C, 68.30; H, 4.74; N, 13.73; Found C, 68.42; H,

4.78 ; N, 13.89.

4.1.7.5. 1-(3-Chlorophenyl)-3-(4-((4-(4-methoxybenzyl)phthalazin-1-yl)amino)phenyl)urea

(7e):

Beige crystals (yield 59%); m.p. 225-227 °C; IR (KBr, ν cm^-1): 3261 (NH), 2956 (CH

1

aliphatic), 1701 (C=O); H NMR (DMSO-d₆)

δ ppm: 3.68 (s, 3H, OCH₃), 4.51 (s, 2H, CH₂),

6.84 (d, 2H, H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 6.98 – 7.03 (m, 1H, H-4 of 3-Cl-

C₆H₄), 7.26 – 7.31 (m, 4H, H-5, H-6 of 3-Cl-C₆H₄and H-2, H-6 of 4-OCH₃-C₆H₄), 7.56 –

7.62 (m, 4H, HN-C₆H₄-NH), 7.72 (s, 1H, H-2 of 3-Cl-C₆H₄), 8.06 – 8.14 (m, 2H, H-6 and H-7

phthalazine), 8.29 (d, 1H, H-5 phthalazine, J= 6.9 Hz), 8.83 (d, 1H, H-8 phthalazine, J= 6.9

13

Hz), 9.53 (s, 1H, NH_urea), 9.63 (s, 1H, NH_urea), 10.61 (s, 1H, NH); C NMR (DMSO-d₆)

δ

ppm: 36.06 (-CH₂-), 54.99 (-OCH₃), 114.02, 116.30, 117.22, 118.80, 120.59, 121.23, 124.45,

125.01, 126.62, 126.75, 129.07, 129.70, 130.33, 130.58, 133.16, 133.61, 134.40, 137.76,

141.39, 152.00, 152.54, 158.05; Anal. Calcd. for C₂₉H₂₄ClN₅O₂: C, 68.30; H, 4.74; N, 13.73;

Found C, 68.47; H, 4.76 ; N, 13.86.

4.1.7.6. 1-(4-Chlorophenyl)-3-(4-((4-(4-methoxybenzyl)phthalazin-1-yl)amino)phenyl)urea

(7f):

Beige crystals (yield 68%); m.p. 223-225 °C; IR (KBr, ν cm^-1): 3244 (NH), 2819 (CH

aliphatic), 1674 (C=O); ¹H NMR (DMSO-d₆)

δ ppm: 3.68 (s, 3H, OCH₃), 4.51 (s, 2H, CH₂),

6.83 (d, 2H, H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 7.26 – 7.33 (m, 4H, H-2, H-6 of 4-

OCH₃-C₆H₄and H-3, H-5 of 4-Cl-C₆H₄), 7.48 – 7.62 (m, 6H, H-2, H-6 of 4-Cl-C₆H₄and 4H

of HN-C₆H₄-NH), 8.07 – 8.15 (m, 2H, H-6 and H-7 phthalazine), 8.30 (d, 1H, H-5

phthalazine, J= 6.9 Hz), 8.89 (d, 1H, H-8 phthalazine, J= 6.9 Hz), 9.64 (s, 1H, NH_urea), 9.66

(s, 1H, NH_urea), 10.85 (s, 1H, NH); ¹³C NMR (DMSO-d₆)

OCH₃), 114.04, 118.71, 119.35, 120.78, 124.71, 125.12, 125.39, 126.79, 126.84, 128.56,

δ ppm: 35.85 (-CH₂-), 55.00 (-

17

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128.82, 129.75, 129.92, 133.86, 134.69, 138.33, 138.84, 151.97, 152.61, 158.08; Anal. Calcd.

for C₂₉H₂₄ClN₅O₂: C, 68.30; H, 4.74; N, 13.73; Found C, 68.44; H, 4.76 ; N, 13.85.

