Design and synthesis of phthalazine-based compounds as potent anticancer agents with potential antiangiogenic activity via VEGFR-2 inhibition

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

019, VOL. 34, NO. 1, 1347–1367

2

https://doi.org/10.1080/14756366.2019.1642883

RESEARCH PAPER

Design and synthesis of phthalazine-based compounds as potent anticancer

agents with potential antiangiogenic activity via VEGFR-2 inhibition

a

b

c

d

e

Salwa Elmeligie , Asmaa M. Aboul-Magd , Deena S. Lasheen , Tamer M. Ibrahim , Tamer M. Abdelghany ,

f

c,g

Sohair M. Khojah and Khaled A. M. Abouzid

a

b

Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt; Pharmaceutical Chemistry Department,

c

Faculty of Pharmacy, Nahda University, Beni Suef, Egypt; Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University,

Cairo, Egypt; Pharmaceutical Chemistry Department, Faculty of Pharmacy, Kafrelsheikh University, Kafr El-Sheikh, Egypt; Pharmacology and

Toxicology Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt; Biochemistry Department, Faculty of science, King Abdulaziz

University, Jedda, Kingdom of Saudi Arabia; Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City,

d

e

f

g

Sadat City, Egypt

ABSTRACT

ARTICLE HISTORY

Received 1 April 2019

Revised 11 June 2019

Accepted 30 June 2019

In the designed compounds, either a biarylamide or biarylurea moiety or an N-substituted piperazine

motif was linked to position 1 of the phthalazine core. The anti-proliferative activity of the synthesised

compounds revealed that eight compounds (6b, 6e, 7b, 13a, 13c, 16a, 16d and 17a) exhibited excellent

broad spectrum cytotoxic activity in NCI 5-log dose assays against the full 60 cell panel with GI50 values

ranging from 0.15 to 8.41 mM. Moreover, the enzymatic assessment of the synthesised compounds against

VEGFR-2 tyrosine kinase showed the significant inhibitory activities of the biarylureas (12b, 12c and 13c)

KEYWORDS

Substituted phthalazines;

VEGFR-2 kinase inhibitors;

anti-proliferative; apoptosis

with IC s of 4.4, 2.7 and 2.5 lM, respectively, and with 79.83, 72.58 and 71.6% inhibition of HUVEC at

5

0

1

0 lM, respectively. Additionally, compounds (7b, 13c and 16a) were found to induce cell cycle arrest at

S phase boundary. Compound 7b triggered a concurrent increase in cleaved caspase-3 expression level,

indicating the apoptotic-induced cell death.

1

. Introduction

transduction, these inhibitors may be broadly categorised into

two main types: type I; inhibitors represent ATP competitors that

generally bind in or around the catalytic site of the kinase in its

active conformation, in the region originally occupied by the

1

Cancer is a leading cause of death worldwide . Epidemiological

studies revealed that cancer accounts for one of every five deaths.

Moreover, it is estimated that the annual number of deaths due

to cancers will increase from 7.6 million in 2008 to 13 million

4

adenine moiety of ATP . On the other hand, type-II inhibitors sta-

2

bilise the inactive conformation of the enzyme derived upon the

movement of the DFG motif (i.e. Asp-Phe-Gly), hence, they exploit

new interactions with the lipophilic pocket revealed in this new

in 2030 .

Apoptosis, or programed cell death, plays a crucial role in

maintaining the normal body health. However, malignant cells are

generally less sensitive to these stresses and tend to evade apop-

tosis. Genetic regulation, initiation and effector mechanisms can

be regarded as stages of apoptosis in its simplest model. Gamma

and ultraviolet irradiation, anticancer drugs, deprivation of survival

factors such as interleukin-1 (IL-1), or other cytokines that activate

5

rearrangement .

In the past few years, a number of VEGFR-2 inhibitors have

proven success as targeted anticancer therapeutics. Among these

is the 1,4-disubstituted phthalazine inhibitor, vatalanib. (PTK-787)

6

(

^I⁾⁽^I^C50 VEGFR-2 ¼ 43 nM) (Figure 1) .

3

The biarylurea-based inhibitor, sorafenib (II) (IC -VEGFR-

“death receptors” are considered as initiators of apoptosis . Hence,

50

2

¼ 90 nM) (Figure 1), has been approved for the treatment of

the identification of apoptosis inducers has evolved as an attract-

ive approach for the development of potential anticancer agents.

From another point of view, angiogenesis, the process of

sprouting of new blood vessel formations from pre-existing ones,

7

advanced renal cell carcinoma . Sorafenib was proved to suppress

tumour proliferation, metastasis and angiogenesis; in addition, it

was also found to be capable of inducing apoptosis, both in vitro

is also regarded as an important hallmark of cancer development. and in vivo, in a host of cancer cells. However, the underlying

8–10

Vascular endothelial growth factor family (VEGFs) are known to be mechanisms seemed to vary depending on cancer cell types

one of the key regulators of angiogenesis. Their biologic effects is Interest in developing novel biarylurea-based VEGFR-2 inhibitors

exerted by binding to extracellular domain.

has been increasingly highlighted with the clinical success of sora-

Multiple reports have detailed several small-molecule inhibitors fenib and its analogs, such as regorafenib (III) .

.

