Towards discovery of novel scaffold with potent antiangiogenic activity; design, synthesis of pyridazine based compounds, impact of hinge interaction, and accessibility of their bioactive conformation on VEGFR-2 activities

Journal of Enzyme Inhibition and Medicinal Chemistry

ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20

Towards discovery of novel scaffold with potent

antiangiogenic activity; design, synthesis of

pyridazine based compounds, impact of hinge

interaction, and accessibility of their bioactive

conformation on VEGFR-2 activities

Maiy Y. Jaballah, Rabah A. T. Serya, Nasser Saad, Sohair M. Khojah, Marawan

Ahmed, Khaled Barakat & Khaled A. M. Abouzid

To cite this article: Maiy Y. Jaballah, Rabah A. T. Serya, Nasser Saad, Sohair M. Khojah,

Marawan Ahmed, Khaled Barakat & Khaled A. M. Abouzid (2019) Towards discovery of novel

scaffold with potent antiangiogenic activity; design, synthesis of pyridazine based compounds,

impact of hinge interaction, and accessibility of their bioactive conformation on VEGFR-2

activities, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 1573-1589, DOI:

10.1080/14756366.2019.1651723

To link to this article: https://doi.org/10.1080/14756366.2019.1651723

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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

2019, VOL. 34, NO. 1, 1573–1589

https://doi.org/10.1080/14756366.2019.1651723

RESEARCH PAPER

Towards discovery of novel scaffold with potent antiangiogenic activity; design,

synthesis of pyridazine based compounds, impact of hinge interaction, and

accessibility of their bioactive conformation on VEGFR-2 activities

Maiy Y. Jaballah^a, Rabah A. T. Serya^a, Nasser Saad^b, Sohair M. Khojah^c, Marawan Ahmed^d, Khaled Barakat^d,e,fand

Khaled A. M. Abouzid^a,g

b

^aFaculty of Pharmacy, Pharmaceutical Chemistry Department, Ain Shams University, Abbassia, Cairo, Egypt; Faculty of Pharmaceutical Sciences

c

and Pharmaceutical Industries, Pharmaceutical Chemistry Department, Future University in Egypt, Cairo, Egypt; Faculty of Science, Biochemistry

d

Department, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia; Faculty of Pharmacy and Pharmaceutical Sciences, University of

e

f

Alberta, Edmonton, AB, Canada; Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB, Canada; Li Ka Shing Applied Virology

g

Institute, University of Alberta, Edmonton, AB, Canada; Faculty of Pharmacy, Department of Organic and Medicinal Chemistry, University of

Sadat City, Menoufia, Egypt

ABSTRACT

ARTICLE HISTORY

Received 27 May 2019

Revised 2 July 2019

Accepted 24 July 2019

Pyridazine scaffolds are considered privileged structures pertaining to its novelty, chemical stability, and

synthetic feasibility. In our quest towards the development of novel scaffolds for effective vascular endo-

thelial growth 2 (VEGFR-2) inhibition with antiangiogenic activity, four novel series of pyridazines were

designed and synthesised. Five of the synthesised compounds; namely (8c, 8f, 15, 18b, and 18c) exhib-

ited potent VEGFR-2 inhibitory potency (>80%); with IC₅₀values ranging from low micromolar to nanomo-

lar range; namely compounds 8c, 8f, 15, 18c with (1.8 mM, 1.3 mM, 1.4 mM, 107nM), respectively.

Moreover, 3-[4-f(6-oxo-1,6-dihydropyridazin-3-yl)oxygphenyl]urea derivative (18b) exhibited nanomolar

potency towards VEGFR-2 (60.7 nM). In cellular assay, the above compounds showed excellent inhibition

of VEGF-stimulated proliferation of human umbilical vein endothelial cells at 10lM concentration. Finally,

an extensive molecular simulation study was performed to investigate the probable interaction with

VEGFR-2.

KEYWORDS:

Pyridazine derivatives;

VEGFR-2 inhibitors;

antitumor agents; HUVEC;

antiangiogenic agents;

hinge interaction

1. Introduction

and these have demonstrated clinical benefits in the treatment of

some tumours with manageable side effects. Many angiogenesis

inhibitors are also being evaluated in clinical trials for the treat-

ment of various cancers.

Angiogenesis is the process of sprouting or splitting of novel capillary

blood vessels from the quiescent pre-existing vasculature¹.

Angiogenesis is normally orchestrated via a finely balanced equilib-

rium between pro-angiogenic and anti-angiogenic factors. However,

aberrant equilibrium between these factors is associated with several

human disorders, like rheumatoid arthritis, psoriasis, and cancer².

The vascular endothelial growth 2 (VEGFR-2), also known as

(KDR) has spurted as an attractive pharmacological target in can-

cer therapy pertaining to its crucial rule in tumorangiogenesis.

Since the binding of VEGF to VEGFR-2 leads to receptor dimerisa-

tion, followed by the autophosphorylation of tyrosine residues in

the intracellular kinase domain, resulting in potent mitogenic and

chemotactic effects on endothelial cells (ECs)³.

The expression of VEGF is up-regulated by tumour-related

changes, such as hypoxia, protooncogene activation, and the

aberration of tumour-suppressor genes. Since the overexpression

of VEGF correlates with poor prognosis and the clinical stage of

patients with solid tumours, VEGF/VEGFR-2 signalling is thought

to be an attractive target for the treatment of cancer.

In the last two decade, pyridazine based scaffold has emerged

as a novel, promising scaffold for the design and development of

potent protein kinase inhibitors. Including either substituted pyri-

dazine ring; or those incorporated in bicyclic ring system; such as

imidazopyridazines. An imidazopyridazine derivative; Ponatinib⁹;

evolved as a potent orally active Pan-inhibitor of (BCR-ABL) kinase,

has reached phase I clinical evaluation of patients with refractory

CML and other hematologic malignancies¹⁰. Moreover, TAK-593;

an imidazo[1,2-b]pyridazine scaffold was designed and synthesised

as a hinge binder^11,12. It inhibits VEGFR1/2/3 (3.2/0.95/1.1 nM) and

PDGFRa/b (4.3/13 nM). Oral administration of TAK-593 is currently

being tested in

a Phase I clinical trial in non-hematologic

advanced cancer¹³(Figure 1).

Despite the various advantages of pyridazines in drug design;

including modulation of the physico-chemical properties, relatively

ADME and toxicity profile, easy and diverse synthetic methods of

access, as well as their affinity for a great number of receptor pro-

teins; the potential inhibitory activity of pyridazine derivatives

Several VEGFR humanised anti-VEGF monoclonal antibody

(bevacizumab) and small-molecule VEGFR-2 kinase inhibitors

(Sorafenib^4,5Sunitinib^6,7, and Pazopanib⁸) have been approved, against VEGFR-2 is yet unravelled. Therefore, pyridazine derivatives

CONTACT Khaled A. M. Abouzid

abouzid@yahoo.com, khaled.abouzid@fop.usc.edu.eg, khaled.abouzid@pharma.asu.edu.eg

Faculty of Pharmacy, University of

Sadat City, 25th District, Sadat City, Menoufia 32897, Egypt

Supplemental data for this article can be accessed here.

ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

1574

M. Y. JABALLAH ET AL.

Sorafenib

Sunitinib

Pazopanib

Ponatinib

TAK593

Figure 1. Structures of VEGFR-2 inhibitors approved for clinical use.

were designed and synthesised in an attempt to elaborate a novel preparation of compound 2 was obtained through reaction of 3-

chemical scaffold for this purpose which could serve as alternative chloro-6-hydrazinylpyridazine with cyanogen bromide. Which was

therapy for the current sorafenib and its analogues.

either reacted with phenyl isocyanate derivative to furnish 3 or

was reacted with morpholine, followed by the reaction with 3,4-

dichloro phenyl isocyanates to afford compounds 5a–c.

Scheme 2 describes the cyclo-condensation of either 4- or 3-

nitroacetophenone derivatives to furnish the corresponding nitro

phenyl pyridazinone derivatives (6a,b) respectively, which were

later subjected to catalytic hydrogenation to afford the corre-

sponding amines (7a,b) in high yield (90%) followed by nucleo-

philic substitution of substituted phenyl isocyanates to furnish

phenyl pyridazinone urea derivatives 8a–f.

On the other hand, in Scheme 3 the (6-chloropyridazin-3-yl)a-

mino)phenyl derivatives were obtained via reflux the key inter-

mediates 10a–f with 3,6-dichloropyridazine in butanol under

nitrogen atmosphere to afford compounds 11a–f.

In Scheme 4, nucleophilic substitution of 3.6-dichloropyridai-

zine with 4-nitroaniline yielded 6-chloro-N-(4-nitrophenyl)pyrida-

zin-3-amine; which was further reacted with sodium methoxide

the 6-chloropyridazin-3-yl)nitro)phenyl derivatives was refluxed

with sodium methoxide to furnish 6-methoxy-N-(4-nitrophenyl)pyr-

idazin-3-amine (14) in high yield. Following catalytic hydrogen-

ation of Compound (14); condensation with substituted phenyl

isocyanate furnished compound 15.

2. Rationale and design

In light of the previous findings; we aimed at designing potent

novel chemotypes as potent VEGFR-2 inhibitors. Thus, an array of

pyridazine derivatives; including the unexplored triazolopyridazine

derivatives through scaffold hopping strategy of the imidazopyri-

dazine moieties were designed (Figure 2). Additionally, bioisoster-

ism strategy was applied for designing pyridazine derivatives as

VEGFR-2 inhibitors by replacing the central pyridine carboxamide

core of sorafenib, a well-known VEGFR-2 inhibitor with IC₅₀value

of 90 nM, with a pyridazine nucleus.

The newly synthesised pyridazine derivatives 3, 5a–c, 8a–f,

11a–f, 15, and 18a–c were evaluated for their potential inhibitory

activity toward VEGFR-2. Also, the most active derivatives were

screened for their pendant antiangiogenic properties. Moreover,

they were screened for their in vitro anticancer activity against a

panel of 56 human cancer cell lines at NCI-USA.

3. Results and discussion

Scheme 5, describes the multistep synthesis of pendant com-

pounds 18a–c. Following nucleophilic substitution of 3,6-dichloro-

3.1. Chemistry

The synthetic strategies adopted for the preparation of the new pyridazine with paracetamol, the furnished compound was

pyridazines are described in Schemes 1–5. In Scheme 1, deacetylated following reflux in concentrated hydrochloric acid.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1575

Figure 2. Design of the pyridazine- urea derivatives through scaffold hopping.