4.1.7.7. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((4-(4-methoxybenzyl)phthalazin-1-

yl)amino)phenyl)urea (7g):

Orange crystals (yield 66%); m.p. 268-270 °C; IR (KBr, ν cm^-1): 3340 (NH), 2833 (CH

1

aliphatic), 1714 (C=O); H NMR (DMSO-d₆)

δ ppm: 3.70 (s, 3H, OCH₃), 4.52 (s, 2H, CH₂),

6.85 (d, 2H, H-3 and H-5 of 4-OCH₃-C₆H₄, J= 8.7 Hz), 7.26 (d, 2H, H-2 and H-6 of 4-OCH₃-

C₆H₄, J= 8.4 Hz), 7.53 – 7.68 (m, 6H, H-5, H-6 of 4-Cl-3-CF₃-C₆H₃and 4H of HN-C₆H₄-

NH), 8.08 – 8.17 (m, 3H, H-2 of 4-Cl-3-CF₃-C₆H₃and H-6, H-7 phthalazine), 8.32 (d, 1H, H-

5 phthalazine, J= 6.6 Hz), 8.83 (d, 1H, H-8 phthalazine, J= 6.6 Hz), 9.50 (s, 1H, NH_urea), 9.84

13

(s, 1H, NH_urea), 10.67 (s, 1H, NH); C NMR (DMSO-d₆)

δ ppm: 36.11 (-CH₂-), 54.99 (-

OCH₃), 114.02, 116.41, 119.04, 120.52, 120.98, 122.10, 122.70, 124.32, 124.60, 124.84,

126.58, 126.72, 129.15, 129.69, 132.00, 133.56, 134.32, 137.32, 139.45, 152.06, 152.53,

158.04; Anal. Calcd. for C₃₀H₂₃ClF₃N₅O₂: C, 62.34; H, 4.01; N, 12.12; Found C, 62.48; H,

4.07 ; N, 12.31.

4.1.7.8. 1-(4-((4-(4-Methoxybenzyl)phthalazin-1-yl)amino)phenyl)-3-(3-methoxyphenyl)urea

(7h):

Beige crystals (yield 70%); m.p. 220-222 °C; IR (KBr, ν cm^-1): 3261 (NH), 2831 (CH

aliphatic), 1683 (C=O); ¹H NMR (DMSO-d₆)

δ ppm: 3.67 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃),

4.45 (s, 2H, CH₂), 6.53 (d, 1H, H-4 of 3-OCH₃-C₆H₄, J= 7.8 Hz), 6.80 (d, 2H, H-3 and H-5 of

4-OCH₃-C₆H₄, J= 9.0 Hz), 6.92 (d, 1H, H-6 of 3-OCH₃-C₆H₄, J= 7.2 Hz), 7.14 – 7.24 (m, 4H,

H-2, H-5 of 3-OCH₃-C₆H₄and H-2, H-6 of 4-OCH₃-C₆H₄), 7.42 (d, 2H, HN-C₆H₄-NH, J= 8.7

Hz), 7.81 (d, 2H, HN-C₆H₄-NH, J= 8.7 Hz), 7.86 – 7.94 (m, 2H, H-6 and H-7 phthalazine),

8.07 (d, 1H, H-5 phthalazine, J= 6.3 Hz), 8.54 – 8.57 (m, 2H, H-8 phthalazine and NH_urea),

8.63 (s, 1H, NH_urea, D₂O exchangable), 9.02 (s, 1H, NH, D₂O exchangable); Anal. Calcd. for

C₃₀H₂₇N₅O₃: C, 71.27; H, 5.38; N, 13.85; Found C, 71.52; H, 5.42; N, 14.02.

4.1.7.9. 1-(4-((4-(4-Methoxybenzyl)phthalazin-1-yl)amino)phenyl)-3-(4-methoxyphenyl)urea

(7i):

Yellow crystals (yield 69%); m.p. 215-217 °C; IR (KBr, ν cm^-1): 3259 (NH), (CH aliphatic),

1666 (C=O); ¹H NMR (DMSO-d₆)

δ ppm: 3.69 (s, 3H, 4-OCH₃), 3.71 (s, 3H, 4-OCH₃), 4.52

(s, 2H, CH₂), 6.84 (d, 4H, H-3, H-5 of 4-OCH₃-C₆H₄-CH₂and H-3, H-5 of 4-OCH₃-C₆H₄-NH

18

ACCEPTED MANUSCRIPT

J= 8.4 Hz), 7.27 (d, 2H, H-2 and H-6 of 4-OCH₃-C₆H₄, J= 8.1 Hz), 7.37 (d, 2H, H-2 and H-6

of 4-OCH₃-C₆H₄-NH, J= 8.7 Hz), 7.52 – 7.61 (m, 4H, HN-C₆H₄-NH), 8.09 – 8.17 (m, 2H, H-

6 and H-7 phthalazine), 8.31 (d, 1H, H-5 phthalazine, J= 8.4 Hz), 8.91 (d, 1H, H-8

13

phthalazine, J= 6.9 Hz), 9.16 (s, 1H, NH_urea), 9.47 (s, 1H, NH_urea), 10.94 (s, 1H, NH); C

NMR (DMSO-d₆)

δ ppm: 35.82 (-CH₂-), 55.00 (-OCH₃), 55.14 (-OCH₃), 113.97, 114.05,

118.55, 119.66, 120.87, 124.81, 125.55, 126.88, 128.77, 129.29, 129.76, 132.87, 133.94,

134.83, 138.97, 151.94, 152.88, 154.35, 158.10; Anal. Calcd. for C₃₀H₂₇N₅O₃: C, 71.27; H,

5.38; N, 13.85; Found C, 71.51; H, 5.44; N, 13.97.

