1-Piperazinylphthalazines as potential VEGFR-2 inhibitors and anticancer agents: Synthesis and in vitro biological evaluation

ACCEPTED MANUSCRIPT

Graphical abstract

1

-Piperazinylphthalazines as potential VEGFR-2 inhibitors and anticancer agents:

Synthesis and in vitro biological evaluation

Sahar M. Abou-Seri, Wagdy M. Eldehna, Mamdouh M. Ali , Dalal A. Abou El Ella

Three novel series of phthalazine derivatives based on the1-piperazinyl-4-phenylphthalazine

scaffold were designed and synthesized as potential VEGFR-2 inhibitors and antitumor

agents.

ACCEPTED MANUSCRIPT

-Piperazinylphthalazines as potential VEGFR-2 inhibitors and anticancer

1

agents: Synthesis and in vitro biological evaluation

a

b,

c

d,

Sahar M. Abou-Seri , Wagdy M. Eldehna *, Mamdouh M. Ali , Dalal A. Abou El Ella *

a

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini

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

b

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,

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

c

Biochemistry Department, Division of Genetic Engineering and Biotechnology, National

Research Centre, Dokki 12622, Giza, Egypt.

d

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo,

Abbassia, P.O. Box 11566, Egypt.

Abstract

In our endeavor towards the development of effective VEGFR-2 inhibitors, three novel series

of phthalazine derivatives based on 1-piperazinyl-4-arylphthalazine scaffold were synthesized. All

the newly prepared phthalazines 16a-k, 18a-e and 21a-g were evaluated in vitro for their

inhibitory activity against VEGFR-2. In particular, compounds 16k and 21d potently inhibited

VEGFR-2 at sub-micromolar IC values 0.35±0.03 and 0.40±0.04µM, respectively. Moreover,

5

0

seventeen selected compounds 16c-e, 16g, 16h, 16j, 16k, 18c-e and 21a-g were evaluated for

their in vitro anticancer activity according to US-NCI protocol, where compounds 16k and 21d

proved to be the most potent anticancer agents. While, compound 16k exhibited potent broad

spectrum anticancer activity with full panel GI (MG-MID) value of 3.62 µM, compound 21d

5

0

showed high selectivity toward leukemia and prostate cancer subpanels [subpanel GI₅₀(MG-

MID) 3.51 and 5.15 µM, respectively]. Molecular docking of compounds16k and 21d into

VEGFR-2 active site was performed to explore their potential binding mode.

Keywords: Synthesis; 1-Piperazinylphthalazines; VEGFR-2 inhibitors; Anticancer activity.

*

Corresponding author: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain

Shams University, Cairo, Abbassia, P.O. Box 11566, Egypt. Tel.: +2-012-2274-5745; fax: +2-02-

2

508-0728. E-mail address: dalal@pharma.asu.edu.eg

*

Corresponding author: Department of Pharmaceutical Chemistry, College of Pharmacy,

Egyptian Russian University, Badr City, Cairo, P.O. Box 11829, Egypt. Tel.: +2-02-2864-3342;

+2-1068837640; fax: +2-02-2864-3332. E-mail address: wagdy2000@gmail.com

1

. Introduction

ACCEPTED MANUSCRIPT

Vascular endothelial growth factors (VEGFs) is a family of members that bind in an

overlapping pattern to three diversified but structurally relative VEGF receptors (VEGFRs),

which are transmembrane proteins possessing tyrosine kinase domain (TK). VEGFR-1 (Flt-1) is

critical for hematopoietic cell development, VEGFR-2 (human KDR or murine Flk-1) for vascular

endothelial cell development and angiogenesis, VEGFR-3 (Flt4) for lymphatic endothelial cell

development [1]. VEGFR-2, a 210−230 kDa glycoprotein expressed in vascular endothelial cells,

is the major regulator of VEGF-driven responses in endothelial cells, including proliferation,

migration, tube formation and vascular permeability. VEGFR-2 is considered to be a pivotal

signal transducer in both physiologic and pathologic angiogenesis [2].

Angiogenesis is the process of sprouting or splitting of novel capillary blood vessels from the

quiescent pre-existing vasculature [3]. Angiogenesis is normally orchestrated via a finely

balanced equilibrium between pro-angiogenic and anti-angiogenic factors. However, aberrant

equilibrium between these factors is associated with several human disorders, like rheumatoid

arthritis, psoriasis, diabetic neuropathy and cancer [4]. Mammalian cells need oxygen and

nutrients for their survival and are therefore located within 100-200 µm of blood vessels, the

diffusion limit for oxygen. To grow beyond this size, tumor cells induce angiogenesis through the

secretion of pro-angiogenic growth factors to ensure their oxygen and nutrients [5]. Thence,

therapeutic strategies based on the inhibition of VEGFR-2, in the current medical era, have stood

out as an attractive approach for discovering therapies for many human malignancies. To date,

seven small molecule VEGFR-2 kinase inhibitors (Sorafenib 1 [6], Regorafenib 2 [7], Sunitinib 3

[

8], Pazopanib 4 [9], Vandetanib 5 [10], Axitinib 6 [11] and cabozantinib 7 [12]) have been

approved for clinical use (Figure 1) [13].

In the last two decades, 1,4-disubstituted phthalazine scaffold has emerged as a promising

scaffold for the design and development of potent VEGFR-2 inhibitors. Vatalanib (PTK787) 8

(Figure 2), an 1-anilino-4-substitutedphthalazino derivative brought to light by Novartis and

Schering AG in 1998, is one of the most potent and selective first-generation VEGFR kinase

inhibitors, with IC value of 43 nM against VEGFR-2 [14, 15]. Currently, Vatalanib 8 is in phase

5

0

III clinical trials for the treatment of advanced colorectal cancer [16] and entered phase II studies

for malignant mesothelioma [17], metastatic gastrointestinal stromal tumor [18] and metastatic

melanoma [19]. Subsequently, extensive studies have been devoted to design and synthesis of

different agents with a general structure of 1-anilino-4-substitutedphthalazine as potential

VEGFR-2 inhibitors such as IM-023911 9, 4-(4-((4-(chlorodifluoromethoxy)phenyl)amino)

phthalazin-1-yl)benzamide, with IC₅₀= 190 nM (Figure 2) [16, 20-24]. Unfortunately, these

2

research efforts failed to develop a clinically approved VEGFR-2 inhibitor based on 1-anilino-4-

ACCEPTED MANUSCRIPT

arylphthalazine scaffold.

Moreover, 1,4-disubstituted phthalazines have attracted considerable attention as effective

anticancer agents. Two studies reported the cytotoxicity of novel series of 1-anilino-4-

(arylsulfinyl /sulfonylmethyl)phthalazines 10 (Figure 2) against Bel-7402 (human liver cancer cell

line) and HT-1080 (human fibro sarcoma cell line) [25, 26]. Recently, Ping Gong and his

coworkers explored the in vitro anticancer activity of different 1-piperazinyl-4-

substitutedphthalazine derivatives against A549 (non-small cell lung cancer cell line), HT-29

(human colorectal cancer cell line) and MDA-MB-231 (breast cancer cell line). Most of the

synthesized derivatives showed superior potency to the positive control drug, vatalanib. Among

them, 1-piperazinyl-4-benzylphthalazine derivative 11 (Figure 2) possessed IC values of 2.48,

5

0

4

.59 and 0.76 µM against A549, HT-29 and MDA-MB-231, respectively, while vatalanib showed

values of 21.16, 22.11 and 57.72 µM, respectively [27-29]. Additionally, many synthetic

piperazinyl derivatives were developed as potent anticancer agents [30-34].

Interestingly, no attention was paid to investigate the potential inhibitory activity of the 1-

piperazinyl-4-substitutedphthalazines towards VEGFR-2.

In view of the previous findings, we herein report the synthesis of three different sets of

phthalazine derivatives 16a-k, 18a-e and 21a-g based on the 1-piperazinyl-4-phenylphthalazine

scaffold (Figure 2), with the prime aim of developing potent VEGFR-2 inhibitors with good

anticancer activity. Firstly, phenyl group was selected to substitute the C-4 of the 1-

piperazinylphthalazine core. Then, three strategies were applied on this core. The first one

depends on the utilization of different linkers, acetamide (-CH -CONH-) in the first series 16a-k

2

and acetyl (-CH -CO-) in second series 18a-e, between 1-piperazinyl and aryl moieties. In the

2

second strategy, a bioisosteric approach was adopted to replace the phenyl ring attached to the

acetamide linker in 16 with 2-thiazolyl moiety in 21a-g. The third strategy focused on the usage

of different substituents on phenyl and 2-thiazolyl moieties. The substitution pattern on the

pendant aryl moieties in 16, 18 and 21 was selected so as to ensure different electronic and

lipophilic environments which could manipulate the activity of the target compounds (Figure 2).

The final synthesized 1-piperazinyl-4-phenylphthalazines 16a-k, 18a-e and 21a-g were

evaluated for their potential inhibitory activity toward VEGFR-2. Also, they were screened for

their in vitro anticancer activity against a panel of 56 human cancer cell lines at NCI-USA.

2

. Results and discussion

2

.1. Chemistry

3

The synthetic strategies adopt r the preparati the new phthalazines are depicted in

A Ce dC Ef oPTED MAN Uo nS Co fR IPT

Schemes 1−4. In Scheme 1,4-phenyl-1-(2H)-phthalazinone 12 was obtained, in 80% yield, by

cyclocondensation of 2-benzoylbenzoic acid with hydrazine sulfate in the presence of sodium

hydroxide [35]. Next, chlorination of the phthalazinone 12 was carried out via heating with excess

of phosphorus oxychloride to furnish 1-chloro-4-phenylphthalazine 13 in 73% yield [36]. The

later reacted with excess of anhydrous piperazine in refluxing propan-2-ol to afford the key

intermediate 4-phenyl-1-(piperazin-1-yl)phthalazine 14 in 85% yield [37].

The first group of the target phthalazines 16a-k was obtained in good yields (59-75%) through

the reaction of the key intermediate 14 with the appropriate 2-chloro-N-(un/substituted

phenyl)acetamide 15a-k in refluxing dry acetone using potassium carbonate as a base (Scheme 2).

Alternatively, potassium carbonate catalyzed coupling of the key intermediate 14 with the

appropriate phenacyl bromide 17a-e at room temperature in dry acetone produced the

corresponding target derivatives 18a-e with 58-81% yield (Scheme 3). Finally, the 2-amino

function of (un)substituted 2-aminothiazoles 19a-g was acylated with 2-chloroacetyl chloride to

furnish intermediates 20a-g (yield, 65-79%), which reacted with the piperazinyl phthalazine 14 in

dry DMF in presence of potassium carbonate to give the respective phthalazine derivatives 21a-g

in 55-75% yield (Scheme 4).

The structures of all newly prepared compounds were confirmed under the basis of spectral

and elemental analyses which were in full agreement with the proposed structures.

2

.2. Biological evaluations

.2.1. VEGFR-2 kinase inhibition assay

All the newly prepared compounds (16a-k, 18a-e and 21a-g) were tested in vitro for their

kinase inhibitory activity against VEGFR-2. Sorafenib was included in the experiments as a

reference drug. The results were reported as a 50% inhibition concentration value (IC ,

5

0

determined in triplicate) Table 1. The tested compounds 16, 18 and 21 displayed moderate to

good inhibitory activity with IC values ranging from 4.68±0.47 to 0.35±0.03 µM. In particular,

5

0

compounds 16k and 21d potently inhibited VEGFR-2 at sub-micromolar IC values (0.35±0.03

5

0

and 0.40±0.04 µM, respectively). Also, compounds 16i, 16j and 21g moderately inhibited

VEGFR-2 with IC values of 1.56±0.16, 1.35±0.14

5

0

and 1.44±0.14 µM, respectively.

2

.2.1.1. Structure activity relationship SAR

The SAR study depends on the comparison between the inhibitory activity against VEGFR-2

and structural variations resulted from applying the previous three strategies. Initially, we focused

4

on the impact of substitution o

Compound 16a with unsubstituted phenyl group exhibited the least inhibitory action on VEGFR-2

IC₅₀= 4.68±0.47 µM) relative to the substituted analogs. Introduction of 4-methyl, 3-

n the terminal phenyl on the

ACCEPTED MANUSCRIP Ta ctivity of the first series 16a-k.