1

of VEGFR-2 acting by binding to the ATP-binding site in the intra-

Moreover, the biarylamide, (AAL993) (IV), a hybrid-design type-

cellular kinase domain resulting in diminished VEGF signal II inhibitor derived from the type-I inhibitor vatalanib, was

CONTACT Khaled A. M. Abouzid

abouzid@yahoo.com, Khaled.abouzid@pharma.asu.edu.eg

Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain

Shams University, Cairo, 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.

1

348

S. ELMELIGIE ET AL.

Figure 1. Structures of some known VEGFR-2 kinase inhibitors.

reported as a potent inhibitor of VEGFR family with VEGFR-2 IC

of 23 nM .

Also, telatinib (V), the furopyridazine derivative, emerged as a

the hydrophobic pocket revealed by the movement of the DFG

motif of the kinase in its inactive conformation .

5

0

1

2

20

Furthermore, two other series of vatalanib analogs (series D,

potent and orally available inhibitor of VEGFR-2, VEGFR-3 with E) were designed having a 4-chloroaniline moiety at position 1 of

IC s 6 and 4 nM, respectively. It is currently in clinical trials for the phthalazine and an anilino or phenoxy motif linked to the

5

0

13

gastric and colorectal cancer .

position 4 via a methylene spacer.

From another point of view, the piperazinyl moiety, the small

In addition, a sixth series of substituted piperazine-based deriv-

rigid heterocyclic motif, has been successfully incorporated in sev- atives (series F) was prepared as well, via linking the two major

14–17

eral potent kinase inhibitors

. The aryl piperazine-based deriva- fragments: the phthalazine core recognised as a kinase-privileged

tive (VI) (Figure 1) was reported to exhibit potent single-digit fragment, to the piperazine moiety aiming to offer more rigidity

18,19

15

nanomolar inhibitory activity on specific kinases

.

and to facilitate deriving spatial–activity relationships .

2

. Rationale and design

3. Chemistry

Based on the above findings, three series of phthalazine-based The pathways adopted for the synthesis of the new substituted

derivatives linked to a biarylamide (series A) or biarylurea tail phthalazine derivatives are depicted in Schemes (1–3). The key

(

series B, C) at position 1 of the phthalazine core via an amino or intermediate 1-aryl-3–(4-hydroxyphenyl)urea (1a–c), were prepared

2

1

ether linkage were designed and synthesised as potent anti-prolif- following the literature methods as illustrated in (Scheme 1(a)) .

erative derivatives as well as apoptosis inducers in tumour cells. On the other hand, cyclisation of the respective o-substituted ben-

Different substitution patterns were introduced to the terminal zoic acid derivative with hydrazine hydrate afforded the corre-

aromatic ring aiming to better occupy the hydrophobic pocket sponding phthalazinones (2a,b). Chlorination of 2a,b with

revealed by the kinase in its DFG-out conformation (Figure 2). The phosphorous oxychloride afforded the corresponding 1-chloroph-

design of these series was based on attaining new hybrid struc- thalazines (3a,b), which were used to prepare the target series of

tures from sorafenib and vatalanib while maintaining the structure 1-substituted phthalazines via reaction with various nucleophiles.

activity relationships as well as common pharmacophoric features Thus, the reaction of 3a,b with p-phenylenediamine in butanol via

shared by several VEGFR inhibitors which involve the following the displacement of the chloro atom of phthalazine with the suit-

three features; (1) a flat heteroaromatic ring system to better able nitrogen nucleophile such as p-phenylenediamine afforded

1

occupy the ATP-binding region of the kinase, (2) a hydrogen the N -(phthalazin-1-yl)benzene-1,4-diamines (4a,b). The latter

bond donor–acceptor pair represented by the urea or amide moi- compounds (4a,b) were further reacted with the respective ben-

eties that usually form hydrogen bonds with Glu 885 and Asp zoyl chloride in acetonitrile, in the presence of triethylamine to

1

046 residues, (3) a substituted terminal aryl moiety to occupy afford the target phthalazines bearing the biarylamide tail (5a–d)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1349

Figure 2. Strategy for the design of the target compounds.

(

series A). Moreover, the reaction of (4a,b) with different aryliso- Bethesda, Maryland, USA. In vitro NCI anticancer screening

cyanates in DMF yielded the biarylureas (6a–f) (series B). On the involves two-stage process, the first stage started with the evalu-

other hand, 1-aryl-3–(4-(4-substituted phthalazin-1-yloxy)phenyl)ur- ation of the selected candidates against the full NCI 60 cell lines

eas (7a–f) (series C) were obtained via reacting the respective panel representing leukaemia, non-small cell lung cancer, melan-

1

-chlorophthalazines (3a,b) with the appropriate intermediates oma, colon, CNS, breast, ovarian, renal and prostate cancers at a

(

1a–c) in refluxing acetonitrile. This latter reaction proceeds via single dose of 10 mM. NCI selection is based on degree of struc-

ipso addition by the nucleophile that gives an anion with a highly ture variation and application of computer modelling techniques

delocalised charge then followed by leaving of the chlorine atom to prioritise compounds based on their ability to add diversity to

of the respective 1-chlorophthalazines (3a,b) to afford the alkox-

yphthalazines (7a–f). Refluxing the 1-chlorophthalazines (3a,b)

with the appropriate piperazine derivative in ethanol yielded the

target phthalazines bearing the substituted piperazinyl tail (8a–j)

the NCI small molecule compound collection.