4.2. Measurement of inhibitory activity against VEGFR-2

The kinase activity of VEGFR-2 was carried out in the National Research Centre, Giza,

Egypt and measured by use of an anti-phosphotyrosine antibody with the Alpha Screen

system (PerkinElmer, USA) according to manufacturer’s instructions. Enzyme reactions were

performed in 50 mM Tris–HCl pH 7.5, 5 mM MnCl₂, 5 mM MgCl₂, 0.01% Tween-20 and 2

mM DTT, containing 10 µM ATP, 0.1 µg/mL biotinylated poly-GluTyr (4:1) and 0.1 nM of

VEGFR-2 (Millipore, UK). Prior to catalytic initiation with ATP, the tested compounds at

final concentrations ranging from 0-100 µg/mL and enzyme were incubated for 5 min at room

temperature. The reactions were quenched by the addition of 25 µL of 100 mM EDTA, 10

µg/mL Alpha Screen streptavidine donor beads and 10 µg/mL acceptor beads in 62.5 mM

HEPES pH 7.4, 250 mM NaCl, and 0.1% BSA. Plate was incubated in the dark overnight and

then read by ELISA Reader (PerkinElmer, USA). Wells containing the substrate and the

enzyme without compounds were used as reaction control. Wells containing biotinylated

poly-GluTyr (4:1) and enzyme without ATP were used as basal control. Percent inhibition

was calculated by the comparison of compounds treated to control incubations. The

concentration of the test compound causing 50% inhibition (IC₅₀) was calculated from the

concentration–inhibition response curve (triplicate determinations) and the data were

compared with Sorafenib as standard VEGFR-2 inhibitor.

4.3. Molecular Docking study

All the molecular modeling studies were carried out using Molecular Operating

Environment (MOE, 10.2008) software. All minimizations were performed with MOE until

an RMSD gradient of 0.05 kcalꢁmol⁻¹Å⁻¹with MMFF94x force field and the partial charges

were automatically calculated. The X-ray crystallographic structure of VEGFR-2 co-

19

ACCEPTED MANUSCRIPT

crystallized with sorafenib as inhibitor (PDB ID: 4ASD) [38] was downloaded from the

protein data bank [39]. The receptor was prepared for docking study using Protonate 3D

protocol in MOE with default options followed by water molecules removal. The co-

crystalized ligand was used to define the active site for docking. Triangle Matcher placement

method and London dG scoring function were used for docking. Docking setup was first

validated by re-docking of the co-crystallized ligand (Sorafenib) in the vicinity of the active

site of the receptor with energy score (S) = −15.19 kcal/mol and RMSD of 0.470Å. The

validated setup was then used in predicting the ligands receptor interactions at the active site

for the synthesized and designed compounds.

4.4. In vitro cytotoxic activity

The cytotoxicity assays were performed at National Cancer Institute (NCI), Bethesda,

USA (against 60 cell lines). The human tumor cell lines of the cancer screening panel were

grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For

a typical screening experiment, cells were inoculated into 96 well microtiter plates in 100 µ at

plating densities ranging from 5000 to 40,000 cells/well depending on the doubling time of

o

individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 C, 5%

CO₂, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs.

After 24 h, two plates of each cell line were fixed in situ with trichloroacetic acid (TCA), to

represent a measurement of the cell population for each cell line at the time of drug addition

(T_z). Experimental drugs were solubilized in dimethyl sulfoxide at 400-fold the desired final

maximum test concentration and stored frozen prior to use. At the time of drug addition, an

aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test

concentration with complete medium containing 50 µg/ml gentamicin. Aliquot of 100 µl of

the drug dilution was added to the appropriate microtiter wells already containing 100 µl of

medium, resulting in the required final drug concentration (10 µM). Triplicate wells were

prepared for each individual dose. Following drug addition, the plates were incubated for an

o

additional 48 h at 37 C, 5% CO₂, 95% air, and 100% relative humidity. For adherent cells,

the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle

addition of 50 µl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for

60 min at 4 ^oC. The supernatant was discarded, and the plates were washed five times with tap

water and air dried. Sulforhodamine B (SRB) solution (100 µl) at 0.4% (w/v) in 1% acetic

acid was added to each well, and plates were incubated for 10 min at room temperature. After