(

trifluoromethyl, 4-cyano or 2/3/4-chloro or 2,6-dichloro substituent, compounds 16b-h, led to

non-significant or slight improvement of activity (IC ranging from 4.35±0.45 to 3.39±0.35 µM).

5

0

Conversely, appending a 3-OCH or 4-OCH substituent, compounds 16i and 16j (IC =

50

3

1

.56±0.16 and 1.35±0.14 µM, respectively), resulted in about 3 fold increase in enzyme inhibition

relative to the unsubstituted analog 16a. Likewise, incorporation of 4-ethyl carboxylate

functionality afforded the most active VEGFR-2 inhibitor 16k (IC = 0.35±0.03 µM), hinting that

5

0

substitution with large lipophilic group is more favorable to the enzyme inhibitory activity.

The same trend was observed in the second series 18a-e, as the unsubstituted phenyl

derivative 18a (IC = 4.54±0.36 µM) exhibited the lowest activity. Grafting a 4-methyl or 4-

5

0

chloro substituent in compounds 18b and 18c, did not result in significant change of potency (IC₅₀

4.26±0.44 and 4.29±0.43 µM, respectively), while introduction of a methoxy group at 3- or 4-

position, compounds 18d and 18e, slightly enhanced the VGFR-2 suppressive activity (IC =

=

5

0

3

.42±0.36 and 2.25±0.22 µM, respectively).

Comparing the activity of compounds 16a-k and 18a-e revealed that, the acetamide linker

seemed to show better activity than acetyl linker for compounds bearing 3-OCH or 4-OCH₃

3

substituent (16i and 16j versus 18d and 18e). On the other hand, the type of linker did not show

significant effect in case of unsubstituted, 4-CH and 4-Cl derivatives (16a, 16b, 16g versus 18a-

3

c).

Finally, we explored the effect of bioisosteric replacement of the pendant phenyl in compound

1

6a with 2-thiazolyl group in 21a. Both compounds displayed similar levels of activity (IC =

50

4

.68±0.47, 4.16±0.35 µM, respectively), indicating that a 2-thiazolyl group could effectively

replace the phenyl ring. Concerning the influence of different substituents on the activity of the

third series 21a-g, substitution at 5- and /or 4-postion of the thiazole ring with different groups

proved to be beneficial for activity. The order of activity of the 4-substituted thiazoles 21e-g was

4

-methoxyphenyl 21g > phenyl 21e > 4-chlorophenyl 21f (IC₅₀= 1.44±0.14, 2.38±0.24,

.11±0.31 µM, respectively). The potency of 4,5-disubstituted thiazole derivatives 21b-d

3

increased parallel to the increase in lipophilicity of substituent at 5-position, and the order of

activity was phenyl carboxamide 21d > ethyl carboxylate 21c > acetyl 21b (IC = 0.40±0.04,

5

0

3

.20±0.29, 3.51±0.30 µM, respectively). Highlighting the significance of grafting large lipophilic

group on the pendant aryl rings in the first, 16, and third, 21, series.

5

2

.2.2. In vitro anticancer activit

Ay .CCEPTED MANUSCRIPT

The structures of the all synthesized compounds (16a-k, 18a-e and 21a-g) were submitted to

the National Cancer Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.nih.gov).

Seventeen compounds 16c-e, 16g, 16h, 16j, 16k, 18c-e and 21a-g were selected 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 [38-40].

-5

2

.2.2.1. Primary single high dose (10 M) screening.

The selected compounds were first evaluated at primary anticancer assay against a panel of

-5

fifty six cancer lines at concentration 10 M. The human tumor cell lines were derived from nine

different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and breast

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

applied to estimate the cell viability and growth [41]. The mean percentages growth inhibition (GI

%) of the tested compounds over the full panel of cell lines are illustrated in Figure 3.

The obtained data revealed that, compounds containing acetamide linker 16c-k displayed fair

anticancer activity (mean % GI = 11-24) except for compound 16k featuring a lipophilic 4-ethyl

carboxylate functionality on the pendant phenyl which elicited the highest activity (mean % GI =

7

3). Replacing the acetamide linker of the first series with acetyl linker in the second series 18c-e

did not result in significant change of growth inhibitory activity (mean % GI = 14-26 %). On the

other hand, replacing the terminal phenyl group in the first series with 2-thiazolyl group in the

third series 21a-g improved the growth inhibitory activity (mean % GI = 23-55 %). The 4-methyl-

5

-phenyl carboxamide disubstituted thiazole analog 21d (mean % GI = 55) emerged as the second

most potent member in this work.

2

.2.2.2. In vitro 5 dose full NCI 56 cell panel assay.

The preliminary screening results revealed that compounds 16k (NSC: D-771612/1) and 21d

(

NSC: D-771617/1) are the most active members of this study, showing effectiveness toward

numerous cell lines that belong to different tumor subpanels (Figure 4). Accordingly, compounds

6k and 21d were selected by the NCI for further evaluation at five-dose assay [0.01–100 µM].

1

The calculated response parameters for both compounds – GI₅₀_,TGI and LD – against each cell

50

line are presented in Tables 2 and 3, respectively. Where, GI represents the growth inhibitory

5

0

level of effect; TGI reflects cytostatic activity while LC is viewed as the cytotoxicity parameter

5

0

for compounds. Furthermore, subpanel and full panel mean graph midpoints (MG-MID) were

calculated for GI parameter, giving an average activity parameter over individual subpanels and

5

0

6

full panel cell lines toward each compound (Table 4). T results of sorafenib as a reference

ACCEPTED MANUSC Rh eI PT

compound were obtained from NCI Data Warehouse index [42], and inserted in Table 2 and 3.

In general, compound 16k exhibited superior anti-proliferative activity against almost the

entire panel of tumor cell lines with GI₅₀range 1.29-9.28 µM (Table 2), but showed non-

significant or weak cytostatic activity against the majority of cell lines except for non-small cell

lung cancer (NCI-H522), melanoma (SK-MEL-2 and SK-MEL-5) and breast cancer (T-47D) cell

lines (TGI =5.03, 6.13, 2.76 and 7.82 µM, respectively) (Table 3). Meanwhile, compound 21d

displayed potent growth inhibitory activity at single digit micro-molar concentration against 35

cell lines belonging to various cancer subpanels (Table 2), but had no cytostatic effect against

most of the tested cell lines (TGI > 100 µM) (Table 3). Moreover, both compounds proved to be

non-lethal agents where the LC values for most cell lines exceeded 100 µM.

5

0

Regarding sensitivity of different cell lines, compound 16k had comparatively homogenous

anti-proliferative effect throughout the whole NCI panel, with effective growth inhibition full

panel GI (MG-MID) value of 3.62 µM and subpanel GI (MG-MID) range of 2.37 – 5.47 µM

5

0

50

(Table 4). Among the tested cancer subpanels, the leukemia, melanoma and breast cancer

subpanels were the most susceptible to the influence of 16k [GI (MG-MID) = 2.37, 2.97 and

5

0

2

.54 µM, respectively] (Table 4). The highest activity was observed against leukemia (SR), Non-

small cell lung cancer (NCI-H522), melanoma (SK-MEL-5), breast cancer (T-47D) (GI = 1.75,

5

0

1

.41, 1.36 and 1.29 µM, respectively) (Table 2). On the other hand, compound 21d showed

different levels of anti-proliferative activity with GI (MG-MID) ranging from 3.51 to >100 µM

5

0

(Table 4). Leukemia was the most sensitive subpanel while renal cancer subpanel was refractory

to 21d (Table 4). Although, compound 21d showed decreased growth inhibitory potential

compared to 16k, it possessed significant specific influence on some cancer cell lines. It was

found especially effective against breast cancer (T-47D) at sub-micromolar level (GI = 0.60

5

0

µM) and revealed a distinctive effectiveness on leukemia (HL-60, MOLT-4 and RPMI-8226),

melanoma (SK-MEL-5), ovarian cancer (OVCAR-4) and breast cancer (MDA-MB-231/ATCC

and MDA-MB-468) (GI range 1.65 ̶ 2.96 µM) (Table 2).

5

0

Moreover, the target compounds 16k and 21d exhibited superior potency over sorafenib

against 10 and 3 cell lines, respectively (Table 2). Of special interest, sorafenib was interrogated

for treatment of breast and non-small cell lung cancers showing significant clinical improvement

[

43-47]. In this regard, compound 16k showed anticancer activity overcoming sorafenib towards

breast (BT-549 and T-47D) as well as NSCL (A549/ATCC and NCI-H522) cancer cells, while

compound 21d was 2.6 times more active than sorafenib against T-47D, offering an additional

merit rather sorafenib. Besides, sorafenib is currently involved in clinical trials for treatment of

7

different hematological malig

Lymphoblastic Leukemia (ALL) [48-51]. Therefore, it is noteworthy to mention that compound

6k significantly surpassed the activity of sorafenib against SR and MOLT-4 cell lines (Table 2).

nanci such Acu Myeloid Leukemia (AML), Acute

ACC Ee sPTED Ma sA NUSt eC RIPT

1

On the other hand, combination of sorafenib with different therapies did not result in significant

clinical benefit for treatment of CNS cancer [52,53]. Accordingly, it is important to point out that

1

6k exhibited higher anti-proliferative activity against CNS cancer cells SNB-19 and SNB-75

than sorafenib (GI = 2.33 and 2.84 µM, respectively) (Table 2).

5

0

Concerning selectivity, the index obtained by dividing the full panel MG-MID (µM) of the

compounds by their individual subpanel MG-MID (µM) is considered as a measure of compound

selectivity. Ratios between 3 and 6 refer to moderate selectivity, ratios greater than six indicate

high selectivity toward the corresponding cell line, while compounds not meeting either of these

criteria are rated as nonselective [54]. In this context, compound 16k proved to be nonselective

with broad spectrum anticancer activity against all tumor subpanels tested at GI level, with

5

0

selectivity ratios ranging from 0.66 to 1.52. On the other hand, compound 21d showed high

selectivity toward leukemia and prostate cancer subpanels with selectivity indexes of 9.31 and

6

.34 respectively (Table 4). These results suggest that, replacement of the terminal substituted

phenyl in 16 with substituted thiazolyl group in 21 may represent a tool to manipulate selectivity

of such class of compounds towards different cancer cells.

3

. Molecular docking

Docking simulation of compounds 16k and 21d showed that they fit into the enzyme active

site almost at the same position of sorafenib with comparable docking scores (-15.19 kcal/mol for

sorafenib, -16.95 and -17.49 kcal/mol for compounds 16k and 21d, respectively) (Figure 5A and

5

C).

Inspection of the top docking poses of the target compound 16k revealed that the N–H of the

acetamide linker formed one hydrogen bond to the carboxylate of Glu885 (2.81 Å), while the

amide carbonyl oxygen identified to be involved in hydrogen bond with the backbone NH of

Asp1046 (2.87 Å). Moreover, the complex is stabilized by arene interactions between the terminal

phenyl and Asp1046, the piperazine ring and Phe1047 as well as two interactions between 4-

phenylphthalazine and Gly922 and Leu840 (Figure 5B). On the other hand, docking of compound

2

1d revealed a binding motif in which the N–H of the acetamide linker acted as essential

hydrogen bond donor for Glu885 in the active site (2.78 Å). Additionally, 21d formed arene

interactions, two of which occurred between the phthalazine moiety and Leu840, while the

piperazine ring interacted with Phe1047. The other two arene interactions occurred between the

8

thiazolyl rest and Ile1044 and Asp1046, which justifies the fact that the terminal phenyl ring in 16

ACCEPTED MANUSCRIPT

can be effectively replaced with thiazole ring in 21 (Figure 5D).

Interestingly, the terminal 4-ethyl carboxylate and 4-phenylcarboxamide functionalities of

compounds 16k and 21d, respectively were accommodated within a lipophilic pocket comprised

of the side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019, Cys1024 and Ile1044,

that may explain their superiority over the other synthesized derivatives in biological activity.