Eighteen of the submitted phthalazines (5b, 5d, 6b, 6e, 7b,

7e, 8a, 8d, 8f, 8g, 8h, 8i, 13a, 13b, 13c, 16a, 16d, and 17a)

were selected by NCI under drug discovery programme for screen-

ing against the full NCI 60 cell panel at single dose 10 mM. Among

the investigated 18 compounds, eight compounds (6b, 6e, 7b,

(

series F) in good yields (Scheme 1(b)).

As for the synthesis of the biarylureas (12a–c and 13a–c) hav-

ing a chloro substituent at 4 position of the phthalazine (Scheme

), it was started by refluxing phthalic anhydride with hydrazine

hydrate in acetic acid, followed by reflux with POCl to give the

,4-dichlorophthalazine intermediate (10). Thereafter, the target

1

3a, 13c, 16a, 16d and 17a) were selected by NCI for further

2

screening at 5-log dose molar range due to their prominent cell

growth inhibition against various cell lines.

3

1

compounds (12a–c and 13a–c) were obtained in a manner similar

to that adopted in Scheme 1(b).

4

.1.1.1. Primary in vitro antineoplastic single-dose assay. Primary

in vitro single-dose anticancer assay was evaluated against full NCI

0 cell panel. Results for each compound were presented as a

Compounds (16a–d, and 17a,b) (series D, E) were synthesised

via initial bromination of the key intermediate (14) with NBS in

presence of catalytic amount of dibenzoyl peroxide. The reaction

of the bromo derivative (15) with the respective aniline or phenol

in acetone then afforded the titled compounds (Scheme 3).

6

mean graph of the growth per cent of the treated cells compared

to the untreated control cells. Eight of the investigated phthala-

zine-based derivatives (6b, 6e, 7b, 13a, 13c, 16a, 16d and 17a)

showed a distinctive pattern of sensitivity against various NCI cell

lines (Tables S1 and S2 in Supplementary material). Thus, the biar-

ylurea-based derivatives having a terminal 4-chloro substituent

4

. Results and discussion

(6b, and 6e) exhibited remarkable broad spectrum cell growth

4.1. Biological evaluation

inhibition above 90% against various cell lines including leukae-

mia, non-small cell lung cancer, melanoma, prostate cancer and

4

.1.1. In vitro anticancer screening at full NCI 60 cell panel

Aiming to evaluate the general antitumour activity of the synthes- breast cancer cell lines. Compound 6e even exhibited lethal effect

ised phthalazines, the structures of the final compounds were sub- (above 100% inhibition) against several leukaemia, melanoma,

mitted to National Cancer Institute “NCI” (www.dtp.nci.nih.gov), renal cancer cell lines. Interestingly, it showed highly potent

1

350

S. ELMELIGIE ET AL.

(

a)

(

b)

ꢀ

Scheme 1. (a) Reagents and conditions: (i) dioxane, rt, 1 h. (b) Reagents and conditions: (i) NH

amine, butanol, 110 C, 1 h, (iv) benzoyl chlorides, acetonitile, triethylamine, 6 h (v) arylisocyanates, DMF, reflux, 8 h, (vi) 1a–c, Cs

2

NH

2

99%, propanol, 2 h (ii) POCl

3

, reflux 70 C, 2 h (iii) p-phenylenedi-

, acetonitrile, reflux, 6 h, (vii) piper-

ꢀ

2

CO

3

2 3

azines, K CO , KI, ethanol, reflux, 3 h.

inhibitory or lethal effects against most of the tested leukaemia spectrum inhibitory or lethal activity against most of the tested

and melanoma cell lines. NCI cell lines representing all the nine tumour subpanels specially

Furthermore, their analogue (7b) linked to the phthalazine those of leukaemia, colon, melanoma and breast cancer cell lines,

nucleus via an ether linkage also showed excellent broad with mean growth inhibition of 106.99%. However, its analogue

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1351

ꢀ

Scheme 2. Reagents and conditions: (i) NH

NH

2 2

acetic, reflux, 3 h (ii) POCl

3

, reflux 110 C, 1 h (iii) p-phenylenediamine, butanol, 110 C, 1 h, (iv) phenylisocyanates,

DMF, reflux, 8–10 h, (v) 1a–c, Cs CO , acetonitrile, reflux, 6–8 h.

2

3

2 3 2 3

Scheme 3. Reagents and conditions: (i) aniline derivatives, butanol, K CO , KI, reflux, 3 h (ii) NBS, dibenzoyl chloride, reflux, 24 h (iii) aniline derivatives, acetone, K CO ,

KI, reflux, 6 h (iv) phenol derivatives, NaH, acetone, reflux, 8 h.

(

7e) having a methyl substituent at 4-position of phthalazine core cancer, melanoma and breast cancer cell lines. Interestingly, its

only showed moderate cell growth inhibition against leukaemia disubstituted analogue (13c) exhibited an excellent broad spec-

and breast cell lines (Table S3 in Supplementary material ).

trum lethal activity against most of the tested NCI cell lines repre-

Thus, the unsubstituted biarylurea derivative (13a) displayed senting all the nine tumour subpanels specially those of non-small

broad spectrum cell growth inhibition above 90% against various lung cancer, colon, CNS and melanoma cell lines, with mean

cell lines including non-small cell lung cancer, melanoma, CNS growth inhibition of 145.09%. Surprisingly, the 4-chloro

1

352

S. ELMELIGIE ET AL.

substituted derivative (13b ) did not show any significant cell line required for 50% inhibition of cell viability and are provided in

growth inhibition (Table S3 in Supplementary material).