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staining, unbound dye was removed by washing five times with 1% acetic acid and the plates

were air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the

absorbance was read on an automated plate reader at a wavelength of 515 nm. For suspension

cells, the methodology was the same except that the assay was terminated by fixing settled

cells at the bottom of the wells by gently adding 50 µl of 80% TCA (final concentration, 16%

TCA). Using the seven absorbance measurements [time zero (Tz), control growth (C), and test

growth in the presence of drug (T_i)], the percentage growth was calculated at the drug

concentration level. Percentage growth inhibition was calculated as:

[(T_i– T_z) / (C – T_z)] x 100 for concentrations for which T_i≥ T_z

[(T_i– T_z) / T_z] x 100 for concentrations for which T_i< T_z

Acknowledgment

The authors would like to extend their sincere appreciation to the Deanship of Scientific

Research at King Saud University for its funding of this research through the Research Group

Project no. RGP-1436-038. Department of Pharmaceutical Chemistry, Faculty of Pharmacy,

Cairo University, Cairo, Egypt, is highly appreciated for supporting this research. The authors

are grateful to National Cancer Institute (NCI) Developmental Therapeutic Program

(www.dtp.nci.nih.gov) for screening the anticancer activity of the newly synthesized

compounds in vitro.

3D Molecular graphics in the discussion and results section were generated using the

UCSF Chimera package [40]. Chimera is developed by the Resource for Biocomputing,

Visualization, and Informatics at the University of California, San Francisco, supported by

grants from the National Institutes of Health National Center for Research Resources

(2P41RR001081) and National Institute of General Medical Sciences (9P41GM103311).

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FIGURE CAPTIONS

Figure 1. Structures of VEGFR-2 inhibitors (I-III) and the target phthalazines

4a-j and 7a-i.

Figure 2. 2D schematic diagram representing vatalanib plausible binding mode in the

VEGFR-2 active site.

Figure 3. (A) Superimposition of the co-crystallized (blue) and the docking pose (red)

of sorafenib in the VEGFR-2 active site with RMSD of 0.470Å. (B) 2D

interaction diagram showing sorafenib docking pose interactions with the

key amino acids in the VEGFR-2 active site. (Distances in Å)

Figure 4. 2D diagram (A) and 3D representation (B) of compound 4g showing its

interaction with the VEGFR-2 receptor active site.

Figure 5. 2D diagram (A) and 3D representation (B) for compound 4j showing its

interaction with the VEGFR-2 receptor active site.

Figure 6. 2D diagram (A) and 3D representation (B) for compound 7h showing its

interaction with the VEGFR-2 receptor active site.

Figure 7. The most sensitive cell lines towards the target compounds 4a-j and 7a-i

according to the GI %.

TABLE CAPTIONS

Table 1. VEGFR-2 kinase inhibitory activity of the synthesized compounds.

Table 2. Docking energy scores (S) in kcal/mol for compounds 4 and 7.

Table 3. Percentage growth inhibition (GI %) of in-vitro subpanel tumor cell lines at

10 µM concentration for seventeen compounds.

SCHEME CAPTIONS

Scheme 1. Reagents and conditions: (i) CH₃COONa / heating 200 °C 5 h; (ii)

NH₂NH₂.H₂SO₄/ NaOH / EtOH / reflux 15 h; (iii) POCl₃/ N,N-

dimethylaniline / reflux 10 h (iv) Anilines / acetone / reflux 3 h.

Scheme 2. Reagents and conditions: (i) THF / Et₃N / rt 15 h; (ii) H₂/Pd/C / MeOH

/ rt 3 h; (iii) Acetone / reflux 2 h.

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Table 1.VEGFR2 kinase inhibitory activity of the synthesized compounds.