Moreover, the involvement of the piperazinyl moiety of both compounds in interaction with

Phe1047 within the active site suggested that this ring may positively contribute to VEGFR2

inhibitory activity.

4

. Conclusion

In summary, we have synthesized three novel series of 1-piperazinyl-4-phenylphthalazine

derivatives 16a-k, 18a-e and 21a-g and evaluated their potential inhibitory action against

VEGFR-2. Compounds 16k and 21d emerged as the most potent VEGFR-2 inhibitors in this

study with IC values of 0.35±0.03 and 0.40±0.04µM, respectively. Whereas, compounds 16i,

5

0

1

6j and 21g moderately inhibited VEGFR-2 with IC₅₀values of 1.56±0.16, 1.35±0.14 and

1

.44±0.14 µM, respectively. The SAR and molecular docking studies pointed that, substitution on

the pendant aryl moiety with large lipophilic group seems to be the most important factor

affecting the activity of a series of N-(aryl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-

yl]acetamide derivatives 16a-k and their N-(substitutedthiazol-2-yl) bioisosteres 21a-g.

Furthermore, seventeen selected compounds 16c-e, 16g, 16h, 16j, 16k, 18c-e and 21a-g were

evaluated for their in vitro anticancer activity according to US-NCI protocol. The results revealed

that compound 16k exhibited potent broad spectrum anticancer activity with full panel GI (MG-

5

0

MID) value of 3.62 µM. Meanwhile, compound 21d showed high degree of differential selectivity

toward leukemia and prostate cancer subpanels [subpanel GI (MG-MID) 3.51 and 5.15 µM,

5

0

respectively] (Table 4). Therefore, compounds 16k and 21d can be considered as interesting

candidates for further development of more potent anticancer agents.

5

. Experimental

5

.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

-1

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

Varian Gemini-300BB at 300 MHz (Varian Inc., Palo Alto, CA) or Bruker spectrophotometer at

1

13

4

00 MHz. H NMR spectra were run at 300 or 400 MHz, while C NMR spectra were run at 75

or 100 MHz in deuterated dimethyl sulfoxide (DMSO-d ) or deuterated chloroform (CDCl ).

6

3

9

Chemical shifts (δ ) are reported lative to TMS as internal standard. All coupling constant (J)

ACCEPTED MANUSCRIPT

re

H

values are given in hertz. Chemical shifts (δ ) are reported relative to DMSO-d as internal

C

6

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 e.V.

Elemental analyses was carried out at the Regional Center for Microbiology 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.

5

.1.1. 4-Phenyl-1(2H)-phthalazinone 12.

o

Compound 12 was prepared according to reported procedure [35] (m.p. 239-241 C, as reported).

5

.1.2. 1-Chloro-4-phenylphthalazine 13.

o

Compound 13 was prepared according to reported procedure [36] (m.p. 158 C, as reported).

5

.1.3. 4-Phenyl-1-(piperazin-1-yl)phthalazine 14.

o

Compound 14 was prepared according to reported procedure [37] (m.p. 176 - 178 C, as reported).

5

.1.4. 2-Chloro-N-(phenyl/substituted phenyl) acetamide derivatives 15a-k.

Compounds 15a-k were prepared according to reported procedures [55-59].

5

.1.5. General procedure for preparation of target compounds 16a-k.

To a stirred mixture of 1-phenyl-4-(1-piperazinyl)phthalazine 14 (0.58 g, 2.0 mmol) and

anhydrous potassium carbonate (0.55 g, 4.0 mmol) in dry acetone, a solution of the appropriate 2-

chloro-N-(substituted phenyl) acetamide derivative 15a-k (2.0 mmol) in dry acetone was added.

The mixture was heated under reflux for 5 h, filtered while hot and the residue was washed with

hot acetone. The combined filtrate and wash were evaporated under vacuum. The residue was

collected, washed with water, dried and crystallized from an appropriate solvent.

5

.1.5.1. N-Phenyl-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamide (16a):

-1

Crystallized from ethanol/water mixture (yield 71 %); m.p.187-189 °C; IR (KBr, ν cm ): 3251

1

(

NH), 3028 (aromatic CH), 1689 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.88 ( s, br, 4H,

6

∗

2

CH piperazine) , 3.30 (s, 2H, COCH ), 3.55 ( s, br, 4H, 2CH piperazine),7.04 ( t, 1H, H-4 of

2

NH-C H , J= 8.1 Hz), 7.29 ( t, 2H, H-3 and H-5 of NH-C H , J= 8.4 Hz), 7.56 – 7.61 (m, 3H, H-3,

6

5

6

5

∗

In the present study, the piperazine protons were detected as two broad singlet signals instead of two triplets. This is

1

in agreement with the published H NMR spectral data of other piperazine derivatives [28, 29].

1

0

H-4 and H-5 of C H ), 7.66 – 7. ( m, 4H, H-2, H-6 of C H and H-2, H-6 of NH-C H ), 7.89 (d,

ACCEPTED MANUSCRIPT

70

H, H-5 phthalazine, J= 7.5 Hz), 7.95 – 8.01 (m, 2H, H-6 and H-7 phthalazine), 8.17 (d, 1H, H-8

6

5

6 5 6 5

1

3

phthalazine, J= 7.8 Hz), 9.83 (s, 1H, NH, D O exchangable); C NMR (DMSO-d , 100 MHz) δ:

2

6

5

1.18 (2CH piperazine), 53.12 (2CH piperazine), 62.25 (COCH ), 119.97, 121.23, 123.87,

2 2 2

1

25.04, 126.65, 126.96, 128.93, 129.13, 129.30, 130.15, 132.24, 132.60, 136.85, 139.12, 156.15,

+

1

59.38, 168.69 (C=O); MS, m/z (%): M 423 (6.3); Anal. Calcd. for C H N O: C, 73.74; H, 5.95;

2

6

25

5

N, 16.54; Found C, 73.78; H, 5.96 ; N, 16.59.

5

.1.5.2. 2-[4-(4-Phenylphthalazin-1-yl)piperazin-1-yl]-N-4-tolylacetamide (16b):

-1

Crystallized from ethanol/water mixture (yield 68 %); m.p.211-213 °C; IR (KBr, ν cm ): 3248

1

(

NH), 3039 (aromatic CH), 1689 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm :2.26 (s, 3H,

6

CH ), 2.87 (s, br, 4H, 2CH piperazine), 3.25 (s, 2H, COCH ), 3.54 (s, br, 4H, 2CH piperazine),

3

2

7

.11 (d, 2H, H-3 and H-5 of 4-CH -C H , J= 8.4 Hz), 7.54 – 7.63 (m, 5H, H-3, H-4, H-5 of C H

3 6 4 6 5

and H-2, H-6 of 4-CH -C H ), 7.66 – 7.70 (m, 2H, H-2 and H-6 of C H ), 7.89 (d, 1H, H-5

3

6

4

6

5

phthalazine, J= 7.2 Hz), 7.92 – 8.01 (m, 2H, H-6 and H-7 phthalazine), 8.17( d, 1H, H-8

1

3

phthalazine, J= 7.8 Hz), 9.73 (s, 1H, NH, D O exchangable); C NMR (DMSO-d , 100 MHz) δ:

2

6

2

0.92 (CH ), 51.18 (2CH piperazine), 53.13 (2CH piperazine), 62.22 (COCH ), 119.98, 121.23,

3 2 2 2

1

25.06, 126.67, 126.97, 128.94, 129.31, 129.50, 130.16, 132.27, 132.64, 132.78, 136.61, 136.85,

+

1

56.16, 159.39, 168.44 (C=O); MS, m/z (%): M 437 (7.9); Anal. Calcd. for C H N O: C, 74.17;

2

7

27

5

H, 6.22; N, 16.01; Found C, 74.17; H, 6.21 ; N, 16.05.

5

.1.5.3.

2-[4-(4-Phenylphthalazin-1-yl)piperazin-1-yl]-N-(3-(trifluoromethyl)phenyl)acetamide

(16c):

-1

Crystallized from ethanol (yield 61 %); m.p. 108-111 °C; IR (KBr, ν cm ): 3369 (NH), 3074

1

(

aromatic CH), 1685 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.88 (s, br, 4H, 2CH

6

2

piperazine), 3.33 (s, 2H, COCH ), 3.56 (s, br, 4H, 2CH piperazine), 7.41 (d, 1H, H-4 of 3-CF -

2

3

C H , J= 7.5 Hz), 7.54 – 7.63 (m, 4H, H-3, H-4, H-5 of C H and H-5 of 3-CF -C H ), 7.67 (d, 2H,

6

4

6

5

3

6

4

H-2 and H-6 of C H , J= 7.8 Hz), 7.88 – 7.92 (m, 2H, H-6 of 3- CF -C H and H-5 phthalazine),

6

5

3

6

4

7

.95 – 8.02 (m, 2H, H-6 and H-7 phthalazine), 8.17 – 8.20 (m, 2H, H-2 of 3-CF -C H and H-8

3 6 4

13

phthalazine), 10.18 (s, 1H, NH); C NMR (DMSO-d , 75 MHz) δ: 50.59 (2CH piperazine), 52.56

6

2

(

2CH piperazine), 61.67 (COCH ), 115.61, 120.73, 123.09, 124.53, 126.14, 126.46, 128.39,

2

1

28.77, 129.61, 129.80, 131.73, 132.10, 136.34, 139.39, 155.63, 158.87, 168.93 (C=O); MS, m/z

+

(

%): M 491 (3.2); Anal. Calcd. for C H F N O: C, 65.98; H, 4.92; N, 14.25; Found C, 66.03; H,

2

7

24

3

5

4

.95; N, 14.33.

1

5

.1.5.4. N-(4-Cyanophenyl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamide (16d):

ACCEPTED MANUSCRIPT

-1

Crystallized from ethanol (yield 75 %); m.p. 140-141 °C; IR (KBr, ν cm ): 3302 (NH), 2225

1

(

C≡N), 3034 (aromatic CH), 1685 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.89 (s, br, 4H,

6

2

3

4

CH piperazine), 3.27 (s, 2H, COCH ), 3.57 (s, br, 4H, 2CH piperazine), 7.55 – 7.62 (m, 3H, H-

2 2 2

, H-4, H-5 of C H ), 7.66 ( d, 2H, H-2 and H-6 of C H , J= 7.8 Hz), 7.76 (d, 2H, H-3 and H-5 of

6

5

6

5

-CN-C H , J= 9.0 Hz), 7.87 (m, 3H, H-2, H-6 of 4-CN-C H and H-5 phthalazine), 7.92 – 7.99

6

4

6

4

1

3

(

m, 2H, H-6 and H-7 phthalazine), 8.16 (d, 1H, H-8 phthalazine, J= 7.8 Hz), 10.21 (s, 1H, NH); C

NMR (DMSO-d , 75 MHz) δ: 50.58 (2CH piperazine), 52.53 (2CH piperazine), 61.70 (COCH ),

6

2

1

05.11 (C≡N), 118.96, 119.48, 120.71, 124.50, 126.12, 126.45, 128.39, 128.76, 129.61, 131.70,

+

1

32.07, 133.11, 136.33, 142.82, 155.62, 158.84, 169.13 (C=O); MS, m/z (%): M 448 (3.5); Anal.

Calcd. for C H N O: C, 72.30; H, 5.39; N, 18.74; Found C, 72.32; H, 5.38; N, 18.78.