(Table S6 in supplementary material ).

For the vatalanib analogues having a 4-anilino (16a, and 16d)

Investigation of the results of the growth inhibition of the

or 4-phenoxy substituent (17a) linked to the phthalazine via tested molecules revealed that the biarylurea-based phthalazines

methylene spacer; It was found that all the three tested deriva- (series B and C) generally exhibited moderate to significant cyto-

tives displayed remarkable broad spectrum cell growth inhibition toxicity on both cell lines. This was relatively in accordance with

above 90% against various cell lines including leukaemia, melan- the NCI findings for the same series of compounds.

oma and breast cancer cell lines, with the meta methoxy analogue

Further analysis of the inhibitory results within the biarylurea-

(

16d) showing the most prominent inhibition against most of the based derivatives linked to phthalazine core via an NH-linker (ser-

tested cell lines with mean growth inhibition of 116.00%.

ies B) revealed that, the 4-chloro-3-trifluoromethyl derivatives (6c

Neither of the biarylamides (5b, and 5d) nor the piperazines and 6f), bearing an electron withdrawing substitution pattern

8a, 8d, 8f, 8g, 8h and 8i) showed any significant cell line growth similar to that of sorafenib, displayed enhanced growth inhibition

(

inhibition under the same test conditions (Table S3 in on both cell lines compared to their unsubstituted analogues (6a

Supplementary material ).

and 6d) (IC s ¼ 6.2, 6.0 and 3.2, 3.1 mM compared to 67.6, 91.2

5

0

and 67.6 and 57.5 mM respectively).

As for the biarylureas bearing an ether linker (series 7), the 4-

chloro-3-trifluoromethyl derivative (7c) similarly displayed signifi-

cant inhibiton on both cell lines, (IC50s ¼ 2.5 and 2.6 mM).

Surprisingly, its methyl analogue (7f) demonstrated weaker activ-

ity (IC50s ¼ 31 and 49 mM) compared to its unsubstituted analogue

4

.1.1.2. In vitro 5 log dose full NCI 60 cell panel assay. Eight com-

pounds (6b, 6e, 7b, 13a, 13c, 16a, 16d and 17a) were selected

by NCI for further 5 log dose screening against full NCI 60 cell

panel. All the 60 cell lines, representing nine tumour subpanels

(leukaemia, non-small cell lung cancer, colon cancer, CNS cancer,

(

7d, IC s ¼ 7.8 and 7.2 mM). However, the amide-based deriva-

prostate cancer, melanoma, ovarian cancer, renal cancer and

breast cancer) which were incubated at five different concentra-

tions (0.01, 0.1, 1, 10 and 100 mM) of each of the test compounds.

The outcomes were used to create log concentration vs % growth

inhibition curves, then three response parameters (GI 50 , TGI and

LC 50 ) were calculated for each cell line, (Tables S4 and S5 in

Supplementary material ).

50

tives (series A) and the piperazine-based derivatives have not

shown any interesting cell line inhibition which was also consist-

ent our previous results.

Unfortunately, neither the 4-chlorophthalazines (12a–c) nor the

vatalanib analogues (series D and E ) showed any significant cell

line inhibition (Table S6 in Supplementary Material ).

All investigated biarylureas (6b, 6e, 7b, 13a, and 13c) and

vatalanib analogues (16a, 16d, and 17a) revealed potent antipro-

liferative activity against most of the tested cell lines representing

the nine different subpanels with GI₅₀values between

4

.1.3. In vitro VEGFR-2 tyrosine kinase activity

.1.3.1 VEGFR-2 kinase activity at single-dose 10 lM concentra-

tion. The VEGFR-2 tyrosine kinase assay was performed in an

attempt to investigate the mechanism of potent antiproliferative

activity exerted by the synthesised compounds. The VEGFR tyro-

sine kinase assays were performed at BPS Bioscience (www.

bpsbioscience.com ). All the synthesised compounds representing

the six series were evaluated for their ability to inhibit VEGFR-2

0

.15–8.41 mM, except for 16a which was insensitive to prostate

and MCF-7 breast cancer cell lines.

With regard to the sensitivity against the tested cell lines, com-

pound (7b and 13c) exhibited the highest broad spectrum submi-

cromolar inhibitory activity against most of the tested cell lines

specially those of leukaemia, colon, melanoma, breast cancer and

renal (13c only) cell panels with GI₅₀values of 0.15–2.81 mM and tyrosine kinase at single dose of 10 mM.

Investigation of the results of VEGFR-2 inhibitory activities

among the synthesised phthalazines revealed that: Within the

biaryl ureas (series B, C): derivatives having a 4-chloro-3-trifluoro-

0

1

.2–2.66 mM for 7b and 13c, respectively. It is worth noting that

3c showed submicromolar GI50s against all tested leukaemia and

melanoma cell lines.

As for the selectivity of the test compounds towards some spe- methyl substitution pattern on the terminal phenyl ring similar to

cific tumour subpanels, which is calculated based on the ratio that of sorafenib (6c, 6f, 7c, 12c and 13c) tended to exhibit

obtained by dividing the full panel MID (the average sensitivity of enhanced VEGFR-2 inhibitory activity as excepted from cell line

all cell lines towards the test agent) by their individual subpanel results compared to their monosubstituted or unsubsti-

MID (the average sensitivity of all cell lines of a particular subpa- tuted analogues.