R; a: 3-OCH₃, b: 3,4-diCl, c:

3-CF₃, 4-Cl

Scheme 1. (a) NH₂NH₂.H₂O (100%), ethanol, reflux, 3 h. (b) CNBr, ethanol, r.t. 24 h. (c) Substituted phenyl isocyanates, methylene chloride, TEA, r.t. 24 h.

(d) Morpholine, n-butanol, reflux, 6 h. (e) Substituted phenyl isocyanates, dry THF, r.t. 24 h.

Scheme 2. (a) i) Glyoxalic acid, glacial acetic acid, reflux, 48 h, then, ii) NH₂NH₂.H₂O, reflux, 3 h, 45% (b) H₂, Pd/C, Ethanol, 6 h. (c) Substituted phenyl isocyanates, dry

THF, r.t. 24 h.

1576

M. Y. JABALLAH ET AL.

Scheme 3. (a) Dry THF, rt, 24 h. (b) H₂, Pd/C, EtOH, 120min. (c) 3,6-dichloropyridazine, n-butanol, reflux, N₂, 48h, 35%

Scheme 4. (a) 4-Nitroaniline, isopropanol, reflux, 6 h. (b) Sodium methoxide, DMF, reflux, 6 h. (c) H₂, Pd/C, methanol, 6 h. (d) (3-trifloromethyl, 4- chloro phenyl ioscya-

nate), dry THF, r.t. 48 h.

R; a: 3-CH₃, b:3-CF₃, 4-Cl, c:3,4-

diCl

Scheme 5. (a) Paracetamol, isopropanol, reflux, anhydrous K₂CO₃, 24 h, 75%. (b) i) Conc HCl, reflux, 1 h; ii) glacial acetic acid, reflux, 6 h. (c) Substituted phenyl isocya-

nates, dry THF, r.t. 24 h.

The resulting 3-chloropyridazine derivative was then refluxed in caused by the tested compounds against VEGFR-2 kinases was

glacial acetic acid to produce the 3-pyridazinone derivative (17) in evaluated compared to reference kinase inhibitor staurosporine at

excellent yield (85%). Following condensation with phenyl iso- a single concentration of 10 mM. The tested compounds depicted

cyanate derivatives, compounds 18a–c were obtained in good weak to excellent inhibitory activity against VEGFR-2 kinases

yields (60–75%). The proposed structures of all newly prepared (Table 1).

compounds were confirmed with spectral and elemental analyses.

3.2.1.1. SAR. The SAR study depends on the comparison between

the inhibitory activity against VEGFR-2 and structural variations of

pyridazine containing compounds.

3.2. Biological evaluations

Compounds in series 1 (compounds 3, 5a–c) exhibited weak

inhibitory activity (3–7%) against VEGFR-2 at 10 mM concentration.

3.2.1. VEGFR-2 kinase inhibition assay

Initial screening at single dose of 10 mM concentration:

All the newly prepared compounds (3, 5a–c, 8a–f, 11a–f, 15, Those compounds belong to the relatively short biaryl moieties.

and 18a–c) were tested in vitro for their kinase inhibitory activity The rigidification of pyridazine containing compounds as well as

against VEGFR-2. The percent inhibition of the enzymatic activity substitution on the 6- position of the pyridazinyl moiety either

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1577

Table 1. VEGFR-2% inhibition, and IC₅₀of test compounds.

VEGFR-2% inhibition^a

VEGFR-2IC₅₀

a

Cpd no.

R1

R2

Cl

R3

Cl

X

-NH position

3

Cl

–

4

3

4

3

4

3.06 0.45

5.07 0.61

7.07 1.11

–

5a

5b

Morphilino

OCH₃

Cl

H

Cl

5c

8a

Morphilino

CF₃

H

Cl

13.10 1.71

22.20 1.85

35.06 2.87

100.0 4.61

21.97 2.73

27.03 3.29

89.03 2.97

21.93 2.42

29.93 4.12

22.07 3.47

11.93 2.25

20.13 2.08

27.1 1.93

–

8b

8c

–

Cl

CF₃

Cl

1.3 0.14 lM

8d

–

H

Cl

–

8e

–

Cl

8f

11a

11b

–

CF₃

OCH₃

Cl

OCH₃

Cl

–

1.8 0.16 lM

Cl

NH

O

–

11c

11d

11e

Cl

CF₃

OCH₃

Cl

OCH₃

Cl

–

11f

15

18a

18b

Cl

OCH₃

O

CF₃

CH₃

CF₃

Cl

H

Cl

–

1.4 0.12 lM

–

60.7 0.03 nM

107 0.04 nM

–

97.1 4.13

15.06 1.89

96.03 3.85

100.27 4.11

90.47 4.53

O

18c

Cl

Strausporine

Sorafenib⁴

90 nM^b

11a-f

3, 5a-c

15, 18a-c

8a-f

^aData are presented as Mean SD, n ¼ 3.

^bReported IC₅₀values.

with chloro or morpholino group (compounds 5a–c) respectively phenyl ring is di-substituted in the 3- and 4-positions (3, 4-

did not improve the activity against VEGFR-2.

dichloro; 3-trifloromethyl,4-chloro) substitution in 18b and 18c,

respectively. Mono substitution results in a significant drop in

activity, i.e. (18a with percentage inhibition of 32%).

In series 2, (compounds 8a–f) exhibited an improvement of

activity was observed since two compounds (8c, 8f) of this series

exhibited potent inhibition of VEGFR-2 kinase activity (100% and

89%, respectively). It is well-observed that incorporation of a cen-

tral phenyl ring; directly attached to the pyridazine ring at the 3-

position; as well as substitution of 6-position of the pyridazine

moiety with oxygen atom, the activity markedly increased upon

substituting the terminal aromatic side chain (3-trifloromethyl, 4-

chloro) regardless the position of the urea linker (3- or 4-position

as exhibited in compounds 8c and 8f, respectively). Interestingly,

only (3-trifloromethyl, 4-chloro) substitution in the terminal phenyl

ring is well-tolerated; whilst other substitutions (e.g. 3,4-di chloro

in 8b) had weak inhibition activity on VEGFR2 enzyme.

In series 3, further extension of the molecule via incorporation

of extra linker NH linker (compounds 11a–f) did not improve the

VEGFR inhibitory activity; since the compounds exhibited weak to

moderate inhibition of the activity; ranging from (10–35%); whilst

replacement of chlorine atom in the 6-position of the pyridazine

ring in the above series with methoxy moiety (as in compound

15) results in marked increase in the potency (89%).

3.2.1.2. Measurement of potential enzyme inhibitory activity (IC₅₀).

The tested compounds (8c, 8f, 15, 18b, and 18c), which dis-

played VEGFR-2 inhibition percent above 75% at 10 mM concentra-

tion were further investigated for their dose-related VEGFR-2

enzymatic inhibition at 5 different concentrations (1 nM - 10 nM -

100 nM - 1 mM - 10 mM) to subsequently calculate their IC₅₀values

(Table 1). Compounds 18b and 18c, potently inhibited VEGFR-2 at

nanomolar IC₅₀values (60.7 0.03 and 107 0.04 nM, respectively).

Also, compounds 8c, 8f, and 15 moderately inhibited VEGFR-2

with IC50 values of, 1.3 0.14, 1.8 0.16, and 1.4 0.12 lM,

respectively. Figures representing IC₅₀are provided in Supporting

Information (Supplemental Figure S1)

These impressive results encouraged us to pursue further

investigations regarding the activity of these compounds

(Table 1).

3.2.2. In vitro human umbilical vein endothelial cells (HUVEC)

anti-proliferative assay

Replacement of the 6-chloropyridazine with pyridazinone moi-

ety; as well as incorporation of an ether linkage (as observed in

compounds 18b,c) lead to tremendous increase in the percentage To Further investigate the potential anti-angiogenic properties of

inhibition (96% and 98%, respectively); provided that the terminal the investigated compounds; compounds that showed potent IC₅₀

1578

M. Y. JABALLAH ET AL.

values against VEGFR-2; where subjected to HUVEC cell line anti- cancer types: leukaemia, melanoma, lung, colon, CNS, ovarian,

proliferative assay.

renal, prostate, and breast cancers.

Angiogenesis process involves EC sprouting from the parent

A 48 h drug exposure protocol was used and sulforhodamine B

vessel, followed by migration, proliferation, alignment, tube forma- (SRB) protein assay was applied to estimate the cell viability and

tion, and anastomosis to other vessels. Several in vitro models growth¹⁸. The mean percentages GI (GI%) of the tested com-

have been attempted to recreate this complex sequence of pounds over the full panel of cell lines are illustrated in Figure 4.

events¹⁴. HUVECs have played a major role as a model system for

The obtained data revealed that, the tested compounds from

series 1 and 2 displayed fair anti-cancer activity (mean % GI ¼

the study of the regulation of EC function and the role of the

11–24) except for compound 11f featuring a lipophilic 3-trifloro-

methyl 4-chloro on the pendant phenyl which elicited the highest

activity (mean % GI ¼ 45). It exhibited broad spectrum and potent

endothelium in the response of the blood vessel wall to stretch,

shear forces, and the development of atherosclerotic plaques and

angiogenesis

Compounds 8c, 8f, 15, 18b, and 18c were selected to be anti-proliferative activity against several NCI cell panel, namely;

tested for their ability to in vitro inhibit VEGF-induced HUVEC cell the leukaemic HL-60 (TB), MOLT-4, and SR cancer cell lines with GI

line proliferation, using doxorubicin as a control.

The given test compounds manifested potent anti-proliferative

activities against HUVEC cell line. Compound (8f) (VEGFR-2 IC₅₀

1827 nM) showed the highest growth inhibition (GI) percent

(99.82%). The rest of compounds manifested moderate to high

inhibition percent.

However, despite their potent VEGFR-2 inhibitory activity, com-

pounds (15) exhibited moderate anti-proliferative activity against

HUVEC cell line (35.5%; Table 2; Figure 3).

81.7%, 70.1%, and 81.2%, respectively, the non-small cell lung can-

cer A549/ATCC, NCI-H226, and NCI-H322M cell lines with cell GI

58.4%, 69.1%, and 71%, respectively, the colon cancer HCT-15,

SW-620 cell line with 69.8% and 68.2% inhibition, respectively, the

melanoma SK-MEL-5 and UACC-62 cell line with 64.72% inhibition,

respectively. The ovarian cancer OVCAR-3 with 67% and the pros-

tate cancer PC-3 cell line with 77.2%. Finally, it showed broad

spectrum cell GI against the breast cancer MCF7, BT-549, T47D,

and MDA-MB-468 with 71%, 68%, 83.4%, and 70.1%, respectively

(Figure 5).