2

7

24

6

5

.1.5.5. N-(2-Chlorophenyl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamide (16e):

-1

Crystallized from ethanol (yield 71 %); m.p. 184-187 °C; IR (KBr, ν cm ): 3315 (NH), 3032

1

(

aromatic CH), 1695 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.93 (s, br, 4H, 2CH

6

2

piperazine), 3.36 (s, 2H, COCH ), 3.59 (s, br, 4H, 2CH piperazine), 7.15 (t, 1H, H-4 of 2-Cl-C H ,

2

6

4

J= 7.8 Hz), 7.37 (t, 1H, H-5 of 2-Cl-C H , J= 8.4 Hz), 7.53 (d, 1H, H-3 of 2-Cl-C H , J= 8.4 Hz),

6

4

6

4

7

1

.57 – 7.66 (m, 3H, H-3, H-4 and H-5 of C H ), 7.67 – 7.70 (m, 2H, H-2 and H-6 of C H ), 7.88 (d,

6 5 6 4

H, H-5 phthalazine, J= 7.8 Hz), 7.94 – 8.02 (m, 2H, H-6 and H-7 phthalazine), 8.19 (d, 1H, H-8

phthalazine, J= 7.8 Hz), 8.28 (d, 1H, H-6 of 2-Cl-C H , J= 8.4 Hz), 10.01 (s, 1H, NH, D O

6

4

2

1

3

exchangable); C NMR (DMSO-d , 100 MHz) δ: 51.56 (2CH piperazine), 53.16 (2CH

2

6

2

piperazine), 61.78 (COCH ), 121.25, 121.76, 123.27, 125.03, 125.47, 126.71, 127.00, 128.40,

2

1

28.95, 129.34, 129.76, 130.15, 132.34, 132.70, 134.91, 136.83, 156.32, 159.34, 168.84 (C=O);

+

MS, m/z (%): M 457 (4.7), M +2 459 (1.4); Anal. Calcd. for C H ClN O: C, 68.19; H, 5.28; N,

2

6

24

5

1

5.29; Found C, 68.21; H, 5.30; N, 15.38.

5

.1.5.6. N-(3-Chlorophenyl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamide (16f):

-1

Crystallized from ethanol (yield 74 %); m.p. 129- 131 °C; IR (KBr, ν cm ): 3325 (NH), 3062

1

(

aromatic CH), 1689 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.87 (s, br, 4H, 2CH

6

2

piperazine), 3.31 (s, 2H, COCH ), 3.56 (s, br, 4H, 2CH piperazine), 7.11 (d, 1H, H-4 of 3-Cl-

2

C H , J= 8.4 Hz), 7.32 (t, 1H, H-5 of 3-Cl-C H , J= 8.4 Hz), 7.56 – 7.61 (m, 4H, H-3, H-4, H-5 of

6

4

6

4

C H and H-6 of 3-Cl-C H ), 7.67 – 7.70 (m, 2H, H-2 and H-6 of C H ), 7.88 – 7.92 ( m, 2H, H-5

6

5

6

4

6

5

phthalazine and H-2 of 3-Cl-C H ), 7.95 – 8.01 (m, 2H, H-6 and H-7 phthalazine), 8.17( d, 1H, H-

6

4

1

3

8

phthalazine, J= 8.1 Hz), 10.02 (s, 1H, NH); C NMR (DMSO-d , 75 MHz) δ: 50. 59 (2CH

6 2

piperazine), 52.56 (2CH piperazine), 61.66 (COCH ), 117.89, 119.00, 120.73, 123.04, 124.51,

2

1

2

1

26.12, 126.46, 128.39, 128.75, 129.61, 130.26, 131.70, 132.06, 132.95, 136.35, 140.05, 155.61,

ACCEPTED MANUSCRIPT

+

1

58.85, 168.66 (C=O); MS, m/z (%): M 457 (3.3), M +2 459 (1.3); Anal. Calcd. for

C H ClN O: C, 68.19; H, 5.28; N, 15.29; Found C, 68.21; H, 5.31; N, 15.34.

26

24

5

.1.5.7. N-(4-Chlorophenyl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamide (16g):

-1

Crystallized from ethanol (yield 71 %); m.p.213-215 °C; IR (KBr, ν cm ): 3244 (NH), 3032

1

(

aromatic CH), 1693 (C=O); H NMR (CDCl , 300 MHz) δ ppm: 2.96 (s, br, 4H, 2CH

3

2

piperazine), 3.31 (s, 2H, COCH ), 3.69 (s, br, 4H, 2CH piperazine), 7.31 (d, 2H, H-3 and H-5 of

2

4

7

1

-Cl-C H , J= 8.7 Hz), 7.55 – 7.59 (m, 5H, H-3, H-4, H-5 of C H and H-2, H-6 of 4-Cl-C H ),

6 4 6 5 6 4

.73 – 7.70 (m, 2H, H-2 and H-6 of C H ), 7.80 – 7.87 (m, 2H, H-6 and H-7 phthalazine), 8.04 (d,

6

5

H, H-5 phthalazine, J= 8.1 Hz), 8.12 (d, 1H, H-8 phthalazine, J= 8.1 Hz), 9.26 (s, 1H, NH, D O

2

1

3

exchangable); C NMR (DMSO-d , 100 MHz) δ: 51.14 (2CH piperazine), 53.10 (2CH

2

6

2

piperazine), 62.22 (COCH ), 121.23, 121.57, 125.06, 126.68, 126.97, 127.43, 128.94, 129.02,

2

+

1

4

6

29.31, 130.15, 132.27, 132.64, 136.85, 138.11, 156.16, 159.38, 168.93 (C=O); MS, m/z (%): M

+

57 (5.6), M +2 459 (2.6); Anal. Calcd. For C H ClN O: C, 68.19; H, 5.28; N, 15.29; Found C,

2

6

24

5

8.23; H, 5.27; N, 15.38.

5

.1.5.8. N-(2,6-dichlorophenyl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamide (16h):

-1

Crystallized from ethanol (yield 75 %); m.p.215-217 °C; IR (KBr, ν cm ): 3269 (NH), 3066

1

(

aromatic CH), 1707 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.94 (s, br, 4H, 2CH

6

2

piperazine), 3.26 (s, 2H, COCH ), 3.59 (s, br, 4H, 2CH piperazine), 7.32 (t, 1H, H-4 of 2,6-Cl -

2

C H , J= 7.8 Hz), 7.53 ( d, 2H, H-3 and H-5 of 2,6-Cl -C H , J= 7.8 Hz), 7.57 – 7.62 (m, 3H, H-3,

6

3

2

6

3

H-4 and H-5 of C H ), 7.66 – 7,70 (d, 2H, H-2 and H-6 of C H ), 7.87 ( d, 1H, H-5 phthalazine, J=

6

5

6

5

7

.8 Hz), 7.94–8.01 (m, 2H, H-6 and H-7 phthalazine), 8.18 (d, 1H, H-8 phthalazine, J= 7.8 Hz),

1

3

9

.76 (s, 1H, NH); C NMR (DMSO-d , 75 MHz) δ: 50. 55 (2CH piperazine), 52.76 (2CH

2

6

2

piperazine), 61.07 (COCH ), 120.73, 124.55, 126.12, 126.46, 128.36, 128.40, 128.77, 129.05,

2

+

1

29.61, 131.72, 132.09, 133.05, 133.75, 136.33, 155.61, 158.87, 168.40 (C=O); MS, m/z (%): M

+

4

91 (4.33), M +2 493 (2.63), M +4 495 (0.51); Anal. Calcd. for C H Cl N O: C, 63.42; H, 4.71;

26 23 2 5

N, 14.22; Found C, 63.46; H, 4.74; N, 14.29.

5

.1.5.9. N-(3-methoxyphenyl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamide (16i):

-1

Crystallized from ethanol/water mixture (yield 59 %); m.p. 187-188 °C; IR (KBr, ν cm ): 3313

1

(

NH), 3062 (aromatic CH), 1681 (C=O); H NMR (CDCl , 300 MHz) δ ppm: 2.92 (s, br, 4H, 2CH

3

2

piperazine), 3.26 (s, 2H, COCH ), 3.64 ( s, br, 4H, 2CH piperazine), 3.79 (s, 3H, OCH ), 6.63 (d,

2

3

1

H, H-4 of 3-OCH -C H , J= 7.8 Hz), 7.02 (d, 1H, H-6 of 3-OCH -C H , J= 7.8 Hz), 7.21 (t, 1H,

3 6 4 3 6 4

1

3

H-5 of 3-OCH -C H , J= 7.8 Hz 7.33 (s, 1H, H-2 of 3-OCH H ), 7.50 – 7.56 (m, 3H, H-3, H-4

ACCEPTED MANUSCRI P- CT

),

and H-5 of C H ), 7.67 (d, 2H, H-2 and H-6 of C H , J= 7.2 Hz), 7.72 – 7.86 (m, 2H, H-6 and H-7

3

6

4

3 6 4

6

5

6

5

phthalazine), 7.99 (d, 1H, H-5 phthalazine, J= 8.1 Hz), 8.10 (d, 1H, H-8 phthalazine, J= 7.2 Hz),

1

3

9

.17 (s, 1H, NH); C NMR (DMSO-d , 100 MHz) δ: 51.17 (2CH piperazine), 53.10 (2CH

6 2 2

piperazine), 55.49 (OCH ), 62.26 (COCH ), 105.68, 109.38, 112.18, 121.23, 125.06, 126.67,

3

2

1

26.97, 128.94, 129.31, 129.93, 130.15, 132.28, 132.64, 136.85, 140.30, 156.16, 159.39, 159.98,

+

68.77 (C=O); MS, m/z (%): M 453 (3.6); Anal. Calcd. for C H N O : C, 71.50; H, 6.00; N,

2

7

27

5

2

5.44; Found C, 71.52; H, 6.03; N, 15.51.

5

.1.5.10. N-(4-Methoxyphenyl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamide (16j):

-1

Crystallized from ethanol/water mixture (yield 60 %); m.p.188-190 °C; IR (KBr, ν cm ): 3313

1

(

NH), 3035 (aromatic CH), 1681 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.87 (s, br, 4H,

6

2

CH piperazine), 3.26 (s, 2H, COCH ), 3.56 (s, br, 4H, 2CH piperazine), 3.73 (s, 3H, OCH ),

2 2 2 3

6

.88 (d, 2H, H-3 and H-5 of 4-OCH -C H , J= 6.9 Hz), 7.57 – 7.62 (m, 5H, H-3, H-4, H-5 of C H

5

3

6

4

6

and H-2, H-6 of 4-OCH -C H ), 7.67 (d, 2H, H-2 and H-6 of C H , J= 7.2 Hz), 7.89 (d, 1H, H-5

3

6

4

6

5

phthalazine, J= 7.2 Hz), 7.94 – 8.00 (m, 2H, H-6 and H-7 phthalazine), 8.16 (d, H, H-8

1

3

phthalazine, J= 7.8 Hz), 9.66 (s, 1H, NH); C NMR (DMSO-d , 75 MHz) δ: 50.63 (2CH₂

6

piperazine), 52.62 (2CH piperazine), 55.12 (OCH ), 61.67 (COCH ), 113.73, 120.71, 121.08,

2

3

2

1

24.52, 126.13, 126.50, 128.39, 128.76, 129.61, 131.74, 132.08, 136.34, 155.32, 155.61, 158.85,

+

67.65 (C=O); MS, m/z (%): M 453 (9.5); Anal. Calcd. for C H N O : C, 71.50; H, 6.00; N,

2

7

27

5

2

5.44; Found C, 71.53; H, 6.04; N, 15.51.

5

.1.5.11. Ethyl 4-{2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamido}benzoate (16k):

-1

Crystallized from ethanol (yield 69 %); m.p.175-177 °C; IR (KBr, ν cm ): 3275 (NH), 3030

1

(

aromatic CH), 1750 (C=O ester), 1689 (C=O amide); H NMR (DMSO-d , 300 MHz) δ ppm:

6

1

.29 (t, 3H, CH CH , J= 7.2 Hz), 2.89 (s, br, 4H, 2CH piperazine), 3.34 (s, 2H, COCH ), 3.56 (s,

2 3 2 2

br, 4H, 2CH piperazine), 4.26 (q, 2H, CH CH , J= 7.2 Hz), 7.54 – 7.65 (m, 3H, H-3, H-4, H-5 of

2

3

C H ), 7.67 (d, 2H, H-2 and H-6 of C H , J= 7.8 Hz), 7.82 (d, 2H, H-2 and H-6 of 4-COOEt-

6

5

6

5

C H , J= 9.0 Hz), 7.88 (d, 1H, H-5 phthalazine, J= 7.8 Hz), 7.92 (d, 2H, H-3 and H-5 of 4-

6

4

COOEt-C H , J= 9.3 Hz), 7.95 –8.01 (m, 2H, H-6 and H-7 phthalazine), 8.17 (d, 1H, H-8

6

4

1

3

phthalazine, J= 7.8 Hz), 10.16 (s, 1H, NH, D O exchangable); C NMR (DMSO-d , 100 MHz) δ:

2

6

1

4.69 (CH CH ), 51.16 (2CH piperazine), 53.06 (2CH piperazine), 60.91 (CH CH ), 62.22

2 3 2 2 2 3

(

COCH ), 119.29, 121.23, 124.85, 125.06, 126.68, 126.97, 128.94, 129.31, 130.15, 130.62,

2

+

1

32.28, 132.64, 136.84, 143.49, 156.17, 159.38, 165.79 (C=O), 169.41 (C=O); MS, m/z (%): M

1

4

1

N O : C, 70.28; H, 5.90; N, 14.13; Found C, 70.31; H, 5.89; N,

95 (7.4); Anal. Calcd. for C HA CCEPTED MANUSCRIPT

29 5 3

2

9

4.20.