24,25

nel towards the test agent)

pounds were regarded to be more selective against leukaemia, the introduction of a 4-chloro group to the phthalazine core

renal, melanoma, colon and breast cancer subpanels.

replacing the 4-CH or unsubstituted derivatives, resulted in

. As per this criterion, the test com-

In accordance with cell lines results, among these biaryl ureas,

3

favourable increase in VEGFR-2 inhibitory activity (12a–c, 13a–c

compared to 6a–f, 7a–f), which might be attributed to enhanced

.1.2. In vitro cytotoxic activity against MCF-7, HCT-116 and lipophilicity of the chloro group. Thus, compounds 6c, 12b, 12c

4

HepG-2 cancer cell lines

and 13c showed 70–100% inhibition at 10 mM concentration

The growth inhibition of the rest of synthesised compounds (not (Table 1).

selected by NCI) (5a, 5c, 6a, 6c, 6d, 6f, 7c, 7d, 7f, 8b, 8c, 8e

However, the vatalanib analogues (16a–d and 17a,b) (series

and 8j) was also evaluated against two specific cell lines, namely D, E), they generally exhibited weak to moderate activity, with

MCF-7 and HCT-116 using doxorubicin as a reference drug. In the derivatives having unsubstituted anilino or phenoxy moiety

addition, compounds (12a, 12b, 12c, 16b, 16c and 17b) were (16a, 17a) showing relatively better activity than their meta sub-

evaluated against a third cell line (HepG-2) These first two cell stituted analogues (Table 2).

lines were selected based on the sensitivity of the previously NCI-

As expected from the cell lines results, neither of the amide-

tested derivatives towards them amongst the 60 NCI panel. The based derivatives (5a–d) (series A) nor the piperazines (8a–j)

growth inhibition is expressed as the median growth inhibitory (series F) showed any significant VEGFR-2 inhibition in accordance

concentration (IC50) which corresponds to the concentration with cell lines results.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1353

Table 1. The VEGFR-2 inhibition per cent of the synthesised phthalazines (series A, B, C) at 10 lM concentration.

Cpd No

X

R

1

R

2

% inhibition

5

6

1

7

1

a

b

c

d

a

b

c

d

e

f

2a

2b

2c

a

b

c

d

e

–

H

CH

H

CH

Cl

H

Cl

H

Cl

H

Cl

H

Cl

H

Cl

H

Cl

H

–

4

8

–

H

CF

H

CF

H

CF

H

CF

H

CF

H

CF

3

12

15

13

20

70

15

19

32

26

70

78

5

13

47

10

14

21

24

38

100

–

3

NH

O

3

Cl

H

CH

Cl

3

Cl

H

Cl

f

3a

3b

3c

Staurosporin

The bold values are the most active compounds.

Table 2. The VEGFR-2 inhibition per cent of the synthesised phthalazines (series

D, E, F) at 10 lM concentration.

Table 3. The IC₅₀values for compounds (6c,

12 b, 12c, 13c).

Compound ID

VEGFR-2 IC50

6

1

c

13.4 lM

4.4 lM

2.7 lM

2.5 lM

2b

2c

3c

4

.1.3.2. Measurement of potential VEGFR-2 inhibitory activity

IC50). Further investigation for the promising candidates, (6c,

2b, 12c, 13c), which exhibited VEGFR-2 inhibition percent above

% inhibition 70% at 10 mM concentration, were evaluated at five different

(

1

Cpd No

Y

X

R

1

8

6a

6b

6c

6d

7a

7b

a

b

c

d

e

NH

O

–

H

Cl

CH

OCH

H

CH

–

H

–

47

21

19

24

31

15

13

8

9

5

9

14

13

9

6

10

concentrations (0.01, 0.1, 1, 10 and 100 mM) to calculate their IC₅₀

values (Table 3 and Figure 3).

Results confirmed the significant inhibition of 4-chlorophthala-

zines bearing a biarylurea tail with a terminal 4-chloro substitution

(12b) or a terminal 4-chloro-3-CF₃substitution (12c, 13c) with

IC50s of 4.4, 2.7 and 2.5 mM respectively.

3

–

ethoxycarbonyl

phenyl

2-fluorophenyl

pyridyl

2-furoyl

ethoxycarbonyl

phenyl

2-fluorophenyl

pyridyl

H

4.1.4. In vitro HUVEC anti-proliferative assay

f

g

h

i

CH

–

3

In order to further assess the antiangiogenic activity of the prom-

ising candidates, three compounds (12b, 12c and 13c) showing

best VEGFR-2 IC s were selected to be tested for their ability to

5

0

j

2-furoyl

in vitro inhibit VEGF-induced HUVEC cell line proliferation at single

dose of 10 mM concentration.

Staurosporin

–

100

The bold values are the most active compounds.