¼

NCI selected compounds belonging to Series 4; i.e. pyridazin-

6-one series linked to biarylureas via an ether linkage (18b,c),

both exhibited weak inhibitory activity against most of the test

cell lines. The weak inhibitory activity of compound (18b) further

confirms the selectivity against VEGFR-2 enzyme.

3.2.3. In vitro anti-cancer activity

The structures of the all synthesised compounds (3, 5a–c,8a–f,

11a–f, 15, and 18a–c) were submitted to the National Cancer

Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.

nih.gov).

Seven compounds were selected to be screened for their anti-

cancer activity in vitro. The anticancer assays were performed in

accordance with the protocol of the Drug Evaluation Branch, NCI,

3.2.3.1. Molecular modelling studies. As discussed in the methods

section, three ligands were selected to perform the extensive in

silico analysis. These ligands are, Sorafenib (SORA), 11c, and 18b.

Bethesda^15–17

.

The selected compounds were evaluated at primary anticancer

assay against a panel of 56 cancer lines at concentration 10–5 M.

The human tumour cell lines were derived from nine different

50

40

30

20

10

0

Table 2. Anti-proliferative activity against HUVEC cell line.

Compound (10 mM)

HUVEC proliferation (%)

No compound

8c

8f

15

18c

18b

102.58 1.158

14.75 0.059

0.18 0.019

64.50 2.017

3.25 0.435

2.13 0.425

5a

8e

11c

11e

11f

18a 18b

Data are presented as Mean SD, n ¼ 3.

Figure 4. Mean % GIs of the tested compounds over NCI 56 cell line panel.

No

Compound

120

100

80

60

40

20

0

15

8c

18c

18b

8f

HUVEC Proliferaꢀon (%)

Figure 3. % HUVEC cell growth following treatment with compounds 8c, 8f, 15, 18b, and 18c compared to untreated control.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1579

11f

120

100

80

60

40

20

0

Leukemia

NSCLC

Colon

CNS

Melanoma

Ovarian

Renal prostate Breast

Figure 5. % Growth exerted by compound 11f at 10 lM concentration over NCI 56 cell line panel.

(SORA)

Figure 6. The chemical structures for the three compounds under the in-depth structural investigation, SORA, 11c, and 18b. SORA and 18b possess an ether linker

ꢁ

whereas 11c has an amine linker. The torsional space around these linkers was scanned at the B3LYP/6–31 þ G level of theory.

As shown in Figure 6, the 4-chloro-3-(trifluoromethyl)phenyl urea was the reason for selecting this compound to perform the in-

depth structural investigation. To avoid redundancy with the

already discussed SAR analysis, we will limit the discussion here to

the predicted binding modes of compounds 11c and 18b com-

pared to SORA. The predicted binding modes of SORA, 11c, and

18b are shown in Figure 7. As shown in the Figure 7(a,c), SORA

and 18b are capable of forming strong H-bond with hinge resi-

dues, CYS919, and GLU917. On the other hand, the predicted

binding conformation of compound 11c lacks this H-bond as

shown in Figure 7(b). As discussed in many studies, the formation

of this specific H-bond is crucial for the activity of all reported

competitive kinase inhibitors^19–22. Interestingly, both SORA and

compound 18b form dual H-bonds at this region, which offers a

significant boost to their affinity against VEGFR-2. Whereas, SORA

forms these two H-bonds with CYS919, 18b engages with GLU917

backbone amide bond in addition to CYS919. Our hydrogen bond

occupancy analysis for the hinge H-bond (H-bond criteria 3.2 Å for

the distance and 120^ꢀfor the angle) with CYS919 showed conser-

moiety is common between all ligands. The major structural differ-

ence between the three ligands is as follows. While SORA and

18b are O-linked (ether linker) to the terminal hetero-aromatic

ring, compound 11c possesses an N-linker (amine linker). In the

following sections, we will discuss how the nature of this linker

affected the predicted binding mode of the three compounds and

how this was reflected on the measured inhibitory effects of the

compounds (Figure 6).

3.3. Detailed analysis of the potential binding modes of

Sorafenib, 11c and 18b; analysing the binding modes and the

simulation trajectories

The synthesised molecules were tested in enzymatic inhibition

assay against VEGFR-2, in comparison to the positive control

Strausporine. As shown in Table 1, compound 18b shows the best

enzymatic inhibition activity with an IC₅₀value of 60.7 nM. This vation for this H-bond for approximately 86% of the simulation

1580

M. Y. JABALLAH ET AL.

Figure 7. The ligand interaction diagrams of the compounds under study. The upper panel shows the three-dimensional binding modes whereas the lower panel

shows the 2D diagrams of (a) Sorafenib, (b) 11c and (c) 18b.

time in VEGFR-2 complex with SORA, and 98.4% for com- crystal conformers for 452 molecules²⁷. Interestingly, the report

found that in 50% of the cases, the crystal conformer lies within

< 0.6 kcal/mol energy difference from the gas phase conformer.

The study also found that it is only 10% of the cases the crystal-

lised state of the molecule can adopt high-energy conformational

structures, and this was mostly for highly polar compounds, such

as sugars. Furthermore, recent SAR analysis studies from several

research groups indicated that there was a strong correlation

between the biological activity of the small molecule ligands and

the relative abundance of the bioactive (bound-like) conformation

in the free state²⁸. These observations suggest that the critical

examination of the proposed binding modes and the relative

abundance of ligand bioactive conformation are essential parts of

any meaningful SAR analysis.

pound 18b.

The second half of the three molecules, the 4-chloro-3-(trifluor-

omethyl)phenylcarbamoylamino moiety forms an optimal inter-

action with several residues at the VEGFR-2 pocket. As shown in

Figure 7, the urea moiety forms two strong H-bonds with GLU885

and ASP996. The 4-chloro-3-(trifluoromethyl)phenyl substitution

occupies the allosteric hydrophobic pocket of VEGFR-2. The pres-

ence of this pocket is a signature for the majority of type-II kinase

inhibitors^23–25; the DFG-out kinase inhibitors. The pocket is pre-

dominantly lipophilic, and thus only lipophilic substitution at this

site region of the molecule can show stable binding. As also

shown in Figure 7, residues ILE994, VAL898, ILE892, LEU969,

ILE888, and LEU898 form the pocket.

In the current study, we were interested to investigate whether

the nature of the linker between the two parts of the molecules,

the 4-chloro-3-(trifluoromethyl)phenylurea moiety and the terminal

hetero-aromatic ring system has an impact on the observed activ-

ity. Therefore, we conducted a relaxed PES scan for the torsion

around this linker. The PES scan was conducted at the B3LYP/

3.4. The potential energy surface (PES) scan for the compounds

under investigation; the impact of the ether/amine linker on the

preferred bioactive conformation

For typical molecular docking software, two steps are necessary,

which include a conformational sampling step followed by a scor-

ing of the predicted binding poses. In general, an internal library

of torsional angles can ensure that the conformational sampling

algorithm generate energetically favourable ligand conformations

based on its internal potential energy function²⁶. In an ideal scen-

ario, the sampled conformations of the small molecule will corres-

pond to a minima on its PES, but not necessarily the global

minimum. Theoretically, the introduction of additional interactions

of the small molecule with the binding site residues, or any crystal

contacts in general, should overcome small energy barriers against

a less preferred conformation for this small molecule. However, a

recent rigorous analysis report by Zheng et al has attempted to

6–31 þ G level of theory via rotating the X-C torsional by 10^ꢀ

ꢁ

increment (X is an oxygen in SORA and 18b, but a nitrogen in

11c). The PES scans were performed for 36 steps to cover the full

torsional space around this bond. We started the scan from a

SORA like bound conformations for the three molecules, SORA,

11c, and 18b. Upon completion, the three PES scans were plotted

together taken the global energy conformation of each molecule

as a reference (0 kcal/mol) for its own PES. The PES scans of the

three molecules are given in Figure 8.

As illustrated in the PES scans in Figure 8, the minimum

energy conformers of compounds 18b and SORA correspond to

the bioactive, bound like conformations of these two molecules.

The PES scan also indicated that the conformational preferences

determine the conformational variations between gas phase and of SORA and compound 18b are very similar. The ether linker

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1581

Figure 8. The relaxed PES scan of the torsional angel of the selected linker. The scan was initiated from a sorafenib bound-like conformation for all ligands such that

they start and end approximately at the same value of the torsional angle. Representative conformations for the most stable (global energy minimum) conformation

are given in the figure. For SORA and 18b, this conformation corresponds to the bioactive conformation, whereas for 11c, this conformation (planar) is not the bio-

active one, i e, not the conformation required to form an H-bond with the hinge residues. The carbon atoms of each ligand are coloured similarly to the scanning

ꢁ

steps. The scan was performed at the B3LYP/6–31þ G level of theory.

on these two molecules favours a perpendicular (non-coplanar) 3.5. Molecular mechanics (MM)-GBSA binding energy and per-

orientation of the terminal hetero-aromatic ring with respect to residue binding energy decomposition

the phenyl-carbamoylamino moiety. This perpendicular conform-

Analysing the generated MD trajectories of the three ligands

ation corresponds to a global energy minimum on the PES sur-

under investigation showed that the bound conformations of

faces of these two molecules. For compound 11c, however, the

SORA and compound 18b exhibited a much more stable trajec-

planar orientation of the two terminal rings is favoured by

tory over the simulation time (25 ns). The observed average ligand

RMSD values for all ligands are within 1 Å, with compound 11c

approximately 3 kcal/mol from the perpendicular, SORA like con-

formation of compound 11c. Our attempt to re-optimise the

being the highest (ꢂ0.99 Å), that is slightly higher than SORA

geometry the perpendicular conformation of compound 11c at

(ꢂ0.64 Å) and 18 b (ꢂ0.69 Å). The maximum ligand RMSD values

ꢁꢁ

the M062X/6–311þþG

level of theory again generated the

are also exhibited by compound 11c (ꢂ1.54 Å), compared to

SORA (ꢂ1.39 Å) and compound 18b (ꢂ1.29 Å). The average recep-

tor RMSD values for all ligands lie within ꢂ1.5 Å, and the max-

imum receptor RMSD value is below 2.5 Å for all trajectories.