5

.1.6. substituted 2-bromo-1-phenylethanone 17a-e.

Compounds 17a-e were prepared according to reported procedures [60].

5

.1.7. General procedure for preparation of target compounds 18a-e.

To a stirred mixture of 1-phenyl-4-(1-piperazinyl)phthalazine 14 (0.58 g, 2.0 mmol) and

anhydrous potassium carbonate (0.55 g, 4.0 mmol) in dry acetone, a solution of the appropriate 2-

bromo-1-phenylethanone derivative 17a-e (2.0 mmol) in dry acetone was added. The reaction

mixture was stirred for 3 h at room temperature. The solid product was collected by filtration,

washed with water, dried and crystallized from an appropriate solvent.

5

.1.7.1. 1-Phenyl-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]ethanone (18a):

-

1

Crystallized from ethanol (yield 77 %); m.p.186-188 °C; IR (KBr, ν cm ): 3057 (aromatic CH),

1

697 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.90 (s, br, 4H, 2CH piperazine), 3.51 (s, br,

6

2

4

H, 2CH piperazine), 4.00 (s, 2H, COCH ), 7.52 – 7.63 (m, 6H, H-3, H-4, H-5 of C H and H-3,

2

6

5

H-4, H-5 of COC H ), 7.66 (d, 2H, H-2 and H-6 of C H , J= 7.8 Hz), 7.87 (d, 1H, H-5 phthalazine,

6

5

6

5

J= 7.8 Hz), 7.95 – 8.00 (m, 2H, H-6 and H-7 phthalazine), 8.04 (d, 2H, H-2 and H-6 of COC H ,

6

5

13

J= 9.0 Hz), 8.17 (d, 1H, H-8 phthalazine, J= 8.4 Hz); C NMR (DMSO-d , 75 MHz) δ: 50.74

6

(

2CH piperazine), 52.49 (2CH piperazine), 63.64 (COCH ), 120.70, 124.50, 126.13, 126.46,

2 2 2

1

28.05, 128.38, 128.57, 128.75, 129.61, 131.71, 132.07, 133.19, 135.92, 136.35, 155.60, 158.82,

+

96.98 (C=O); MS, m/z (%): M 408 (14.0); Anal. Calcd. for C H N O: C, 76.45; H, 5.92; N,

2

6

24

4

3.72; Found C, 76.49; H, 5.94; N, 13.79.

5

.1.7.2. 2-[4-(4-Phenylphthalazin-1-yl)piperazin-1-yl]-1-4-tolylethanone (18b):

-1

Crystallized from ethanol (yield 79 %); m.p.181-183 °C; IR (KBr, ν cm ): 3051 (aromatic CH),

1

693 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.39 (s, 3H, CH ), 2.85 (s, br, 4H, 2CH

6

3

2

piperazine), 3.49 (s, br, 4H, 2CH piperazine), 3.97 (s, 2H, COCH ), 7.33 (d, 2H, H-3 and H-5 of

2

4

6

-CH -C H , J= 7.8 Hz), 7.55 – 7.62 (m, 3H, H-3, H-4 and H-5 of C H ), 7.66 (d, 2H, H-2 and H-

3 6 4 6 5

of C H , J= 7.5 Hz), 7.87 (d, 1H, H-5 phthalazine, J= 7.8 Hz), 7.94 – 8.01 (m, 4H, H-6, H-7

6

5

1

3

phthalazine and H-2, H-6 of 4-CH -C H ), 8.16 (d, 1H, H-8 phthalazine, J= 8.1 Hz); C NMR

3

6

4

(DMSO-d , 100 MHz) δ: 21.65 (CH ), 51.27 (2CH piperazine), 53.04 (2CH piperazine), 64.12

6 3 2 2

(

COCH ), 121.21, 125.01, 126.64, 126.95, 128.70, 128.91, 129.28, 129.62, 130.14, 132.21,

2

1

5

+

1

32.57, 133.91, 136.85, 144.08

, 156.11, 159.33, 196.94 (C=O); MS, m/z (%): M 422 (12.3);

ACCEPTED MANUSCRIPT

Anal. Calcd. for C H N O: C, 76.75; H, 6.20; N, 13.26; Found C, 76.74; H, 6.21; N, 13.37.

2

7

26

4

5

.1.7.3. 1-(4-Chlorophenyl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]ethanone (18c):

-

1

Crystallized from ethanol (yield 81 %); m.p.205-207 °C; IR (KBr, ν cm ): 3064 (aromatic CH),

1

695 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.88 (s, br, 4H, 2CH piperazine), 3.51 (s, br,

6

2

4

H, 2CH piperazine), 3.97 (s, 2H, COCH ), 7.57 – 7.61 (m, 5H, H-3, H-4, H-5 of C H and H-3,

2

6

5

H-5 of 4-Cl-C H ), 7.66 (d, 2H, H-2 and H-6 of C H , J= 7.8 Hz), 7.89 (d, 1H, H-5 phthalazine, J=

6

4

6

5

7

.8 Hz), 7.94 – 7.99 (m, 2H, H-6 and H-7 phthalazine), 8.07 (d, 2H, H-2 and H-6 of 4-Cl -C H , J=

6 4

1

3

8

.4 Hz), 8.16 (d, 1H, H-8 phthalazine, J= 8.1 Hz); C NMR (DMSO-d , 75 MHz) δ: 50.70 (2CH

6

2

piperazine), 52.46 (2CH piperazine), 63.76 (COCH ), 120.71, 124.48, 126.12, 126.47, 128.38,

2

1

28.67, 128.74, 129.60, 130.07, 131.69, 132.06, 134.54, 136.36, 138.09, 155.60, 158.80, 196.13

+

(

C=O); MS, m/z (%): M 442 (13.2), M +2 444 (4.8); Anal. Calcd. for C H ClN O: C, 70.50; H,

26 23 4

5

.23; N, 12.65; Found C, 70.56; H, 5.28; N, 12.74.

5

.1.7.4. 1-(3-Methoxyphenyl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]ethanone (18d):

-1

Crystallized from ethanol/water mixture (yield 68 %); m.p.103-106 °C; IR (KBr, ν cm ): 1681

1

(

C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.89 (s, br, 4H, 2CH piperazine), 3.51 (s, br, 4H,

6

2

CH piperazine), 3.84 (s, 3H, OCH ), 4.02 (s, 2H, COCH ), 7.02 (d, 1H, H-4 of 3-OCH -C H ,

2 3 2 3 6 4

J= 8.4 Hz), 7.44 (t, 1H, H-5 of 3-OCH -C H , J= 8.4 Hz), 7.56 – 7.61 (m, 4H, H-3, H-4, H-5 of

3

6

4

C H and H-6 of 3-OCH -C H ), 7.63 (s, 1H, H-2, of 3-OCH -C H ), 7.66 (d, 2H, H-2 and H-6 of

6

5

3

6

4

3

6

4

C H , J= 7.2 Hz), 7.89 (d, 1H, H-5 phthalazine, J= 7.8 Hz), 7.9 – 8.00 (m, 2H, H-6 and H-7

6

5

1

3

phthalazine), 8.17 (d, 1H, H-8 phthalazine, J= 7.8 Hz); C NMR (DMSO-d , 100 MHz) δ: 51.29

6

(

2CH piperazine), 52.98 (2CH piperazine), 55.79 (OCH ), 64.23 (COCH ), 113.23, 119.66,

2 2 3 2

1

21.04, 121.21, 125.03, 126.67, 126.97, 128.93, 129.30, 130.15, 130.29, 132.26, 132.62, 136.85,

+

1

37.79, 156.14, 159.35, 1593.80, 197.29 (C=O); MS, m/z (%): M 438 (12.5); Anal. Calcd. for

C H N O : C, 73.95; H, 5.98; N, 12.78; Found C, 74.01; H, 6.00; N, 12.75.

27

26

4

2

5

.1.7.5.

1-(4-Methoxyphenyl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]ethanone

(18e):

-1

Crystallized from ethanol/water mixture (yield 58 %), m.p.155-157 °C; IR (KBr, ν cm ): 3070

1

(

aromatic CH), 1687 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.87 (s, br, 4H, 2CH

6

2

piperazine), 3.50 (s, br, 4H, 2CH piperazine), 3.86 (s, 3H, OCH ), 3.91 (s, 2H, COCH ), 7.05 (d,

2

3

2

7

8

H, H-3 and H-5 of 4-OCH -C H , J= 8.7 Hz), 7.56 – 7.62 (m, 3H, H-3, H-4 and H-5 of C H ),

3 6 4 6 5

.66 (d, 2H, H-2 and H-6 of C H , J= 7.8 Hz), 7.87 (d, 1H, H-5 phthalazine, J= 7.8 Hz), 7.95 –

6

5

.08 (m, 2H, H-6 and H-7 phthalazine), 8.04 (d, 2H, H-2 and H-6 of 4-OCH -C H , J= 9.0 Hz),

3

6

4

1

6

1

3

ACCEPTED MANUSCRIPT

.16 (d, 1H, H-8 phthalazine, J= 7.8 Hz); C NMR (DMSO-d , 100 MHz) δ: 51.28 (2CH

6 2

8

piperazine), 53.08 (2CH piperazine), 55.99 (OCH ), 64.12 (COCH ), 114.28, 121.21, 125.03,

2

3

2

1

5

26.66, 126.96, 128.93, 129.30, 129.32, 130.15, 131.01, 132.25, 132.61, 136.85, 156.13, 159.35,

+

63.64, 195.85 (C=O); MS, m/z (%): M 438 (14.0); Anal. Calcd. for C H N O : C, 73.95; H,

2

7

26

4

2

.98; N, 12.78; Found C, 73.78; H, 5.97; N, 12.83.

5

.1.8. Substituted 2-aminothiazoles 19a-g.

Compounds 19a-g were prepared according to reported procedures [61-63].

5

.1.9. 2-Chloro-N-(substituted thiazol-2-yl)acetamides 20a-g.

Compounds 20a-c and 20e-g were prepared according to reported procedures [63-66].

5

.1.9.1. 2-(2-Chloroacetamido)-4-methyl-N-phenylthiazole-5-carboxamide (20d):

A mixture of 2-amino-4-methyl-N-phenylthiazole-5-carboxamide 19d (0.47 g, 2.0 mmol) and

anhydrous potassium carbonate (0.55 g, 4.0 mmol) in dry toluene (15 ml) was stirred at room

temperature, while 2-chloroacetyl chloride (0.23 g, 0.16 mL, 2.0 mmol) was added dropwise. The

reaction mixture was heated under reflux for 3 h, solvent was then removed in vacuo and the solid

obtained was triturated with water, filtered, dried and recrystallized from methanol to produce 20d

-1

(

yield 75 %); m.p.180-182 °C; IR (KBr, ν cm ): 3170 (NH), 3039 (aromatic CH), 1705 (C=O

1

amide); H NMR (DMSO-d , 400 MHz) δ ppm: 2.54 (s, 3H, CH ), 4.42 (s, 2H, COCH ), 7.06 (t,

6

3

2

1

H, H-4 of NHC H , J= 7.36 Hz), 7.30 (t, 2H, H-3 and H-5 of NH-C H , J= 8.0 Hz), 7.67 (d, 2H,

6 5 6 5

1

3

H-2 and H-6 of NH-C H , J= 7.76 Hz), 9.99 (s, 1H, NH), 12.75 (s, 1H, NH); C NMR (DMSO-d ,

6

5

6

1

00 MHz) δ: 17.52 (CH ), 42.75 (COCH ), 119.91, 120.87, 124.21, 128.92, 129.02, 139.28,

3 2

1

52.04, 157.32, 160.96 (C=O), 165.97 (C=O).