Compound

4a

R

H

IC₅₀(µM)

5.76±0.58

Compound

7a

R

H

IC₅₀(µM)

0. 473±0.03

3-CF₃

4-CN

3.97±0.43

5.12±0.54

3.68±0.40

4.67±0.49

3.87±0.39

1.34±0.16

0.754±0.07

3.45±0.34

0.636±0.06

3-CF₃

4-CN

0.460±0.05

0.381±0.04

0.141±0.02

0.118±0.01

0.114±0.01

0.086±0.01

0.083±0.01

0.086±0.01

0.090±0.01

4b

4c

4d

4e

4f

7b

7c

2-Cl

7d

3-Cl

7e

4-Cl

7f

3-CF₃-4-Cl

3-OCH₃

4-OCH₃

4-SO₂NH₂

3-CF₃-4-Cl

3-OCH₃

4-OCH₃

4g

4h

4i

7g

7h

7i

4j

Sorafenib

Data were expressed as Mean ± Standard error (S.E.) of three independent experiments.

ACCEPTED MANUSCRIPT

Table 2. Docking energy scores (S) in kcal/mol for compounds 4 and 7.

Compound

Energy score

Compound

Energy score (S)

(S) kcal/mol

kcal/mol

4a

4b

4c

4d

4e

4f

7a

-14.74

-16.09

-15.18

-14.94

-15.11

-15.06

-16.32

-18.18

-19.27

-18.08

-18.75

-17.85

-18.23

-20.25

-19.12

7b

7c

7d

7e

7f

4g

4h

4i

7g

7h

-15.51

-15.24

7i

-19.86

-15.19

-15.94

4j

Sorafenib

ACCEPTED MANUSCRIPT

Table 3. Percentage growth inhibition (GI %) of in-vitro subpanel tumor cell lines at 10 µM

concentration forseventeen compounds.

Compound^a

Subpanel

4a

4b

4c

4e

4f

4g

4h

4i

7a

7b

7c

7d

7e

7f

7g

7h

7i

Leukemia

CCRF-CEM

K-562

MOLT-4

RPMI-8226

SR

39

49

60

87

52

-

46

48

69

54

52

60

69

94

43

-

77

35

-

31

-

34

-

30

46

-

35

-

37

42

34

47

50

56

95

43

-

33

-

41

30

-

31

-

42

-

44

75

40

55

43

50

66

40

-

43

-

40

-

47

91

39

-

58

30

45

37

89

43

34

Non-Small Cell Lung Cancer

A549/ATCC

HOP-62

HOP-92

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

-

49

-

57

-

63

40

42

-

36

-

45

-

63

35

-

34

50

40

76

34

39

45

38

62

-

35

53

37

-

63

-

55

-

48

35

-

56

36

33

-

40

54

-

33

35

-

57

33

-

48

-

30

36

68

-

33

-

36

58

43

40

37

74

49

-

50

45

-

110

43

Colon Cancer

COLO 205

HCC-2998

HCT-116

HCT-15

HT29

-

49

33

31

-

40

-

34

-

35

-

42

-

36

-

37

-

30

-

31

56

41

49

40

-

42

73

58

48

52

-

66

33

59

30

-

44

36

45

37

-

42

41

34

39

-

53

40

61

51

-

34

39

34

39

-

KM12

SW-620

-

CNS Cancer

SF-268

SF-295

SF-539

SNB-19

SNB-75

U251

-

30

41

-

30

-

30

31

30

-

50

-

45

59

51

-

35

-

32

-

38

36

45

-

33

35

37

32

34

50

46

38

-

49

42

45

-

86

67

43

57

63

52

41

39

49

48

81

49

-

50

49

44

38

59

-

32

Melanoma

LOX IMVI

MALME-3M

M14

MDA-MB-435

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

-

58

-

47

30

-

48

-

30

-

46

42

35

-

30

-

32

-

40

35

-

31

-

35

-

45

33

-

36

-

42

39

60

41

34

-

Ovarian Cancer

IGROV1

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-

RES

-

37

48

37

-

35

40

-

34

-

36

40

33

-

35

48

32

59

45

33

40

50

33

-

46

-

53

78

60

30

66

67

-

32

30

-

SK-OV-3

-

37

-

38

-

Renal Cancer

786-0

A498

ACHN

-

35

-

33

40

31

-

30

-

34

31

43

53

47

41

46

45

-

47

-

36

-

41

-

41

83

54

48

41

44

-

35

-

39

58

32

46

62

-

50

42

-

63

-

32

Article Doi

Increasing the binding affinity of VEGFR-2 inhibitors by extending their hydrophobic interaction with the active site: Design, synthesis and biological evaluation of 1-substituted-4-(4-methoxybenzyl)phthalazine derivatives

DOI: 10.1016/j.ejmech.2016.02.029

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2016.02.029

Full text of DOI:10.1016/j.ejmech.2016.02.029

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