Ligand and receptors RMSD figures are given in the Supporting

Information (Supplemental Figure S3).

planar conformation as the most stable geometry of this mol-

ecule. This data clearly indicates that the planar conformation

of compound 11c is much more favourable than the perpen-

dicular conformation. Taken all the interactions that compound

11c should form with the VEGFR-2 binding site residues, such

as hinge, GLU885 and ASP996 as well as the lipophilic inter-

action with the allosteric pocket, only the perpendicular con-

formation of compound 11c could potentially form these

interactions. As this conformation is very unlikely to happen,

given the high strain energy penalty, compound 11c is

expected to exhibit a very weak inhibition of VEGFR-2 (23% at

10 lM, see Table 1). We also expanded our analysis to com-

pound 15 (amine linker) which has a methoxy substituted pyri-

dazine ring and it was clear from the PES scan that a SORA

like conformation is indeed favourable, only 1 kcal/mol higher

energy than the global energy minimum structure. This may

explain the good activity of compound 15 (IC₅₀¼ 1406 nm,

%inhibition 98% at 10 lM) compared to compound 11c

(%inhibition 23% at 10 lM) in which the proposed bioactive

conformation is 3 kcal/mol higher energy than the global energy

conformer. The PES scan of 15 is given in Supporting

Information (Supplemental Figure S2).

The predicted binding affinities of the molecules have been

estimated using the AMBER/MM-GBSA method as implemented in

AMBER14. The numerical values are listed in Table 2, together

with the predicted docking scores and H-bond occupancies over

the generated trajectories. As shown in Supplemental Table S1,

the predicted AMBER/MM-GBSA free energy of binding is nicely

correlating with the measured inhibitory activities of the com-

pounds. The AMBER/MM-GBSA free binding energy of SORA is

given by –46.87 kcal/mol, approximately 3.49 kcal/mol better than

that of compound 18b (–43.38 kcal/mol) and 9.90 kcal/mol better

than compound 11c (–36.97 kcal/mol). The table also demon-

strated the enhanced contribution of the AMBER/MM-GBSA lipo-

philic van der Waals (vdW) interaction (DG_MM-GBSA/vdW) for all

ligands compared to the electrostatic contributions (DG_MM-GBSA/

_ELE) to the free binding energy. For example, the DG_MM-GBSA/vdWof

SORA is –59.72 kcal/mol, –51.61 kcal/mol for compound 18b, and

–51.84 kcal/mol for compound 11c. The enhanced lipophilic

1582

M. Y. JABALLAH ET AL.

interaction of SORA compared to compounds 18b and 11c is

arguably due to the presence of terminal methyl amino group

that can form additional interaction with PHE918 and LEU840 resi-

dues at the ATP site. The lipophilic energy decomposition for the

favourable residues is given in Supporting Information

Supplemental Figure (S4). The DG_MM-GBSA/ELEelectrostatics inter-

action term showed consistencies with the reported H-bond occu-

pancies. As shown in Supplemental Table S1, the DG_MM-GBSA/ELE

Compounds 9a–f were prepared according to reported

procedures^34–37

.

1–(3-Aminophenyl)-3-substituted phenyl urea (10a–f)

Compounds 10a–f were prepared according to reported

procedures^15,37–40

6-Chloro-N-(4-nitrophenyl)pyridazin-3-amine (12)¹⁶

Compound 12 was prepared according to reported proced-

ure¹⁶(m.p. 182–183 ^ꢀC as reported).

6-Methoxy-N-(4-nitrophenyl)pyridazin-3-amine (13)¹⁷

Compound 13 was prepared according to reported proced-

ure¹⁷(m.p. 123–125 ^ꢀC, as reported).

for compound 18b is –39.98 kcal/mol, slightly better than

term

SORA (–37.95 kcal/mol), and significantly better than 11c (–29.12).

The predicted docking scores listed in Supporting Information

Supplemental Table S1 also show a similar trend with the AMBER/

MM-GBSA, however, the docking scores strongly favour compound

11c, which emphasise the importance of post processing of the

docking results as mandatory step to achieve better performance.

N¹-(6-methoxypyridazin-3-yl)benzene-1,4-diamine (14)

Compound 14 was prepared according to reported proced-

ure¹⁸(m.p. 132–134 ^ꢀC, as reported).

N-(4-((6-Chloropyridazin-3-yl)oxy)phenyl)acetamide (16)

Compound 16 was prepared according to reported proced-

ure^41,42(m.p. 192–194 ^ꢀC C as reported)

ꢀ

4. Experimental

6–(4-Aminophenoxy)pyridazin-3(2H)-one (17)

Compound 17 was prepared according to reported proced-

ure^41,42(m.p. 130 ^ꢀC as reported) General procedure for prepar-

ation of target compounds (3, 5a–c, 8a–f, 11a–f, 15, 18a–c)

To a solution of the corresponding intermediate; compound

2, 4a–c, 7a–f, 10a–f, 14, and 17a–c, respectively (6 mmol) in dry

THF (20 ml), the appropriate isocyanate (6 mmol) was added and

the mixture was stirred at room temperature for 24 h. The formed

solid was collected by filtration, washed with minimal quantities

of dry THF. Crystallisation was accomplished using appropri-

ate solvent.

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

8400Sspectrophotometer and expressed in wave number (cm^–1).

The NMR spectra were recorded by Varian Gemini-300BB at

300 MHz (Varian Inc., Palo Alto, CA) or Bruker spectrophotometer

at 400 MHz. 1H NMR spectra were run at 300 or 400 MHz, while

13 C NMR spectra were run at 75or 100 MHz in deuterated

dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (CDCl3).

Chemical shifts (dH) are reported relative to TMS as internal

standard. All coupling constant (J) values are given in hertz.

Chemical shifts (dC) are reported relative to DMSO-d6 as internal

standard. The abbreviations used are as follows: s, singlet; d,

doublet; m, multiplet. Mass spectra were measured on a GCMS-

QP1000 EX and Helwett Packard 5988 spectrometers at 70 eV.

Elemental analyses were carried out at the Regional Center for

Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt.

Analytical thin layer chromatography on silica gel plates contain-

ing 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.

1-[6-Chloro-[1,

2,

4]triazolo[4,3-b]pyridazin-3-yl)-3–(3,4-

dichlorophenyl)]urea (3)

Acetonitrile giving compound (XVII) as white crystals (0.85 g,

40%); m.p. 173–175 ^ꢀC; ¹HNMR (300 MHz, DMSO-d₆) d 10.00 (s,

1H, NH D₂O exchangeable, NH urea), 9.66 (s, 1H, NH D₂O

exchangeable, NH urea), 8.12(s, 1H, ArH), 8.07 (d, J ¼ 10.7 Hz, 1H,

pyridazinyl H), 7.76 (d, J ¼ 8.8 Hz, 1H, ArH), 7.64 (d, J ¼ 8.8 Hz, 1H,

ArH), 7.36 (d, J ¼ 10.7 Hz, 1H, pyridazinyl H).FT-IR (ύ max, cm²¹):

3288–3272(NH), 3050 (CH aromatic), 2984 (CH aliphatic), 1685

(C ¼ O amide), 1635 (C ¼ N); MS: (Mwt.: 355.97) m/z (% rel. Int.)

357.01 (M^þþ1, 3.31), 355.99 (M^þ, 10.01), 176 (100); Anal. Calcd for

C₁₂H₇Cl₃N₆O C, 40.31; H, 1.97; N, 23.50. Found: C, 40.42; H, 1.94;

N, 23.63.

1-[3,4-Dichlorophenyl)-3–(6-morpholino-[1, 2, 4]triazolo[4,3-

b]pyridazin-3-yl]urea (5a)

4.1.1. 3-Chloro-6-hydrazinylpyridazine (1)

Compound 1 was prepared according to reported procedure²⁹

(m.p. 92 ^ꢀC, as reported).

Crystallised from THF as yellowish white crystals (0.97 g, 40%);

m.p. 145–147 ^ꢀC; ¹HNMR (300 MHz, DMSO-d₆) d 10.00 (s, 1H, NH

D₂O exchangeable), 9.66 (s, 1H, NH D₂O exchangeable), 8.12(s, 1H,

ArH), 8.07 (d, J ¼ 10.7 Hz, 1H), 7.76 (d, J ¼ 8.8 Hz, 1H), 7.64 (d,

J ¼ 8.8 Hz, 1H), 7.36 (d, J ¼ 10.7 Hz, 1H), 3.69 (d, J ¼ 4.2 Hz, 4H, mor-

pholinyl H), 3.53 (d, J ¼ 4.2 Hz, 4H, morpholinyl H).

4.1.2. 6-Chloro[1, 2, 4]triazolo[4,3-b]pyridazin-3-amine (2)

Compound 2 was prepared according to reported procedure³⁰

(m.p. 287 ^ꢀC as reported).

¹³C NMR (DMSO-d6, 100 MHz) d: 47.4 (2CH₂morpholine), 66.1

(2CH₂morpholine), 120.4, 124.0, 125.0, 126.3, 128.3, 128.9, 131.2,

131.9, 139.9, 141.7, 145.1, 155.4, 163

4.1.3. 6-[1, 2, 4]Triazolo[4,3-b]pyridazin-3-amine (4)

Compound 4 was prepared according to reported procedure²⁹

(m.p. 198–200 ^ꢀC, as reported).

FT-IR (ύ max, cm²¹): 3291 (NH), 3122 (CH aromatic), 2988 (CH

aliphatic), 1686 (C ¼ O amide), 1640 (C ¼ N); MS: (Mwt.: 408.24);

Anal. Calcd for C₁₆H₁₅Cl₂N₇O₂C, 47.07; H, 3.70; N, 24.02. Found: C,

47.08; H, 3.73; N, 24.07.

1-[4-Chloro-3-(trifluoromethyl)phenyl)-3–(6-morpholino-[1, 2,

4]triazolo[4,3-b]pyridazin-3-yl]urea (5b)

Crystallised from ethanol as buff crystals (1.19 g, 45%); m.p.

221–223 ^ꢀC; ¹HNMR (300 MHz, DMSO-d₆) d 10.23 (s, 1H, NH D₂O

exchangeable), 9.67 (s, 1H, NH D₂O exchangeable), 8.43(s, 1H,

ArH), 8.22 (d, J ¼ 9.9 Hz, 1H), 7.87 (d, J ¼ 8.8 Hz, 1H), 7.64 (d,

4.1.4. 2–6-(4-Nitrophenyl)pyridazin-3(2H)-one628

Compounds 6 were prepared according to reported procedures³¹.