5

.1.10. General procedure for preparation of target compounds 21a-g.

To a stirred mixture of 1-phenyl-4-(1-piperazinyl)phthalazine 14 (0.58 g, 2.0 mmol) and

anhydrous potassium carbonate (0.55 g, 4.0 mmol) in dry DMF, a solution of the appropriate 2-

chloro-N-(substituted thiazol-2-yl)acetamide derivatives 20a-g (2.0 mmol) in dry DMF was

0

added. The mixture was heated at 100 C for 3 h. The reaction mixture was cooled then poured

over crushed ice. The precipitated product was filtered, washed with water, dried and crystallized

from an appropriate solvent.

5

.1.10.1. 2-[4-(4-Phenylphthalazin-1-yl)piperazin-1-yl]-N-(thiazol-2-yl)acetamide (21a):

1

7

-1

Crystallized from ethanol (yield %); m 233-234 °C; IR

A C6 9C EPTED MANUSCRIPT(KBr, ν cm ): 3284 (NH), 3111

.p.

1

(

aromatic CH), 1695 (C=O); H NMR (CDCl , 300 MHz) δ ppm: 2.91 (s, br, 4H, 2CH

3

2

piperazine), 3.38 (s, 2H, COCH ), 3.67(s, br, 4H, 2CH piperazine), 6.98 (d, 1H, H-5 thiazole, J=

2

3

7

.3 Hz), 7.43 (d, 1H, H-4 thiazole, J= 3.3 Hz), 7.46 – 7.55 (m, 3H, H-3, H-4 and H-5 of C H ),

6 5

.69 (d, 2H, H-2 and H-6 of C H , J= 7.8 Hz), 7.75 – 7.85 (m, 2H, H-6 and H-7 phthalazine), 7.98

6

5

(d, 1H, H-5 phthalazine J= 8.1 Hz), 8.10 (d, 1H, H-8 phthalazine, J= 8.4 Hz), 10.45 (s, 1H, NH);

1

3

C NMR (CDCl , 75 MHz) δ: 51.12 (2CH piperazine), 53.75 (2CH piperazine), 61.47 (COCH ),

3

2

1

6

13.95, 121.96, 124.57, 127.22, 127.66, 128.61, 129.13, 130.14, 131.37, 131.70, 136.70, 137.82,

+

56.76, 159.30, 169.50 (C=O); MS, m/z (%): M +1 431 (12.7); Anal. Calcd. for C H N OS: C,

2

3

22

6

4.16; H, 5.15; N, 19.52; Found C, 64.19; H, 5.14; N, 19.55.

5

.1.10.2.

N-(5-Acetyl-4-methylthiazol-2-yl)-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-

yl]acetamide (21b):

-1

Crystallized from ethanol (yield 72 %); m.p.157-158 °C; IR (KBr, ν cm ): 3298 (NH), 3059

1

(

aromatic CH), 1681 (C=O amide),1658 (C=O ketone); H NMR (DMSO-d , 300 MHz) δ ppm:

6

2

.48 (s, 3H, CH ), 2.55 (s, 3H, COCH ), 2.89 (s, br, 4H, 2CH piperazine), 3.49 (s, 2H, COCH ),

3 3 2 2

3

.53 (s, br, 4H, 2CH piperazine), 7.53 – 7.62 (m, 3H, H-3, H-4 and H-5 of C H ), 7.66 (d, 2H, H-2

2

6

5

and H-6 of C H , J= 7.8 Hz), 7.88 (d, 1H, H-5 phthalazine J= 7.8 Hz), 7.93 – 7.99 (m, 2H, H-6 and

6

5

1

3

H-7 phthalazine), 8.16 (d, 1H, H-8 phthalazine, J= 7.8 Hz), 12.75 (s, 1H, NH); C NMR (DMSO-

d , 75 MHz) δ: 17.92 (CH ), 29.99 (COCH ), 50.66 (2CH piperazine), 52.27 (2CH piperazine),

6

3

2

6

1

0.02 (COCH ), 120.71, 124.49, 125.36, 126.10, 126.47, 131.68, 132.05, 133.49, 136.34, 154.20,

2

+

55.61, 158.81, 158.93, 169.25 (C=O), 190.54 (C=O); MS, m/z (%): M 486 (3.4); Anal. Calcd. for

C H N O S: C, 64.18; H, 5.39; N, 17.27; Found C, 64.23; H, 5.40; N, 17.24.

26

6

2

5

.1.10.3. Ethyl 4-methyl-2-{2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamido} thiazole-5-

carboxylate (21c):

-1

Crystallized from ethanol (yield 75 %); m.p.140-143 °C; IR (KBr, ν cm ): 3169 (NH),

1

3

041(aromatic CH), 1712 (C=O ester), 1676 (C=O amide); H NMR (CDCl , 300 MHz) δ ppm:

3

1

.35 (t, 3H, CH CH , J= 6.9 Hz), 2.67 (s, 3H, CH ), 2.94 (s, br, 4H, 2CH piperazine), 3.42 (s, 2H,

2

3

2

COCH ), 3.71 (s, br, 4H, 2CH piperazine), 4.32 (q, 2H, CH CH , J= 6.9 Hz), 7.53 – 7.60 (m, 3H,

2

3

H-3, H-4 and H-5 of C H ), 7.74 (d, 2H, H-2 and H-6 of C H , J= 7.8 Hz), 7.79 – 7.88 (m, 2H, H-6

6

5

6

5

and H-7 phthalazine), 8.04 (d, 1H, H-5 phthalazine J= 6.9 Hz), 8.10 (d, 1H, H-8 phthalazine, J=

1

3

7

.2 Hz), 10.50 (s, 1H, NH); C NMR (CDCl , 75 MHz) δ: 14.58 (CH CH ), 17.36 (CH ), 51.10

3 2 3 3

(

2CH₂piperazine), 53.80 (2CH₂piperazine), 61.12 (CH CH ), 61.48 (COCH ), 116.54,

2 3 2

1

21.97,124.55, 127.31, 127.68, 128.68, 129.20, 130.19, 131.42, 131.74, 136.68, 156.74, 156.90,

1

8

+

1

58.73, 159.27, 162.92 (C=O)

, 168.94 (C=O); MS, m/z (%): M 516 (3.2); Anal. Calcd. for

ACCEPTED MANUSCRIPT

C H N O S: C, 62.77; H, 5.46; N, 16.27; Found C, 62.81; H, 5.44; N, 16.30.

27

28

6

3

5

.1.10.4. 4-Methyl-N-phenyl-2-{2-[4-(4-phenylphthalazin-1-yl)piperazin-1-yl]acetamido}thiazole-

-carboxamide (21d):

-1

Crystallized from ethanol (yield 69 %); m.p.175-177 °C; IR (KBr, ν cm ): 3242 (NH), 3061

1

(

aromatic CH), 1693 (C=O amide), 1674 (C=O amide); H NMR (DMSO-d , 300 MHz) δ ppm:

6

2

.49 (s, 3H, CH ), 2.90 (s, br, 4H, 2CH piperazine), 3.50 (s, 2H, COCH ), 3.54 (s, br, 4H, 2CH

3 2 2 2

piperazine), 7.09 (t, 1H, H-4 of NHC H , J= 7.2 Hz), 7.35 (t, 2H, H-3 and H-5 of NH-C H , J= 7.8

6

5

6

5

Hz), 7.53 – 7.62 (m, 3H, H-3, H-4 and H-5 of C H ), 7.66 – 7.70 (m, 4H, H-2, H-6 of C H and H-

6

5

6

5

2

, H-6 of NH-C H ), 7.87 (d, 1H, H-5 phthalazine J= 8.1 Hz), 7.94 – 8.00 (m, 2H, H-6 and H-7

6 5

1

3

phthalazine), 8.16 (d, 1H, H-8 phthalazine, J= 7.8 Hz), 9.92 (s, 1H, NH), 12.23 (s, 1H, NH); C

NMR (DMSO-d , 100 MHz) δ: 17.54 (CH ), 51.20 (2CH piperazine), 52.18 (2CH piperazine),

6

3

2

6

1

0.56 (COCH ), 119.42, 120.90, 121.22, 124.14, 125.02, 126.65, 126.96, 128.92, 129.04, 129.29,

2

30.14, 132.23, 132.59, 136.84, 139.38, 151.96, 156.14, 157.31, 159.34, 161.11(C=O), 169.49

+

(

C=O); MS, m/z (%): M 563 (1.5); Anal. Calcd. for C H N O S: C, 66.05; H, 5.19; N, 17.39;

3

1

29

7

2

Found C, 66.10; H, 5.21; N, 17.36.

5

.1.10.5. 2-[4-(4-Phenylphthalazin-1-yl)piperazin-1-yl]-N-(4-phenylthiazol-2-yl)acetamide (21e):

-1

Crystallized from ethanol (yield 63 %); m.p.241-243 °C; IR (KBr, ν cm ): 3356 (NH), 3109

1

(

aromatic CH), 1697 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.90 (s, br, 4H, 2CH

6

2

piperazine), 3.50 (s, 2H, COCH ), 3.52 (s, br, 4H, 2CH piperazine), 7.32 (t, 1H, H-4 of

2

phenylthiazole, J= 8.4 Hz), 7.44 (t, 2H, H-3 and H-5 of phenylthiazole, J= 8.4 Hz), 7.55 – 7.62 (m,

3

8

2

H, H-3, H-4 and H-5 of C H ), 7.66 (s, 1H, H-5 thiazole), 7.67 (d, 2H, H-2 and H-6 of C H , J=

6

5

6

5

.1 Hz), 7.87 – 7.93 (m, 3H, H-5 phthalazine and H-2 and H-6 of phenylthiazole), 7.95 – 8.00 (m,

1

3

H, H-6 and H-7 phthalazine), 8.17 (d, 1H, H-8 phthalazine, J= 7.8 Hz), 12.20 (s, 1H, NH); C

NMR (DMSO-d , 75 MHz) δ: 50.73 (2CH piperazine), 52.33 (2CH piperazine), 60.05 (COCH ),

6

2

1

08.07, 120.71, 124.53, 125.65, 126.12, 126.46, 127.72, 128.39, 128.65, 128.75, 129.61, 131.71,

+

32.07, 134.22, 136.35, 148.82, 155.63, 157.44,158.84, 168.62 (C=O); MS, m/z (%): M 506 (3.4);

Anal. Calcd. for C H N OS: C, 68.75; H, 5.17; N, 16.59; Found C, 68.72; H, 5.20; N, 16.63.

2

9

26

6

5

.1.10.6.

N-[4-(4-Chlorophenyl)thiazol-2-yl]-2-[4-(4-phenylphthalazin-1-yl)piperazin-1-

yl]acetamide (21f):

0

-1

Crystallized from ethanol (yield 63 %); m.p.173-175 C; IR (KBr, ν cm ): 3367 (NH), 3053

1

(

aromatic CH), 1697 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.91 (s, br, 4H, 2CH

6

2

1

9

piperazine), 3.50 (s, 2H, COCH₂), 3.54 (s, br, 4H, 2CH piperazine), 7.47 (d, 2H, H-3 and H-5 of

ACCEPTED MANUSCRIPT

2

4

-Cl-C H , J= 8.1 Hz), 7.56 – 7.62 (m, 3H, H-3, H-4 and H-5 of C H ), 7.67 – 7.70 (m, 3H, H-2,

5

4

6

5

H-6 of C H and H-5 thiazole), 7.89 – 8.10 (m, 5H, H-5, H-6, H-7 phthalazine and H-2, H-6 of 4-

6

5

1

3

Cl-C H ), 8.17 ( d, 1H, H-8 phthalazine, J= 7.8 Hz), 12.04 (s, 1H, NH, D O exchangable); C

5

4

2

NMR (DMSO-d , 100 MHz) δ: 51.26 (2CH piperazine), 52.33 (2CH piperazine), 60.59

6

2

(

COCH ), 109.30, 121.22, 125.00, 126.62, 126.95, 127.85, 128.89, 129.17, 129.26, 130.14, 132.17,

2

+

1

5

6

32.53, 132.72, 133.59, 136.85, 148.09, 156.13, 158.19, 159.34, 169.22 (C=O); MS, m/z (%): M

+

40 (4.2), M +2 542 (1.4); Anal. Calcd. for C H ClN OS: C, 64.37; H, 4.66; N, 15.53; Found C,

2

9

25

6

4.41; H, 4.65; N, 15.50.