1

354

S. ELMELIGIE ET AL.

VEGFR2

(10 m M ATP/0.2 mg/ml poly-Glu-Tyr)

(

10

m

M ATP/0.2 mg/ml poly-Glu-Tyr)

1

20

10

00

1

20

10

00

1

9

0

1

9

0

80

70

60

50

40

8

0

7

0

6

0

5

0

4

0

3

2

1

0

3

0

2

0

1

0

IC =4.4 mM

0

10

-

IC50=13.4

mM

50

-

10

-

0.5

1.5

2.5

3.5

4.5

1.5 -0.5

0.5

1.5

2.5

3.5

4.5

6c [Log(nM)]

12b [Log(nM)]

VEGFR2

10mM ATP/0.2 mg/ml poly-Glu-Tyr)

(

VEGFR2

10m M ATP/0.2 mg/ml poly-Glu-Tyr)

(

1

20

10

00

120

110

100

90

9

8

7

6

5

4

3

2

1

0

80

70

60

5

4

3

2

1

0

IC50=2.7 mM

IC =2.5 mM

5

0

-

10

-10

-

0.5

1.5

2.5

3.5

4.5

-

0.5

1.5

2.5

3.5

4.5

1

2c [Log(nM)]

13c [Log(nM)]

Figure 3. IC50 curves for compounds (6c, 12b, 12c and 13c) against the VEGFR-2 kinase.

Table 4. The effect of Compounds (12 b, 12c, 13c) on HUVEC

proliferation.

120

Compound ID

% Cell growth

% Cell inhibition

1

00

1

2b

2c

3c

20.17

27.42

28.40

79.83

72.58

71.60

8

0

6

The HUVEC anti-proliferative assay was also carried out in BPS

Bioscience Corporation, San Diego, CA, USA (www.bpsbio-

science.com).

40

Angiogenesis process comprises endothelial cell (EC) outgrowth

from the parent vessel, followed by proliferation, migration, tube

formation, alignment, and anastomosis to other vessels. Several in

vitro models have attempted to recreate this complex progression

of events . The development of atherosclerotic plaques and

angiogenesis, the role of the endothelium in the response of the

2

0

2

0

DMSO

12b

12c

13c

blood vessel wall to shear forces and stretch in addition to creat- Figure 4. The bar graphs showing the HUVECs growth percentage after treat-

ment with the target compounds.

ing a model system for the study of the regulation of endothelial

cell function had emphasised the importance of using human

umbilical vein endothelial cells (HUVECs). As most endothelial cell

assays utilise human umbilical vein endothelial cells (HUVECs) or 4.1.5. Cell-cycle analysis

bovine aortic endothelial cells (BAECs) as being relatively easy to In an attempt to elucidate the potential mechanism of action of

harvest from large blood vessels and also as representatives of the synthesised phthalazines that exhibited potent anti-prolifera-

23

vascular endothelial cells in vivo .

tive activity against the NCI cell panel but weak or moderate

The test compounds (12b, 12c and 13c) manifested significant VEGFR-2 inhibitory activity, compounds (7b and 16a) were

inhibition of HUVEC cell lines of 79.83%, 72.58% and 71.60%, selected to investigate their effect on cell-cycle progression, and

respectively, which may support their potential antiangiogenic induction of apoptosis using breast cancer adenocarcinoma cell

activity (Table 4 and Figure 4).

line (MCF-7) and colon cancer cell line (HCT-116), respectively.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1355

(

A)

(B)

(C)

DMSO

7b-24h

7b-48 h

(

D)

1

00

S

G2/M

G0/G1

Pre-G

7

5

50

25

0

DMSO

7b-24h

7b- 48h

Figure 5. Effect of compound 7b on DNA-ploidy flow cytometric analysis of MCF7 cells. MCF-7cells were treated with DMSO 0.01% or compound 7b at its GI50

0.32mM), for 24h and 48 h, and the harvested cells were subjected to Cell-cycle analysis using a FACS Calibur flow cytometer (A,B and C). Bar chart shows percentage

(

of MCF7 cells at each phase of cell cycle in DMSO and compound 7b treated cells.

From another point of view, since the inhibition of VEGF path- time-dependent increase in pre-G cell population (Figures 5 and

way has a greater effect on the induction of endothelial cell apop- 6). 13c showed excellent disruption and even more prominent

tosis and the decrease of vascular tubulogenesis, as the repair of increase in sub-G1 cell population than 7b. The observed increase

endothelial lining defects caused by endothelial apoptosis is coor- in sub-G1 cell population may imply DNA fragmentation and

dinated by growth factors, mainly by vascular endothelial growth apoptosis as a potential mechanism for 7b/13c-induced cancer

factor (VEGF), which conveys survival signals to endothelial cells cell death.

and acts as a potent antiapoptotic, proliferation stimulating fac-

Furthermore, exposure of HCT-116 cells to 16a at GI₅₀concen-

2

6

tor ; thus, the biarylurea derivative (13c) (VEGFR-2 IC ¼ 2.5 mM) tration (1.20 mM) for 24 h and 48 h also seemed to induce a signifi-

50

was selected to test its effect on cell cycle analysis and induction cant action at both S phase and sub-G1 cell population (Figure 7).

of apoptosis using breast cancer adenocarcinoma cell line

(

MCF-7).