6–(4-Aminophenyl)pyridazin-3(2H)-one (7)^32,33

Compounds

6

were prepared according to reported

procedures³²,

1–(3 or 4-Nitrophenyl)-3- substituted phenylurea 9a–f

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1583

J ¼ 8.8 Hz, 1H), 7.21 (d, J ¼ 9.9 Hz, 1H), 3.73 (d, J ¼ 4.2 Hz, 4H, mor- (C ¼ N); MS: (Mwt.: 336.34) Anal. Calcd for C₁₈H₁₆N₄O₃: C, 64.28; H,

4.79; N, 16.66. Found: C, 64.34; H, 4.92; N, 16.72.

pholinyl H), 3.62 (d, J ¼ 4.2 Hz, 4H, morpholinyl H).

FT-IR (ύ max, cm²¹): 3278–3288 (NH), 3112 (CH aromatic),

1-[3,4-Dichlorophenyl)-3–(3-(6-oxo-1,6-dihydropyridazin-3-yl)

phenyl)]urea (8d)

2990 (CH aliphatic), 1695 (C ¼ O amide), 1636 (C ¼ N); MS: (Mwt.:

441.78) m/z (% rel. Int.)443.82 (M^þþ2, 2.87), 442.82 (M^þþ1, 3.31),

Crystallised from methanol as white crystals(0.78g., 35%) m.p.:

232–233 ^ꢀC. ¹HNMR (300 MHz, DMSO-d₆

d 13 (s, 1H, D₂O

441.75 (M^þ, 8.94), 151 (100); Anal. Calcd for C₁₇H₁₅ClF₃N₇O₂C,

46.22; H, 3.42; N, 22.19. Found: C, 46.27; H, 3.48; N, 22.22.

1-[3-Methoxyphenyl)-3–(6-morpholino-[1, 2, 4]triazolo[4,3-b]

pyridazin-3-yl]urea (5c)

Crystallised from acetonitrile as yellowish white crystals (1.1 g,

50%); m.p. 189–192 ^ꢀC; ¹HNMR (300 MHz, DMSO-d₆) d 9.87 (s,

1H, NH D₂O exchangeable), 9.54 (s, 1H, NH D₂O exchangeable),

8.45(s, 1H, ArH), 8.07 (d, J ¼ 9.8 Hz, 1H), 7.82 (d, J ¼ 8.2 Hz, 1H),

7.54 (d, J ¼ 9.8 Hz, 1H), 7.36 (d, J ¼ 8.2 Hz, 1H), 3.78 (d, J ¼ 4.2 Hz,

4H, morpholinyl H), 3.69 (d, J ¼ 4.2 Hz, 4H, morpholinyl H), 3.52(s,

3H, OCH₃).

exchangeable, pyridazinyl NH), 9.19 (s, 1H, D₂O exchangeable, NH

urea), 8.93 (s, 1H, D₂O exchangeable, NH urea), 8.01–7.92(m, 2H,

ArH), 7.71 – 7.52 (m, 3H, ArH), 7.49 (s, J ¼ 8.8 Hz, 1H, pyridazinyl

H), 7.30 – 7.13 (m, 2H, ArH), 6.99 (d, J ¼ 8.8 Hz, 1H, pyridazinyl H).

.FT-IR (ύ max, cm²¹): 3288–3297 (NH), 3118 (CH aromatic),

2994 (CH aliphatic), 1677 (C ¼ O amide), 1631 (C ¼ N); MS:(Mwt.:

374.21) m/z (% rel. Int.) 375.32 (M^þþ1, 5.42), 374.32 (M^þ, 8.94),

151 (100) Anal. Calcd for C₁₇H₁₂Cl₂N₄O₂: C, 54.42; H, 3.22; N, 14.93.

Found: C, 54.43; H, 3.27; N, 14.82.

1-[4-Chloro-3-(trifluoromethyl)phenyl)-3–(3-(6-oxo-1,6-dihy-

dropyridazin-3-yl)phenyl]urea (8e)

FT-IR (ύ max, cm²¹): 3301–3309 (NH), 3130 (CH aromatic),

2988 (CH aliphatic), 1686 (C ¼ O amide), 1643 (C ¼ N); ¹³C NMR

(DMSO-d6, 100 MHz) d: 47.4 (2CH₂morpholine), 66.1 (2CH₂mor-

pholine), 120.4, 124.0, 125.0, 126.3, 128.3, 128.9, 131.2, 131.9,

139.9, 141.7, 145.1, 155.4, 163. MS: (Mwt.: 369.38) m/z (% rel.

Int.)371.42 (M^þþ2, 5.34), 270.39 (M^þþ1, 2.21), 368.38 (M^þ, 10.45),

152 (100); Anal. Calcd for C₁₇H₁₉N₇O₃C, 55.28; H, 5.18; N, 26.54.

Found: C, 55.32; H, 5.19; N, 26.64.

Crystallised from THF as yellowish crystals (1.02 g., 42%) m.p.:

198–200 ^ꢀC. ¹HNMR (300 MHz, DMSO-d₆d 12.22 (s, 1H, D₂O

exchangeable, pyridazinyl NH), 8.99 (s, 1H, D₂O exchangeable, NH

urea), 8.54 (s, 1H, D₂O exchangeable, NH urea),8.32 (s, 1H, ArH),

7.97 (d, J ¼ 9.9 Hz, 1H, pyridazinyl H), 7.40 (m, 1H, ArH), 7.23 – 7.11

(m, 2H, ArH), 6.94 (m, 3H, ArH), 6.82 (d, J ¼ 9.9 Hz, 1H, pyridazinyl

H). FT-IR (ύ max, cm²¹): 3302 (NH), 3028 (CH aromatic), 2942 (CH

aliphatic), 1690 (C ¼ O amide), 1643 (C ¼ N); MS: (Mwt.: 408.72) m/

z (% rel. Int.) 410.72(M^þþ2, 6.23), 411.79, 408.81 (M^þ, 18.65), 194

(100); Anal. Calcd for C₁₈H₁₃F₃N₄O₂C, 57.76; H, 3.50; N, 14.97.

Found: C, 57.82; H, 3.62; N, 14.86.

1-[3,4-Dichlorophenyl)-3–(4-(6-oxo-1,6-dihydropyridazin-3-yl)

phenyl)]urea (8a)

Crystallised from isopropanol as yellowish crystals(1.12g., 50%)

1

m.p.: 232–235 ^ꢀC. HNMR (300 MHz, DMSO-d₆d 12.25 (s, 1H, D₂O

1-[(3-Methoxyphenyl)-3–(3-(6-oxo-1,6-dihydropyridazin-3-yl)

phenyl)]urea (8f)

exchangeable, pyridazinyl NH), 9.19 (s, 1H, D₂O exchangeable, NH

urea), 8.93 (s, 1H,D₂O exchangeable, NH urea), 8.14(s, 1H, ArH),

7.71 (dd, J ¼ 8.8 Hz, 2H, ArH), 7.56(d, J ¼ 9.9 Hz, 1H, pyridazinyl H),

7.49 – 7.30 (m, 2H, ArH), 7.13 (dd, J ¼ 8.8 Hz, 2H, ArH), 6.99 (d,

J ¼ 9.9 Hz, 1H, pyridazinyl H).MS: (Mwt.: 374.03); FT-IR (ύ max,

cm²¹) 3286–3300 (NH), 3125 (CH aromatic), 1690 (C ¼ O amide),

1640 (C ¼ N); Anal. Calcd for C₁₇H₁₂Cl₂N₄O₂: C, 54.42; H, 3.22; N,

14.93; Found: C, 54.65; H, 3.43; N, 14.98.

Crystallised from THF as pale yellow crystals(0.37g., 71%) m.p.:

221–223 ^ꢀC. ¹HNMR (300 MHz, DMSO-d₆d 13.01 (s, 1H, D₂O

exchangeable, pyridazinyl NH), 8.89 (s, 1H, D₂O exchangeable, NH

urea), 8.67 (s, 1H, D₂O exchangeable, NH urea), 7.99 (s, 1H, ArH),

7.86 (d, J ¼ 9.3 Hz, 1H, pyridazinyl H), 7.45 – 7.23 (m, 3H, ArH), 7.08

(m, 3H, ArH), 6.92 (d, J ¼ 9.3 Hz, 1H, pyridazinyl H), 3.63(s, 3H,

OCH₃) 13 C NMR (DMSO-d6, 100 MHz) d:119.8, 120.4, 123, 124,

124.1, 125, 125.1, 126.3, 130.1, 131.2, 131.9, 134.3, 137.4, 139.7,

139.9, 143.9, 154.1, 160.8. FT-IR (ύ max, cm²¹): 3302 (NH), 3038

(CH aromatic), 2945 (CH aliphatic), 1682 (C ¼ O amide), 1625

(C ¼ N); MS: (Mwt.: 336.34); Anal. Calcd for C₁₈H₁₆N₄O₃C, 64.28; H,

4.79; N, 16.66. Found: C, 64.42; H, 4.91; N, 16.72.

1-[(4-Chloro-3-(trifluoromethyl)phenyl)-3–(4-(6-oxo-1,6-dihy-

dropyridazin-3-yl)phenyl)]urea (8b)

Crystallised from THF as orange crystals (1.12 g., 50%) m.p.:

245–246 ^ꢀC. ¹HNMR (300 MHz, DMSO-d₆d 12.35 (s, 1H,D₂O

exchangeable, pyridazinyl NH), 9.22 (s, 1H, D₂O exchangeable, NH

urea), 8.89 (s, 1H,D₂O exchangeable, NH urea), 8.45(s, 1H, ArH),

7.54 (dd, J ¼ 8.3 Hz, 2H, ArH), 7.32(d, J ¼ 9 Hz, 1H, pyridazinyl H),

7.25 – 7.19 (m, 2H, ArH), 7.05 (dd, J ¼ 8.3 Hz, 2H, ArH), 6.89(d,

J ¼ 9 Hz, 1H, pyridazinyl H).

1-[3-((6-Chloropyridazin-3-yl)amino)phenyl)-3–(3,4-dichloro-

phenyl)]urea (11a)

Crystallised from THF to give compound (XXXa) as white crys-

tals (0.8 g, 20%); m.p. 238–240 ^ꢀC; ¹HNMR (300 MHz, DMSO-d₆) d

9.98(s, 1H, NH D₂O exchangeable, NH urea), 9.77(s, 1H, NH D₂O

exchangeable, NH urea), 8.99 (s, 1H, NH D₂O exchangeable, NH

Aryl), 8.74 (s, 1H, ArH), 7.84 (d, J ¼ 9.3 Hz, 1H, pyridazinyl H), 7.61 –

7.45 (m, 2H, ArH), 7.38 (t, J ¼ 6.4 Hz, 1H, ArH), 7.29 (d, J ¼ 6.4 Hz,

1H, ArH), 7.18– 7.02 (m, 2H, ArH), 6.89 (d, J ¼ 9.3 Hz, 1H, pyrida-

zinyl H).