5

.1.10.7.

N-[4-(4-Methoxyphenyl)thiazol-2-yl]-2-[4-(4-phenylphthalazin-1-yl)

piperazin-1-

yl]acetamide (21g):

-1

Crystallized from ethanol (yield 55 %); m.p.163-165 °C; IR (KBr, ν cm ): 3292 (NH), 3088

1

(

aromatic CH), 1691 (C=O); H NMR (DMSO-d , 300 MHz) δ ppm: 2.92 (s, br, 4H, 2CH

6

2

piperazine), 3.49 (s, 2H, COCH ), 3.55 (s, br, 4H, 2CH piperazine), 3.74 (s, 3H, OCH ), 6.98 (d,

2

3

2

H, H-3 and H-5 of 4-OCH -C H , J= 8.7 Hz), 7.44 (s, 1H, H-5 thiazole), 7.56 – 7.62 (m, 3H, H-3,

3 5 4

H-4 and H-5 of C H ), 7.67 (d, 2H, H-2 and H-6 of C H , J= 7.8 Hz), 7.83 (d, 2H, H-2 and H-6 of

6

5

6

5

4

-OCH -C H , J= 8.1 Hz), 7.87 (d, 1H, H-5 phthalazine J= 7.8 Hz), 7.95 – 8.00 (m, 2H, H-6 and

3 5 4

H-7 phthalazine), 8.17 (d, 1H, H-8 phthalazine, J= 7.8 Hz), 11.91 (s, 1H, NH, D O exchangable);

2

1

3

C NMR (DMSO-d , 100 MHz) δ: 51.26 (2CH piperazine), 52.85 (2CH piperazine), 55.59

6

2

(

OCH ), 60.59 (COCH ), 106.55, 114.54, 121.22, 125.04, 126.64, 126.96, 127.50, 127.58, 128.91,

3 2

1

29.28, 130.15, 132.22, 132.58, 136.85, 149.23, 156.15, 157.81, 159.36, 159.45, 169.04 (C=O);

+

MS, m/z (%): M 536 (1.6); Anal. Calcd. for C H N O S: C, 67.14; H, 5.26; N, 15.66; Found C,

30

28

6

2

6

7.20; H, 5.28; N, 15.75.

5

.2 Biological evaluation

.2.1. Measurement of inhibitory activity against VEGFR-2

The kinase activity of VEGFR-2 was measured by use of a 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 MnCl , 5 mM MgCl , 0.01% Tween-

2

0 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

2

0

Alpha Screen streptavidine donor beads and 10 µg/mL acceptor beads in 62.5 mM HEPES pH 7.4,

ACCEPTED MANUSCRIPT

2

50 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 (IC ) was calculated from the concentration–inhibition response curve (triplicate

5

0

determinations) and the data were compared with Sorafenib as standard VEGFR-2 inhibitor.

5

.2.2. In vitro cytotoxic activity

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

(against 56 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 individual

o

cell lines. After cell inoculation, the microtiter plates were incubated at 37 C, 5% CO , 95% air

2

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. Additional four, 10-fold or ½ log serial

dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 µl

of these different drug dilutions were added to the appropriate microtiter wells already containing

1

00 µl of medium, resulting in the required final drug concentrations. 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% CO , 95% air, and 100% relative humidity. For adherent cells, the

2

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

o

C. 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 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

2

1

same except that the assay was terminated by fixing settl cells at the bottom of the wells by

ACCEPTED MANUSC Re dI PT

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 at the

five concentration levels (T )], the percentage growth was calculated at each of the drug

i

concentration levels. Percentage growth inhibition was calculated as:

[

(T – T ) / (C – T )] x 100 for concentrations for which T ≥ T

i z z i z

(T – T ) / T ] x 100 for concentrations for which T < T

z

i

z

i

Three dose response parameters were calculated for each experimental compound: Growth

inhibition of 50% (GI ) was calculated when [(T – T ) / (C – T )] x 100 = 50. The compound

5

0

i

z

concentration resulting in total growth inhibition (TGI) was calculated when T = T . The LC

50

i

z

indicating a net loss of cells following treatment was calculated when [(T – T ) / T ] x 100 = – 50.

i

z

5

.3. Molecular docking

The molecular docking studies were carried out using Molecular Operating Environment

MOE) software version 2010.10 [67]. The two compounds, 16k and 21d, were drawn on MOE.

(

The structures were subjected to energy minimization using Hamiltonian-Force Field-MMFF94x

and the force field partial charges were calculated for each molecule. Stochastic conformational

analysis was run for each molecule using default settings and the four most stable conformers for

each compound were retained. The X-ray crystal structure of VEGFR-2 in complex with

sorafenib was downloaded from http://www.rscb.org/pdb (PDB ID: 4ASD) [68]. The protein-

ligand complex obtained from the protein data bank was prepared for docking as follows: 1-The

enzyme was 3D protonated, where hydrogen atoms were added at their standard geometry, the

partial charges were computed and the system was optimized. 2-Deletion of chain B of the protein

together with co-crystallized water molecules was performed. 3-The binding pocket has been

defined, isolated and then the back bone was hidden.

Flexible ligand-rigid receptor docking of the most stable conformers was done with

MOEDOCK using Alpha triangle placement method and London dG as a scoring function. The

obtained poses were subjected to force field refinement using the same scoring function. Ten of

the most stable docking models for each ligand were retained with the best scored conformation.

In order to validate the docking procedure, sorafenib was re-docked into the active site of 4ASD.

The docking results showed a near perfect alignment with the original ligand and displayed the

same binding interactions as obtained from the X-ray crystallography pdb file.

2

ACCEPTED MANUSCRIPT

Acknowledgment

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.

References

[

1] E. Stuttfeld and K. Ballmer-Hofer, IUBMB Life 61(2009), 915-922.

2] S. Koch, S. Tugues, X. Li, L. Gualandi and L. Claesson-Welsh, Biochem. J. 437 (2011),

1

69-183.

[

3] W. Risau, Nature 386 (1997), 671-674.

4] T. Pralhad, S. Madhusudan and K. Rajendrakumar, J. Pharm. Pharmacol. 55 (2003), 1045-

1

053.

[

5] P. Carmeliet and R.K. Jain, Nature 407 (2000), 249-257.

6] J. Dumas, W. Scott, J. Elting and H. Hatoum-Makdad, WO Patent, 2003, 03/068228 A1.

7] S. Wilhelm, J. Dumas, G. Ladouceur, M. Lynch and W. Scott, US Patent, 2007,

2

0070020704 A1.

[

8] L. Sun, C. Liang, S. Shirazian, Y. Zhou, T. Miller, J. Cui, J.Y. Fukuda, J.-Y. Chu, A.

Nematalla, X. Wang, H. Chen, A. Sistla, T.C. Luu, F. Tang, J. Wei and C. Tang, J. Med.

Chem. 46 (2003), 1116-1119.

9] P.A. Harris, A. Boloor, M. Cheung, R. Kumar, R.M. Crosby, R.G. Davis-Ward, A.H.

Epperly, K.W. Hinkle, R.N. Hunter, J.H. Johnson, V.B. Knick, C.P. Laudeman, D.K.

Luttrell, R.A. Mook, R.T. Nolte, S.K. Rudolph, J.R. Szewczyk, A.T. Truesdale, J.M. Veal,

L. Wang and J.A. Stafford, J. Med. Chem. 51 (2008), 4632-4640.

[

10] L.F. Hennequin, E.S. Stokes, A.P. Thomas, C. Johnstone, P.A. Plé, D.J. Ogilvie, M.

Dukes, S.R. Wedge, J. Kendrew and J.O. Curwen and J. Med. Chem. 45 (2002), 1300-

1

312.

11] R.S. Kania, S.L. Bender, A.J. Borchardt, J.F. Braganza, S.J. Cripps, Y. Hua, M.D.

Johnson, T.O. Johnson, , H.T. Jr. Luu, C.L. Palmer, S.H. Reich, A.M. Tempczyk-russell,

M. Teng, C. Thomas, M.D. Varney and M.B. Wallace, WO Patent, 2001, 01/02369 A2.

12] A.P. Thomas, L.F. Hennequin and P.A. Plé, WO Patent, 1998, 98/13350.

13] P. Wu, T.E. Nielsen and M.H. Clausen, Trends Pharmacol. Sci. 36 (2015), 422-39.

14] G. Bold, J. Frei, P. Traxler, K.-H. Altmann, H. Mett, D.R. Stover and J.M. Wood, EP

Patent, 1998, 98/00764.

[

15] G. Bold, K.-H. Altmann, J. Frei, M. Lang, P.W. Manley, P. Traxler, B. Wietfeld, J.

Brüggen, E. Buchdunger, R. Cozens, S. Ferrari, P. Furet, F. Hofmann, G. Martiny-Baron,

2

3

J. Mestan, J. Rösel, M. S

E. Masso, R. Roth, C. Schlachter, W. Vetterli, D. Wyss and J.M. Wood, J. Med. Chem. 43

2000), 2310-2323.

ills, D. Stover, F. Acemoglu, E. Boss, R. Emmenegger, L. Lässer,

ACCEPTED MANUSCRIPT

(

[

16] J. Dumas and J.A. Dixon, Expert Opin. Ther. Patents 15 (2005), 647-658.

17] T. Jahan, L. Gu, R. Kratzke, A. Dudek, G.A. Otterson, X. Wangb, M. Green, E.E. Vokes

and H.L. Kindler, Lung Cancer 76 (2012), 393-396.

[

18] H. Joensuu, F.D. Braud, G. Grignagni, T. D. Pas, G. Spitalieri, P. Coco, C. Spreafico, S.

Boselli, F. Toffalorio, P. Bono, T. Jalava, C. Kappeler, M. Aglietta, D. Laurent and PG

Casali, Br. J. Cancer 104 (2011), 1686-1690.

19] N. Cook, B. Basu, S. Biswas, P. Kareclas, C. Mann, C. Palmer, A. Thomas, S. Nicholson,

B. Morgan, D. Lomas, B. Sirohi, A.P. Mander, M. Middleton and P.G. Corrie, Eur. J.

Cancer 46 (2010), 2671-2673.

20] E.L. Piatnitski, M.A.J. Duncton, A.S. Kiselyov, R. Katoch-Rouse, D. Sherman, D.L.

Milligan, C. Balagtas, W.C. Wong, J. Kawakamia and J.F. Doody, Bioorg. Med. Chem.

Lett. 15 (2005), 4696-4698.

21] M.A.J. Duncton, E.L. Piatnitski, R. Katoch-Rouse, L.M. Smith, A.S. Kiselyov, D.L.

Milligan, C. Balagtas, W.C. Wong, J. Kawakamia and J.F. Doody, Bioorg. Med. Chem.

Lett. 16 (2006), 1579-1581.

[

22] A.S. Kiselyov,V.V. Semenov and D. Milligan, Chem. Biol. Drug Des. 68 (2006), 308-313.

23] A.S. Kiselyov, M. Semenova, V.V. Semenov and E.L. Piatnitski, Chem. Biol. Drug Des.

6

8 (2006), 250-255.

[

24] M.A.J. Duncton, E.L.P. Chekler, R. Katoch-Rouse, D. Sherman, W.C. Wong, L.M.