Cell cycle is a chain of events that takes place in a cell leading

to its division and replication. G1 phase, S phase (synthesis), G2

4

.1.6. Apoptosis determination

To further investigate the ability of compounds (7b, 13c and 16a)

phase (collectively known as interphase) and M phase (mitosis) to induce apoptosis, an Annexin V (conjugated to FITC) apoptosis

are considered as the four main distinct phases of the cell cycle. detection kit was employed. This assay detects phosphatidylserine

The G1-phase, also called post-mitotic pre-synthesis phase, directly (PS) expressed on the surface of apoptotic cells and fluoresces

follows cell division. The S- or synthesis phase is characterised by green after interacting with the labelled Annexin V. During early

the process of DNA replication. The G2-, premitotic or post-syn- apoptosis, membrane asymmetry is lost, and PS translocates from

thetic phase is the time when the cell prepares to split off in two the cytoplasmic side of the membrane to the external leaflet.

cells, the actual division. Finally, in the M- or mitosis-phase div- Propidium iodide (PI), the counter stain used in this assay, has the

2

7

ision, the doubled DNA organised in chromosomes is separated .

ability to cross only compromised membranes to intercalate into

The effect of compound (7b and 13c) on the normal cell-cycle the DNA. Therefore, PI is used to detect the late stages of apop-

progression was characterised using flow cytometric analysis of tosis by the presence of red fluorescence. Exposure of MCF-7 cells

TM

the DNA ploidy in MCF-7 cells using BD FACSDIVA

software. to 7b at its GI50 (0.32 mM) for 24 h and 48 h increased the percent-

Exposure of MCF-7 cells to either 7b or 13c at their GI₅₀concen- age of Annexin-V positive cells indicating an early (lower right

tration (0.32 mM, 0.57 mM, respectively) for 24 h and 48 h was quadrant) or late (upper right quadrant) apoptosis in a time-

shown to induce a remarkable disruption in cell cycle profile dependent manner compared to DMSO treated cells (Figure 8).

and cell-cycle arrest at S-phase boundary with concurrent However, 13c and 16a increased the percentage of Annexin-V

1

356

S. ELMELIGIE ET AL.

Figure 6. Effect of compound 13c on DNA-ploidy flow cytometric analysis of MCF7 cells. MCF-7cells were treated with DMSO 0.01% or compound 13c at its GI50

0.57 mM), for 24 h and 48 h, and the harvested cells were subjected to cell-cycle analysis using a FACS Calibur flow cytometer (A, B and C). Bar chart shows percent-

(

age of MCF7 cells at each phase of cell cycle in DMSO and compound 13c treated cells.

positive cells in lower right quadrant indicating early apoptosis form by undergoing proteolytic processing. Cleaved caspase-3 fur-

(

Figures 9 and 10).

ther activates a cascade of caspases that induce cell death. Thus,

the ability of compound 7b to increase the expression of cleaved

caspase-3 was studied as a driving force in 7b-induced apoptotic

cell death. Exposure of MCF-7 cells to compound (7b) (0.32 mM)

for 24 h and 48 h resulted in time-dependent increase in cleaved

caspase-3 protein expression, indicating a pivotal involvement of

caspases in the induced cancer cell death (Figure 12).

4

.1.7. Effects of 7b on the cellular and nuclear morphology

To further examine the apoptosis inducing effect of 7b, the cellu-

lar as well as nuclear morphological changes for MCF-7 cells

treated with 7b (0.32 mM) were studied following Dapi staining.

The effect of 7b on cell morphology was initially analysed with

light microscopy. While control cells treated with DMSO exhibited

normal morphological features, cells treated with 7b for 24 and

4

8 h showed deteriorated morphological changes including cell 4.2. Molecular modelling studies

rounding, detachment and cellular fragmentation (Figure 11(A)).

Similarly, fluorescence staining further indicated that, while DMSO-

treated control cells exhibited uniformly dispersed chromatin, 7b-

treated cells showed typical apoptotic characteristics; including

condensation of chromatin (brightly stained cells), blubbing with

the appearance of nuclear fragmentation (arrow heads indicate

apoptotic nucleus) (Figure 11(B)). These results collectively con-

firms the ability of compound (7b) to induce cellular apoptosis in

MCF-7 cells as a mechanism of cancer cell death.

28

Molecular docking study was performed using AutoDock Vina

through docking of the synthesised compounds in the VEGFR-2

kinase active site. Docking study aimed to interpret the VEGFR-2

inhibitory activity of the investigated molecules and gain further

insight into their relative binding affinities and binding interac-

tions with the kinase active site. The coordinates of the VEGFR-2

structure were obtained from the crystal structure of VEGFR com-

plexed with sorafenib as its inhibitor (PDB code 4ASD), which

revealed the hydrogen bond interactions between the NH and CO

motifs of urea moiety with the backbone of Asp1046 and the car-

4

.1.8. Effect of compound 7b treatment on the expression level of boxylic acid moiety of Glu885, respectively, as well as a H-bond

cleaved caspase-3

with Cys919 residue in the hinge region of the kinase

Caspase-3 is a member of the cysteine-aspartic acid protease (cas- active site .

2

0

pase) family. Sequential activation of caspases is considered as

Owing to the fact that the chloro group of the co-crystallised

one of the important keys in cellular apoptosis process. Actually, ligand (sorafenib) is in the far proximity to any halogen-bond

29–31

caspases exists as inactive proenzymes that changed to the active acceptor in the binding site (Figure S7 in Supplementary material ),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1357

Figure 7. Effect of compound 16a on DNA-ploidy flow cytometric analysis of HCT-116 cells. HCT-116 cells were treated with DMSO 0.01% or compound 16a at its GI50

1.20mM), for 24h and 48 h, and the harvested cells were subjected to Cell-cycle analysis using a FACS Calibur flow cytometer (A,B and C). Bar chart shows percentage

(

of HCT-116 cells at each phase of cell cycle in DMSO and compound 16a treated cells.