FT-IR (ύ max, cm²¹): 3299–3310 (NH), 3038 (CH aromatic),

2915 (CH aliphatic), 1682 (C ¼ O amide), 1633 (C ¼ N); MS:(Mwt.:

408.76) m/z (% rel. Int.) 410.76 (M^þþ2, 4.08), 411.79 (M^þþ1, 3.31),

408.81(M^þ, 11.99), 194 (100); Anal. Calcd for C₁₈H₁₂ClF₃N₄O₂: C,

52.89; H, 2.96; N, 13.71; Found: C, 52.72; H, 3.01; N, 13.85.

1-[4–(6-Oxo-1,6-dihydropyridazin-3-yl)phenyl)-3–(3-(methoxy)

phenyl)]urea (8c)

FT-IR (ύ max, cm²¹): 3347–3325 (NH), 3117 (CH aromatic),

2994 (CH aliphatic), 1682 (C ¼ O amide), 1644 (C ¼ N); MS: (Mwt.:

408.67) Anal. Calcd for C₁₇H₁₂Cl₃N₅O: C, 49.96; H, 2.96; N, 17.14.

Found: C, 49.98; H, 2.87; N, 17.25.

4-[Chloro-3-(trifluoromethyl)phenyl)-3–(3-((6-chloropyridazin-

3-yl)amino)phenyl)]urea (11b)

Crystallised from ethanol as buff crystals (1.35 g, 67%) m.p.:

201–203^ꢀ. NMR (300 MHz, DMSO-d₆d 12.45 (s, 1H, D₂O exchange-

able, pyridazinyl NH), 9.21 (s, 1H, D₂O exchangeable, NH urea),

8.89 (s, 1H, D₂O exchangeable, NH urea), 7.46 (d, J ¼ 8.2 Hz, 1H,

pyridazinyl H), 7.37 (dd, J ¼ 9.4 Hz, 2H, ArH), 7.25–7.09 (m, 2H,

ArH), 6.99 (dd, J ¼ 9.4 Hz, 2H, ArH), 6.87 (d, J ¼ 8.2 Hz, 1H, pyrida-

zinyl H), 3.52(s, 3H, OCH₃). ¹³C NMR (DMSO-d6, 100 MHz)d:118.7,

119.7, 122.1, 125.1, 126.3, 127.4, 127.4, 130.4, 132.3, 132.6, 134.3,

137.2, 139.9, 140.1, 154.1,160.8. FT-IR (ύ max, cm²¹): 3293 (NH),

Crystallised from methanol to give compound (XXXb) as buff

1

crystals (1.1 g, 25%); m.p. >250 ^ꢀC; HNMR (300 MHz, DMSO-d₆) d

10.12 (s, 1H, NH D₂O exchangeable, NH urea), 9.86(s, 1H, NH D₂O

exchangeable, NH urea), 8.87 (s, 1H, NH D₂O exchangeable, NH

3089 (CH aromatic), 2954 (CH aliphatic), 1685 (C ¼ O amide), 1638 Aryl), 8.65 (s, 1H, ArH), 8.12 (d, J ¼ 9 Hz, 1H, pyridazinyl H), 7.56 –

1584

M. Y. JABALLAH ET AL.

7.43 (m, 2H, ArH), 7.38 (d, J ¼ 8.4 Hz, 1H, ArH), 7.29 (d, J ¼ 8.4 Hz,

1H, ArH), 7.06– 6.99 (m, 2H, ArH), 6.9(d, J ¼ 9 Hz, 1H, pyrida- zin-3-yl)amino) phenyl) urea 15

Crystallised from isopropanol giving compound XXXVII (1.5 g,

1–(4-Chloro-3-(trifluoromethyl)phenyl)-3–(4-((6-methoxypyrida-

zinyl H).

FT-IR (ύ max, cm²¹): 3338–3314 (NH), 3051 (CH aromatic), 60%); m.p. (>250 ^ꢀC).

¹HNMR (300 MHz, DMSO-d₆) d 9.97(s, 1H, NH D₂O exchange-

able, NH urea), 9.09(s, 1H, NH D₂O exchangeable, NH urea), 8.95(s,

1H, NH D₂O exchangeable, NH Aryl), 8.68(s, 1H, ArH), 8.11(d,

J ¼ 9.2 Hz, 1H, pyridazinyl H), 7.64(dd, J ¼ 9.6 Hz, 2H, ArH),

7.37–7.19(m, 2H, ArH), 7.08 (dd, J ¼ 9.6 Hz, 2H, ArH), 6.99 (d,

J ¼ 9.2 Hz, 1H, pyridazinyl H), 3.92 (s, 3H, OCH₃).

2981 (CH aliphatic), 1664 (C ¼ O amide), 1664 (C ¼ N); MS: (Mwt.:

442.22); m/z (% rel. Int.) 444.24 (M^þþ2, 6.40), 442.35 (M^þ, 31.12),

168.27(100) Anal. Calcd for C₁₈H₁₂Cl₂F₃N₅O: C, 48.89; H, 2.74; N,

15.84. Found: C, 48.92; H, 2.83; N, 15.92.

1-[(3-((6-Chloropyridazin-3-yl)amino)phenyl)-3–(3,5-dimethox-

yphenyl)]urea (11c)

Crystallised from methanol to give compound (XXXc) as

yellowish white crystals (1.39 g, 35%); m.p. 195–197 ^ꢀC; ¹HNMR

(300 MHz, DMSO-d₆) d 10.23 (s, 1H, NH D₂O exchangeable, NH

urea), 9.76(s, 1H, NH D₂O exchangeable, NH urea), 9.25 (s, 1H, NH

D₂O exchangeable, NH Aryl), 8.74 (s, 1H, ArH), 8.56 (d, J ¼ 10.4 Hz,

1H, pyridazinyl H), 8.46 (d, J ¼ 8.2 Hz, 1H, ArH), 8.25 – 8.03 (m, 2H,

ArH), 7.89 (d, J ¼ 8.2 Hz, 1H, ArH), 7.76– 7.53 (m, 2H, ArH), 7.24(d,

J ¼ 10.4 Hz, 1H, pyridazinyl H), 3.65(s, 3H, OCH₃), 3.47(s, 3H, OCH₃).

FT-IR (ύ max, cm²¹): 3347–3357 (NH), 3117 (CH aromatic),

2994 (CH aliphatic), 1682 (C ¼ O amide), 1644 (C ¼ N); MS:(Mwt.:

399.08):m/z (% rel. Int.) 401.11(M^þþ2, 1.39), 399.08(M^þ, 4.18),

219.12 (100); Anal. Calcd for C₁₉H₁₈ClN₅O₃: C, 57.07; H, 4.54; N,

17.52; Found: C, 57.12; H, 4.58; N, 17.59.

FT-IR (ύ max, cm²¹): 3347 (NH), 3117 (CH aromatic), 2994 (CH

aliphatic), 1682 (C ¼ O amide), 1644 (C ¼ N); (Mwt.: 437.80) m/z (%

rel. Int.) 439.89 (M^þþ2, 10.64), 437.82(M^þ, 3.23), 366.10 (41.23),

215.09 (100).Anal. Calcd for C₁₉H₁₅ClF₃N₅O₂C, 52.12; H, 3.45; N,

16.00. Found: C, 52.34; H, 3.52; N, 16.07.

1–(3,4-Dichlorophenyl)-3–(4-((6-oxo-1,6-dihydropyridazin-3-yl)oxy)

phenyl)urea 18a

Crystallised from methanol as yellowish white crystals (1.1 g,

50%); m.p. 189–190 ^ꢀC; ¹HNMR (300 MHz, DMSO-d₆) d 12.25 (s,

1H, D₂O exchangeable, pyridazinyl NH), 9.19 (s, 1H, D₂O exchange-

able, NH urea), 8.93 (s, 1H, D₂O exchangeable, NH urea), 7.82 (d,

J ¼ 9.2 Hz, 1H, pyridazinyl H), 7.71–7.62 (m, 2H, ArH), 7.40 (dd,

J ¼ 8.3 Hz, 2H, ArH), 7.35(dd, J ¼ 8.3 Hz, 2H, ArH), 7.23 – 7.11 (m,

1H, ArH), 6.82 (d, J ¼ 9.9 Hz, 1H, pyridazinyl H).

1-[4-((6-Chloropyridazin-3-yl)amino)phenyl)-3–(3,4-dichloro-

phenyl]urea (11d)

FT-IR (ύ max, cm²¹): 3288–3296 (NH), 3047 (CH aromatic),

2990 (CH aliphatic), 1685 (C ¼ O amide), 1632 (C ¼ N); MS: (Mwt.:

391.21); Anal. Calcd for C₁₇H₁₂Cl₂N₄O₃C, 52.19; H, 3.09; N, 14.32.

Found: C, 52.23; H, 3.17; N, 14.45.

Crystallised from ethanol(0.8 g, 20%); m.p. 252–254 ^ꢀC; ¹HNMR

(300 MHz, DMSO-d₆) d 10.02 (s, 1H, NH D₂O exchangeable, NH

urea), 9.64(s, 1H, NH D₂O exchangeable, NH urea), 9.46 (s, 1H, NH

D₂O exchangeable, NH Aryl), 8.46 (s, 1H, ArH), 8.32 (d, J ¼ 9.2 Hz,

1H, pyridazinyl H), 8.15 (dd, J ¼ 8.8 Hz, 2H, ArH), 8.01–7.87 (m, 2H,

ArH), 7.56 (dd, J ¼ 8.8 Hz, 2H, ArH), 7.24(d, J ¼ 9.2 Hz, 1H, pyrida-

zinyl H).

1–(4-Chloro-3-(trifluoromethyl)phenyl)-3–(4-((6-oxo-1,6-dihydro-

pyridazin-3-yl)oxy)phenyl)urea 18b

Crystallised from ethanol as white crystals (1.1 g, 50%); m.p.