Smith, J.K. Kawakami, A.S. Kiselyov, D.L. Milligan, C. Balagtas, Y.R. Hadari, Y. Wang,

S.N. Patel, R.L. Rolster, J.R. Tonra, D. Surguladze, S. Mitelman, P. Kussie, P. Bohlen and

J. F. Doody, Bioorg. Med. Chem. 17 (2009), 731-740.

[

25] J. Li, Y.F. Zhao, X.Y. Yuan, J.X. Xu and P. Gong, Molecules 11 (2006), 574-582.

26] X. Zhai, J. Li, L. He, S. Zheng, Y.B. Zhang and P. Gong, Chin. Chem. Lett. 19 (2008), 29-

3

2.

27] S.L. Zhang, Y.J. Liu, Y.F. Zhao, Q.T. Guo and P. Gong, Chin. Chem. Lett. 21 (2010),

071-1074.

[

1

28] S. Zhang, Y. Zhao, Y. Liu, D. Chen, W. Lan, Q. Zhao, C. Dong, L. Xia and P. Gong, Eur.

J. Med. Chem. 45 (2010), 3504-3510.

29] Y. Liu, S. Zhang, Y. Li, J. Wang, Y. Song and P. Gong, Arch. Pharm. Chem. Life Sci. 345

(2012), 287-293.

2

4

[

30] A. Foroumadi, S. Emam

Shafiee, Biomed. Pharmacother. 63 (2009), 216-220.

31] M. Tuncbilek, E.B. Guven, T. Onder and R.C. Atalay, J. Med. Chem. 55 (2012), 3058-

065.

i, S. Rajabalian, M. Badinloo, N. Mohammadhosseini and A.

ACCEPTED MANUSCRIPT

3

32] S.-L. Cao, Y. Han, C.-Z. Yuan, Y. Wang, Z.-K. Xiahou, J. Liao, R.-T. Gao, B.-B. Mao,

B.-L. Zhao, Z.-F. Li and X. Xu, Eur. J. Med. Chem. 64 (2013), 401-409.

33] C. Fytas, G. Zoidis, A. Tsotinis, G. Fytas, M. A. Khan , S. Akhtar, K.M. Rahman and D.E.

Thurston, Eur. J. Med. Chem. 93 (2015), 281-290.

34] H.-H. Lin, W.-Y. Wu, S.-L. Cao, J. Liao, L. Ma, M. Gao, Z.-F. Li and X. Xu, Bioorg.

Med. Chem. Lett. 23 (2013), 3304-3307.

35] W.M. Eldehna, H.S. Ibrahim, H.A. Abdel-Aziz, N.N. Farrag and M.M. Youssef, Eur. J.

Med. Chem. 89 (2015), 549-560.

36] Y. Rival, R. Hoffmann, B. Didier, V. Rybaltchenko, J.-J. Bourguignon and C.G. Wermuth,

J. Med. Chem. 41 (1998), 311-317.

37] B.S. Lucas, W. Aaron, S. An, R.J. Austin, M. Brown, H. Chan, A. Chong, R. Hungate, T.

Huang, B. Jiang, M.G. Johnson, J.A. Kaizerman, G. Lee, D.L. McMinn, J. Orf, J.P.

Powers, M. Rong, M.M. Toteva, C. Uyeda, D. Wickramasinghe, G. Xu, Q. Ye and W.

Zhong, Bioorg. Med. Chem. Lett. 20 (2010), 3618-3622.

[

38] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J.

Langley, P. Cronise, A. Vaigro-Wolfe, M. Gray-Goodrich, H. Campbell and M.R. Boyd,

J. Natl. Cancer Inst. 83 (1991), 757-766.

[

39] M.R. Boyd and K.D. Paull, Drug Dev. Res. 34 (1995), 91-109.

40] M.R. Boyd, in: B.A. Teicher (Ed.), Cancer Drug Discovery and Development: Anticancer

Drug Development Guide: Preclinical Screening, Clinical Trials and Approval, Humana

Press, Totowa, NJ, USA, Second edition 2014, Chapter 1, pp. 41-62.

[

41] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. Warren, H.

Bokesch, S. Kenney and M. Boyd, J. Natl. Cancer Inst. 82 (1990), 1107-1112.

42] DTP Data Search: http://dtp.nci.nih.gov/dtpstandard/dwindex/index.jsp. (retrieved on

1

.08.2015)

43] L.A. Mina, M. Yu, C. Johnson, C. Burkhardt, K.D. Miller and R. Zon, Invest. New

drugs, 31 (2013), 1307-1310.

44] T. Luu, P. Frankel, C. Chung, W. Chow, J. Mortimer, A. Hurria and G. Somlo, Clin.

breast cancer, 14 (2014), 94-100.

2

5

[

45] A.-M. C. Dingemans, W

Kunst, E. Thunnissen, D.A.M. Heideman and E.F. Smit, Clin. Cancer Res. 19 (2013), 743-

1.

.W. Mellema, H.J.M. Groen, A. van Wijk, S.A. Burgers, P.W.A.

ACCEPTED MANUSCRIPT

5

[

46] https://clinicaltrials.gov/ct2/show/NCT00863746

47] D.R. Spigel, H.A. Burris III, F.A. Greco, D.L. Shipley, E.K. Friedman, D.M. Waterhouse,

R.C. Whorf, R.B. Mitchell, D.B. Daniel, J. Zangmeister, J.D. Bass and J.D. Hainsworth, J.

Clin. Oncol. 29 (2011), 2582-2589.

[

48] https://clinicaltrials.gov/ct2/show/NCT00131989

49] G. Borthakur, H. Kantarjian, F. Ravandi, W. Zhang, M. Konopleva, J.J. Wright, S. Fader,

S. Verstovsek, S. Mathews, M. Andreeff and J.E. Cortes, Haematologica. 96 (2011), 62-

6

8.

[

50] E. Weisberg and M. Sattler, Haematologica. 97 (2012), 1620-1621.

51] M. Levis, S.H. Faderl, G. Garcia-Manero, E. Jabbour, N.G. Daver, S.E. Dellasala, S.

Pierce, J.A. Burger, J.E. Cortes, H.M. Kantarjian and F. Ravandi, Blood, 122 (2013),

3

934-3934.

[

52] D.M. Peereboom, M.S. Ahluwalia, X. Ye, J.G. Supko, S.L. Hilderbrand, S. Phuphanich,

L.B. Nabors, M.R. Rosenfeld, T. Mikkelsen and S.A. Grossman, Neuro Oncol. 15 (2013),

4

90-496.

53] E. Galanis, S.K. Anderson, J.M. Lafky, J.H. Uhm, C. Giannini, S.K. Kumar, T.K.

Kimlinger, D.W. Northfelt, P.J. Flynn, K.A. Jaeckle, T.J. Kaufmann and J.C. Buckner,

Clin. Cancer Res. 19 (2013), 4816-4823.

[

54] E.M. Acton, V.L. Narayanan, P.A. Risbood, R.H. Shoemaker, D.T. Vistica and M.R.

Boyd, J. Med. Chem. 37 (1994), 2185-2189.

55] I.E. Virsis, D.A. Silaraya, B.A. Grinberga, A.A. Prikulis, I.D. Gerbashevska and M.E.

Orenish, Khim. Farm. Zh. 15 (1981), 23-26.

[

56] Z. Aktürk, F. Kılıç, K. Erol and V. Pabuçcuoğlu, Il Farmaco 57 (2002), 201-206.

57] R. Kumar, M. Kaur, M.S. Bahia and O. Silakari, Eur. J. Med. Chem. 80 (2014), 83-91.

58] R.V. Patel, P.K. Patel, P. Kumari, D.P. Rajani and K.H. Chikhalia, Eur. J. Med. Chem. 53

(2012), 41-51.

[

59] H. Behbehani and H.M. Ibrahim, Molecules 17 (2012), 6362-6385.

60] L.C. King and G.K. Ostrum, J. Org. Chem. 29 (1964), 3459-3461.

61] D.W. Rangnekar, G.J. Kazemi, J.V. Malanker and R. Ramamurthy, Phosphorus Sulfur,

1

41 (1998), 185-190.

[

62] B.S. Kuarm, J.V. Madhav and B. Rajitha, Lett. Org. Chem. 8 (2011), 549-553.

63] A. Geronikakil and G. Theophilidis, Eur. J. Med. Chem. 27 (1992), 709-716.

2

6

[

64] A.C. Conibear, K.A. Lobb, P.T. Kaye, G.L. Blatch and T. Bodill, Bioorg. Med. Chem.

ACCEPTED MANUSCRIPT

Lett. 19 (2011), 1321-1327.

[

65] H.-L. Liu, Z. Li and T. Anthonsen, Molecules 5 (2000), 1055-1061.

66] S.M. El-Messery, G.S. Hassan, F.A.M. Al-Omary and H.I. El-Subbagh, Eur. J. Med.

Chem. 54 (2012), 615-625.

[

67] MOE. Chemical computing Group, Inc., Montereal https://www.chemcomp.com/

68] M. McTigue, B.W. Murray, J.H. Chen, Y.L. Deng, J. Solowiej and R.S. Kania, Proc. Natl.

Acad. Sci. USA,109 (2012), 18281-18289.

[

69] S. Wilhelm, C. Carter, M. Lynch, T. Lowinger, J. Dumas, R.A. Smith, B. Schwartz, R.

Simantov and S. Kelley, Nat. Rev. Drug Discovery 5 (2006), 835-844.

2

7

ACCEPTED MANUSCRIPT

FIGURE CAPTIONS

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

Figure 2. Structures of compounds 8-11 and the target 1-piperazinyl-4-phenylphthalazine

derivatives 16a-k, 18a-e and 21a-g.

Figure 3. Mean % growth inhibitions of the tested compounds over NCI 56 cell line panel.

Figure 4. % Growth inhibition exerted by compounds 16k and 21d at 10 µM concentration over

NCI 56 cell line panel.

Figure 5. The best scored docking model of (A) Compound 16k (in yellow) in 3D style overlaid

with sorafenib (in orange), (B) Compound 16k in 2D style, (C) Compound 21d (in

yellow) in 3D style overlaid with sorafenib (in orange) and (D) Compound 21d in 2D

style; most hydrogen atoms and some residues were omitted for clarity

TABLE CAPTIONS

Table 1. VEGFR-2 kinase inhibitory activity of the target 1-piperazinyl-4-phenylphthalazines.

Table 2. NCI in vitro testing result of GI of compounds 16k (NSC: D-771612/1) and 21d (NSC:

5

0

a,b

D-771617/1) and sorafenib at five dose level.

a

Table 3. TGI values of compounds 16k and 21d and sorafenib over the most sensitive cell lines.

a

Table 4. Median growth inhibitory concentrations (GI , µM) of in vitro subpanel tumor cell

5

0

lines for compounds 16k and 21d and sorafenib.

SCHEME CAPTIONS

Scheme 1. Synthesis of the key intermediate 14. Reagents and conditions: (i)

NH NH .H SO / NaOH / reflux 1h / 80%; (ii) POCl / heat 85 °C 4h / 73%; (iii)

2

4

3

anhydrous piperazine / isopropyl alcohol / heat 75 °C 4h / 85%.

Scheme 2. Synthesis of target phthalazines 16a-k. Reagents and conditions: (i) dioxane / r.t.

2

h / 75-93%; (ii) compound 14 / dry acetone / K CO / reflux 5h / 59-75%.

2 3

Scheme 3. Synthesis of target phthalazines 18a-e. Reagents and conditions: (i) CuBr /

2

CH COOEt and CHCl / reflux 2-4h / 77-85%; (ii) compound 14 / dry acetone /

3

K CO / r.t. 3h / 58-81%.

2

3

Scheme 4. Synthesis of target phthalazines 21a-g. Reagents and conditions: (i) dry toluene /

K CO / reflux 3h / 65-79%; (ii) compound 14 / dry DMF / K CO / heating 3h /

2

3

2

3

55-75%.

2

8

Article Doi

1-Piperazinylphthalazines as potential VEGFR-2 inhibitors and anticancer agents: Synthesis and in vitro biological evaluation

DOI: 10.1016/j.ejmech.2015.10.053

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2015.10.053

Full text of DOI:10.1016/j.ejmech.2015.10.053

Products guided by the article

R&D Labs maybe for 10132-01-1

Relevant to this article

Hot Product