201–203 ^ꢀC; ¹HNMR (300 MHz, DMSO-d₆) d 12.02 (s, 1H, D₂O

exchangeable, pyridazinyl NH), 9.45 (s, 1H, D₂O exchangeable, NH

urea), 8.72 (s, 1H, D₂O exchangeable, NH urea), 8.32 (s, 1H, ArH),

8.02 (d, J ¼ 9.2 Hz, 1H, pyridazinyl H), 7.30 (dd, J ¼ 10.2, 8.3 Hz, 2H,

ArH), 7.21(dd, J ¼ 10.2, 8.3 Hz, 2H, ArH), 7.15 – 7.06 (m, 2H, ArH),

6.99 (d, J ¼ 9.9 Hz, 1H, pyridazinyl H).

FT-IR (ύ max, cm²¹): 3299–3312 (NH), 3040 (CH aromatic),

2980 (CH aliphatic), 1683 (C ¼ O amide), 1638 (C ¼ N); MS: (Mwt.:

408.67); Anal. Calcd for C₁₇H₁₂Cl₃N₅O: C, 49.96; H, 2.96; N, 17.14.

Found. C, 49.99; H, 2.98; N, 17.16.

1-[4-Chloro-3-(trifluoromethyl)phenyl)-3–(4-((6-chloropyrida-

zin-3-yl)amino)phenyl)]urea (11e)

FT-IR (ύ max, cm²¹): 3285–3297 (NH), 3129 (CH aromatic),

2979 (CH aliphatic), 1692 (C ¼ O amide), 1635 (C ¼ N); MS: (Mwt.:

424.06) m/z (% rel. Int.) 426.06 (M^þþ2, 2.55), 427.11 (M^þþ1, 6.56),

424.06 (M^þ, 7.68), 198 (100); Anal. Calcd for C₂₀H₁₈ClF₃N₄O₃C,

50.90; H, 2.85; N, 13.19; Found: C, 50.98; H, 2.87; N, 13.20.

1–(3-Methoxyphenyl)-3–(4-((6-oxo-1,6-dihydropyridazin-3-yl)oxy)-

phenyl)urea 18c.

Crystallised from acetonitrile (1.1 g, 25%); m.p. 149–151 ^ꢀC;

¹HNMR (300 MHz, DMSO-d₆) d 9.77(s, 1H, NH D₂O exchangeable,

NH urea), 9.54 (s, 1H, NH D₂O exchangeable), 9.46 (s, 1H, NH D₂O

exchangeable, NH Aryl), 8.74(s, 1H, ArH), 8.15 (dd, J ¼ 8.9 Hz, 2H,

ArH), 7.84(d, J ¼ 9.3 Hz, 1H, pyridazinyl H), 7.61 – 7.45 (m, 2H, ArH),

7.38(dd, J ¼ 8.9 Hz, 2H, ArH), 7.12 (d, J ¼ 9.3 Hz, 1H, pyridazinyl H).

FT-IR (ύ max, cm²¹): 3291 (NH), 3110 (CH aromatic), 2982

(CH aliphatic), 1690 (C ¼ O amide), 1645 (C ¼ N); MS:(Mwt.: 442.22);

m/z (% rel. Int.) 444.34(M^þþ2, 4.32), 442.26(M^þ, 4.18), 168.56(100)

Anal. Calcd for C₁₈H₁₂Cl₂F₃N₅O: C, 48.89; H, 2.74; N, 15.84. Found:

C, 48.95; H, 2.82; N, 15.89.

Crystallised from acetonitrile as yellowish white crystals (1.1 g,

50%); m.p. 156–157 ^ꢀC; ¹HNMR (300 MHz, DMSO-d₆) d 12.05 (s,

1H, D₂O exchangeable, pyridazinyl NH), 9.09 (s, 1H, D₂O exchange-

able, NH urea), 8.87 (s, 1H, D₂O exchangeable, NH urea), 7.85 (d,

J ¼ 9.2 Hz, 1H, pyridazinyl H), 7.72–7. 65(m, 2H, ArH), 7.40 (dd,

J ¼ 8.3 Hz, 2H, ArH), 7.25(dd, J ¼ 8.3 Hz, 2H, ArH), 7.13 – 7.01 (m,

1H, ArH), 6.22 (d, J ¼ 9.2 Hz, 1H, pyridazinyl H).3.52(s, 3H, OCH₃).

FT-IR (ύ max, cm²¹): 3314 (NH), 3141 (CH aromatic), 2981 (CH

aliphatic), 1661 (C ¼ O amide), 1661 (C ¼ N); MS: (Mwt.: 352.12);

Anal. Calcd for C₁₈H₁₆N₄O₄C, 61.36; H, 4.58; N, 15.90. Found: C,

61.42; H, 4.61; N, 15.95.

1-[(4-((6-Chloropyridazin-3-yl)amino)phenyl)-3–(3,5-dimethox-

yphenyl)]urea (11f)

Crystallised from methanol/water (0.59 g, 15%); m.p.

203–205 ^ꢀC; ¹HNMR (300 MHz, DMSO-d₆) d 10.01(s, 1H, NH D₂O

exchangeable, NH urea), 9.77 (s, 1H, NH D₂O exchangeable), 9.32

(s, 1H, NH D₂O exchangeable, NH Aryl), 8.45(s, 1H, ArH), 8.32 (dd,

J ¼ 8.8 Hz, 2H, ArH),7.76(d, J ¼ 9.3 Hz, 1H, pyridazinyl H), 7.54 – 7.32

(m, 2H, ArH), 7.21(dd, J ¼ 8.8 Hz, 2H, ArH), 6.89 (d, J ¼ 9.3 Hz, 1H,

pyridazinyl H), 3.57(s, 3H, OCH₃), 3.54(s, 3H, OCH₃)

4.2. Biological evaluation

FT-IR (ύ max, cm²¹): 3310–3292 (NH), 3120 (CH aromatic),

2924 (CH aliphatic), 1690 (C ¼ O amide), 1644 (C ¼ N); MS:(Mwt.:

399.11) m/z (% rel. Int.) 401.16 (M^þþ2, 1.39), 399.09(M^þ, 4.18),

219.23 (100); Anal. Calcd for C₁₉H₁₈Cl₂N₅O₃: C, 57.07; H, 4.54; N,

17.52. Found: C, 57.21; H, 4.59; N, 17.62

4.2.1. Measurement of inhibitory activity against VEGFR-2

The kinase activity of VEGFR-2 was measured by use of a phos-

photyrosine antibody with the

Alpha Screen system (PerkinElmer, United States) according to

manufacturer’s instructions. Enzyme reactions were performed in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1585

50 mM Tris–HCl pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01% Tween- 4.2.3. In vitro cytotoxic activity

The cytotoxicity assays were performed at NCI, Bethesda, United

States (against 56 cell lines). The human tumour cell lines of the

cancer screening panel were grown in RPMI 1640 medium con-

taining 5% foetal bovine serum and 2 mM L-glutamine. For a typ-

ical screening experiment, cells were inoculated into 96 well

microtiter plates in 100 l at plating densities ranging from 5000

to 40,000 cells/well depending on the doubling time of individual

cell lines. After cell inoculation, the microtiter plates were incu-

bated 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 (Tz).

20 and 2 mM DTT, containing 10 lM ATP, 0.1 lg/mL biotinylated

poly-GluTyr (4:1), and 0.1 nM of VEGFR-2 (Millipore, United

Kingdom). Prior to catalytic initiation with ATP, the tested com-

pounds at final concentrations ranging from 0 to 100 lg/mL and

enzyme were incubated for 5 min at room temperature. The reac-

tions were quenched by the addition of 25 lL of 100 mM EDTA,

10 lg/mLAlpha Screen streptavidine donor beads and 10 lg/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, United States). Wells containing the

substrate and the enzyme without compounds were used as reac-

tion control. Wells containing biotinylated poly-GluTyr (4:1) and

enzyme without ATP were used as basal control. Percent inhib-

ition 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 concentra-

tion–inhibition response curve (triplicate determinations) and the

data were compared with Sorafenib as standard VEGFR-2 inhibitor.

Experimental drugs were solubilised 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 fro-

zen concentrate was thawed and diluted to twice the desired final

maximum test concentration with complete medium containing

1

50 lg/ml gentamicin. Additional four, 10-fold or = log serial dilu-

2

tions were made to provide a total of five drug concentrations

plus control. Aliquots of 100 ll of these different drug dilutions

were added to the appropriate microtiter wells already con-

taining100 ll of medium, resulting in the required final drug con-

4.2.2. In vitro HUVEC anti-proliferative assay

The assay was performed at single dose concentration of 10 mM,

where HUVEC, human (Life Technologies # C-003-5C) served as centrations. Triplicate wells were prepared for each individual

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

additional 48 h at 37 oC, 5% CO2, 95% air, and 100% relative

humidity. For adherent cells, the assay was terminated by the add-

ition of cold TCA. Cells were fixed in situ by the gentle addition of

50 ll of cold 50% (w/v) TCA (final concentration, 10% TCA) and

incubated for 60 min at 4 ^ꢀC. The supernatant was discarded, and

the plates were washed five times with tap water and air dried.

SRB solution (100 ll) at 0.4% (w/v) in 1% acetic acid was added to

each well, and plates were incubated for 10 min at room tempera-

ture. After staining, unbound dye was removed by washing five

times with 1% acetic acid and the plates were air dried. Bound

stain was subsequently solubilised 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 ll of 80%

TCA (final concentration, 16% TCA). Using the seven absorbance

measurements [time zero (T_z), control growth (C), and test growth

in the presence of drug at the five concentration levels (Ti)], the

percentage growth was calculated at each of the drug concentra-

tion levels. Percentage GI was calculated as: [(Ti – Tz)/(C –

Tz)] ꢃ 100 for concentrations for which Ti ꢄ Tz [(Ti – Tz)/Tz] ꢃ 100

for concentrations for which Ti < Tz.

the cells’ source, in Medium 200 (Life Technologies # M-200–500),

with large vessel endothelial supplement (LVES) (Life Technologies

# A14608-01) and Pen-strep (Hyclone # SV30010). Alamar Blue

(Life Technologies

cent reagent.

# DAL1025) was used as the fluores-

HUVEC cells were cultured in Medium 200 with 2% LVES and

1% Pen-strep. To perform the proliferation assay, HUVEC cells

were seeded at 5000 cells/50 ll/well in a 96-well black clear bot-

tom tissue culture plate. Cells were incubated at 37 ^ꢀC and 5%

CO2 overnight to allow them to recover and reattach. Next day

cells were treated with test compounds for 72 h. After treatment,

cell proliferation was measured by Fluorescent quantitation of ala-

mar Blue reagent. The alamar Blue assay incorporates a fluoromet-

ric/colorimetric growth indicator based on detection of metabolic

activity. Specifically, resazurin, the active ingredient in the alamar

Blue reagent, is blue in colour and virtually non-fluorescent. Upon

entering cells, resazurin is reduced to resorufin, a compound that

is red in colour and highly fluorescent. Continued cell growth

maintains a reduced environment, therefore increasing the overall

fluorescence and colour of the media surrounding cells. The fluor-

escence intensity of alamar Blue reagent was shown to be directly

proportional to cell number. To perform the alamar Blue assay,

10 mM of alamar Blue reagent was added to each well and the

plate was incubated at 37 ^ꢀC for an additional 2 h. Fluorescence

intensity was measured at an excitation of 530 nm and an emis-

sion of 590 nm using a BioTek SynergyTM 2 microplate reader.

Cell proliferation assays were performed in triplicate at each

concentration. The fluorescent intensity data were analysed using

the computer software, Graphpad Prism. In the absence of the

compound, the fluorescent intensity (Ft) in each data set was

defined as 100%. In the absence of cells, the fluorescent intensity

(Fb) in each data set was defined as 0%. The percent cell in the

presence of each compound was calculated according to the fol-

lowing equation: % Cell ¼ (F – Fb)/(Ft – Fb), where F ¼ the fluor-

escent intensity in the presence of the compound, Fb ¼ the

fluorescent intensity in the absence of cells, and Ft ¼ the fluores-

cent intensity in the absence of the compound.

Three dose response parameters were calculated for each

experimental compound: GI of 50% (GI50) was calculated when

[(Ti – Tz)/(C – Tz)] ꢃ 100 ¼ 50.

4.3. Methods and computational details

4.3.1. Protein and ligand preparation

The atomic coordinates of the kinase domain of VEGFR-2 co-crys-

tallised with the FDA approved competitive kinase inhibitor,

Sorafenib⁴³, were retrieved from the protein data bank (PDB ID:

3WZE)⁴⁴. The missing non-terminal segments of the protein were

built using MODELLER⁴⁵using the standard protocol. The struc-

ture with the lowest z-DOPE score was selected for further struc-

tural investigation and simulation. The z-DOPE score is

a

normalised atomic distance-dependent statistical potential based

1586

M. Y. JABALLAH ET AL.

on known protein structures. The selected structure was further the Particle Mesh Ewald method⁵⁵. The cutoff distance for the

prepared using the protein preparation wizard in Maestro⁴⁶. In

electrostatic and vdW energy term was set at 9.0 Å. To avoid edge

protein preparation, original hydrogen atoms, if any, were deleted

and new hydrogen atoms were added. This was followed by

adjusting the bond orders for the amino acid residues and the lig-

and. In adding hydrogen atoms, the protonation states of titrable

residues were assigned at pH of 7 using PROPKA51. Finally, a

restrained minimisation with convergence of heavy atoms to an

RMSD of 0.3 Å was performed utilising the OPLS3⁴⁷force-

field parameters.

effects in all calculations, Periodic boundary conditions were

applied. To allow for an integration time step of 2 fs, the SHAKE

algorithm⁵⁶was applied for all bonds involving hydrogen atoms.

Trajectory frames were recorded every 4 ps throughout all produc-

tion runs. All analysis script was written in python and bash and

performed mainly through the CPPTRAJ utility in AMBER⁵⁷.

4.3.4. The AMBER/MM-GBSA binding energy calculation

For the ligand preparation step, the chemical structures of 11c

and 18b were drawn in Maestro. The structures were prepared

through the ligprep module of Schrodinger⁴⁸. Ligand preparation

includes adding missing hydrogen atoms and assigning proper

The final 25 ns MD simulation trajectory was used to estimate the

free binding energy using the AMBER/MM-GBSA method⁵⁸. A total

number of 3101 frames with an 8 ps interval were selected to per-

bond orders and protonation states at pH 7. This was followed by form the binding energy analysis. All waters and counter ions

energy minimisation of the compounds in the OPLS3 force field.

were stripped prior to carrying out the binding energy calcula-

tions. The MM-GBSA protocol was carried out similarly for all pro-

tein-ligand complexes. In summary, the MMG-GBSA protocol

approximates the free binding energy (DG_bind) as follow:

4.3.2. Docking and scoring simulations

Three molecules were selected for molecular docking simulations;

namely, Sorafenib, compound 11c, and compound 18b. The

molecular docking simulations were performed using Smina, a ver-

sion of AutoDock Vina optimised to support high-throughput

minimisation and scoring, and also provides enhanced control

over docking parameters^49,50. The docking search space was cen-

tred on the centre of mass of the original ligand of the 3WZE

structure, Sorafenib, and extended to 20 Å ꢃ 20 Å ꢃ 20 Å box

dimension. The best 5 poses for each ligand were rescored

through three different scoring functions, including NNscore 2⁵¹,

RFscore⁵², and DLSCORE⁵³. For each ligand, the pose that showed

the smallest heavy atoms RMSD compared to the co-crystallised

ligand, Sorafenib was selected to carry out the MD simulation and

binding energy calculations.

DG_bind¼ G_complex–ðG_proteinþ G_ligand

DG_bind¼ DE_MMþ DG_solv–TDS

Þ

¼ DE_vdWþ DE_ELEþ DG_polþ DG_nonpol–TDS

In the previous formulas, G_complexis the protein-ligand complex

free energy, G_proteinis the protein free energy, and G_ligandis the

ligand free energy. DG_bindis approximated by summing the

changes in the MM (DE_MM) energy, the solvation energy (DG_solv),

and the entropic contributions (TDS). DE_MMis further divided by

lipophilic vdW (DE_vdW) and electrostatic (DE_ELE) components. The

solvation energy (DG_solv) is further divided into polar (DG_pol) and

non-polar (DG_nonpol) contributions. The vdW (DE_vdW) and electro-

static (DE_ELE) components were extracted from the MD simulation

trajectory using the same forcefield parameters. The polar compo-

nent (DG_pol) of the solvation energy was calculated using the

GBSA module of AMBER. The non-polar component was estimated

as DG_nonpol¼ cSASA þ b, SASA is the solvent-accessible surface

area, c and b are two constants that were set to 0.0072 kcal/mol/Å

and 0.0 kcal/mol, respectively. For practical reasons, the last term

(TDS) was ignored.

4.3.3. Classical molecular dynamics simulations

All MD simulations were carried out in AMBER14 software package

for the ligand pose that showed the smallest RMSD value to the

co-crystallised ligand (see above). Each complex was immersed in

a cubic box of TIP3P water model and neutralised by counter

ions. Final salt concentration was brought to 150 mM NaCl con-

centration to mimic physiological conditions. Each solvated system

was minimised through four steps: first, a harmonic constraint

potential of 100 kcal/mol/Å²was applied to all non-hydrogen

atoms of the protein and the ligand. In the following minimisation

stages, the restrains were reduced to 50, 5, and 0 kcal/mol/Å². In

each step, minimisation was performed by the steepest descent

method for the first 1000 steps and the conjugated gradient

method for the subsequent 4000 steps. Each system was then

gradually heated in the NVT ensemble from 0 to 300 K in 100 ps

using a Langevin thermostat with a coupling coefficient of 1.0/ps

with a force constant 5.0 kcal/mol/Å²on the atoms of the protein

and the ligand, using a 1 fs integration time step. And then add-

itional two rounds of MD (50 ps each at 300 K) were performed

with decreasing protein/ligand heavy atoms restraint weights

from 1, 0.5 to 0.1 kcal/mol/Å². Finally, each system was again equi-

librated for 1000 ps by releasing all restrains. Actual MD produc-

tion run was performed for 6 ꢃ 5 ns in the NPT ensemble for each

system giving a total production simulation of 30 ns. The first 5 ns

was discarded from our analysis and considered as a final equili-

bration phase. All equilibration and production simulations were

performed at 300 K with Berendsen temperature coupling⁵⁴and

1 atm with isotropic molecule-based scaling and a 2fs integration

4.3.5. Relaxed PES scan

The three-dimensional structure of compounds 11c and 18b were

superposed on top of the bioactive, co-crystallised conformation

of Sorafenib. Upon docking, we observed that the majority of the

generated docked poses of 18b indeed suggests that this con-

formation as the optimal binding pose of 18b. For compound

11c, however, none of the generated poses exhibited this con-

formation. At this stage, it was not clear whether this was a short-

coming from the sampling algorithm of the docking or simply

because this pose was very energetically unfavourable. This may

explain the low activity of compound 11c even though it bears

the required H-bond acceptor site at the terminal hetero-aromatic

ring. To investigate this effect in more details, we ran a relaxed

2D PES scan for the terminal ring torsion depicted in Figure 6 (C-

C-X-C, where X is an O atom in SORA and 18b, and N in 11c).

Performing a PES scan at the MM level of theory is impractical

given the inherent approximations at this level of theory.

Alternatively, we decided to perform the PES scan at the QM level

of theory using a Density Functional Theory Based model. The

starting conformation for the PES scans for all ligands was the

Sorafenib bound like ligand conformation. PES scan was per-

ꢁ

time step. Long-range Coulombic interactions were handled using formed at the B3LYP/6–31 þ G level of theory, as employed in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1587

Gaussian 16 program package⁵⁹. Selected torsional angle was

rotated by 360^ꢀover 36 scan steps; i.e. 10^ꢀincrement for each

step. For each ligand, identified minima on the PES were further

Funding

This work was funded by STDF (project no. 5251).

ꢁꢁ

optimised at the M062X/6–311þþG level of theory. Frequency

ꢁꢁ

calculations at the same model (M062X/6–311þþG ) showed

that all frequencies are positive, which indicates that the identified

minima are true minima on the PES surface.

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Detailed analysis of the binding modes and low energy con-

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inhibition activity. Further investigations are required to determine

the mechanism of anticancer activity of 11f.

Acknowledgements

The authors are grateful to the Center of Excellence for Drug

Discovery and Development Research at the Faculty of Pharmacy,

Ain Shams University, for performing the NMR spectroscopy of the

compounds. Also, we are thankful to NCI, Maryland, United States

for carrying out cytotoxicity study and BPS bioscience San Diego,

CA, United States for performing enzymatic and HUVEC antiproli-

ferative activity.

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Article Doi

Towards discovery of novel scaffold with potent antiangiogenic activity; design, synthesis of pyridazine based compounds, impact of hinge interaction, and accessibility of their bioactive conformation on VEGFR-2 activities

DOI: 10.1080/14756366.2019.1651723

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Article abstract of DOI:10.1080/14756366.2019.1651723

Full text of DOI:10.1080/14756366.2019.1651723

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