Figure 8. Effect of compound 7b treatment on induction of apoptosis. MCF-7 cells were treated with DMSO 0.01% or compound 7b at its GI50 (0.32 mM) for 24 or

4

8h, the harvested cells were stained with Annexin V-FITC apoptosis detection kit and Analyzed on a Flow cytometer.

we excluded investigating halogen-bond contacts in our docking-

based assessment.

The relative higher inhibitory activity of 6c compared to similar

analogues of 6a and 6b is most probably attributable to the fact

The best-scored pose of the biarylurea derivative (6c) repro- that 6c possessing additional halogen group (trifluoromethyl

duced the key interactions of the co-crystallised ligand (sorafenib) group) on the phenyl moiety which contributes to an additional

which includes H-bonding interactions of Glu885 and Asp1046 hydrophobic interaction, this also agrees with the predicted score

2

8

with the urea group of 6c. In addition, other hydrophobic interac- of the best poses by AutoDock Vina (Table 5). More information

tions can be observed for the hydrophobic side chains of Ile892, about rationalising the docking poses of the 6d, 6e and 6f can

Leu1019 and Val898 with the halogen groups (chloro and trifluor- be found in (Figure S8 in Supplementary material ).

omethyl) of the phenyl moiety of 6c. Nevertheless, losing the

Unlike 6c, the predicted best scored poses of both biarylurea

H-bonding interaction of the Cys 919 with the terminal amide derivatives 12b and 12c showed a flip of 180 degrees compared

group of sorafenib can explain the lower activity of 6c compared to sorafenib and biarylurea compound (6c) (Figure 13(B)). This

to sorafenib (Figure 13(A)).

implies that the chlorophthalazine moiety of both derivatives 12b

1

358

S. ELMELIGIE ET AL.

Figure 9. Effect of compound 13c treatment on induction of apoptosis. MCF-7 cells were treated with DMSO 0.01% or compound 13c at its GI50 (0.57 mM) for 24 or

8h, the harvested cells were stained with Annexin V-FITC apoptosis detection kit and Analyzed on a Flow cytometer.

4

Figure 10. Effect of compound 16a treatment on induction of apoptosis. HCT-116 cells were treated with DMSO 0.01% or compound 16a at its GI50 (1.20 mM) for 24

or 48h, the harvested cells were stained with Annexin V-FITC apoptosis detection kit and Analyzed on a Flow cytometer.

and 12c is directed towards the 4-chloro-3-trifluoromethylphenyl 5. Conclusion

moiety of sorafenib. Avoiding a possible clash of the chloro group

Four series of phthalazine-based derivatives bearing biarylamides

with the backbone of Cys 919 can explain this flipping behaviour

(

5a–d), biarylurea (6a–f, 7a–f, 12a–c and 13a–c) or a substituted

(

Figure S9 in Supplementary material ). The flipped pose showed

piperazine moiety at position 1 of the phathalazine nucleus

8a–j), in addition to two other series of vatalanib analogs

16a–d, and 17a,b) were designed, synthesised as anticancer

interaction pattern in the binding site that maintained the H-

bonding interaction of Glu885, however, in this case with the

amino group of 12b and 12c. Also, chlorophthalazine ring was

packed in the hydrophobic region of Ile892, Val898 and Leu1019.

Similarly, the chlorophenyl moiety of 12b and the 4-chloro-3-tri-

fluoromethylphenyl moiety of 12c showed hydrophobic contacts

with the side chains of Phe918 and Leu840 (Figure 13(B,D)).

Owing to the existence of additional hydrophobic contacts of 12c

(

agents exerting potent anti-proliferative activity as well as apop-

tosis inducers in tumour cells. Eight of the phthalazines deriva-

tives; namely the biarylureas (6b, 6e, 7b, 13a and 13c) and the

vatalanib analogues (16a, 16d and 17a) exhibited excellent broad

spectrum anti-proliferative activity in NCI 5-log dose screening

against 60 cell panel with GI₅₀values ranging from 0.15 to

(extra halogens) compared to 12a and 12b, the predicted scores

8

.41 mM, especially on leukaemia, renal, melanoma and breast can-

cer cell lines. Accordingly, the three compounds (7b, 13c and

6a) were selected to further investigate the potential underlying

showed relative improvement (Table 5). This also agrees with the

marginal improvement of the observed inhibitory activity of 12c

compared to similar analogs. Additional Figures of the docked

poses of 12a, 12b and 12c can be found in the (Figure S10 in

Supplementary material ).

1

mechanisms that induced cancer cell death. The latter three com-

pounds were found to induce cell-cycle arrest with concurrent

increase in pre-G cell population and increase the apoptotic cell

Like 12c, the predicted pose of 13c showed the same flip and population in time-dependent manner. Moreover, compound (7b)

relatively the same interaction pattern. Only exception is that the was found to disrupt the normal cellular and nuclear morphology

isosteric ether link in 13c showed H-bonding interaction with and increase the expression of cleaved caspase-3, the pro-apop-

Asp1046 instead of Glu885 with 12c as shown in (Figure 13(C)). totic protein, in MCF-7 cell lines which collectively indicate the

This agrees with the biological activity observed for both 12c involvement of these compounds in apoptotic-induced cell death.

and 13c.

Moreover, all the synthesised phthalazines were evaluated for

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1359

(

A)

DMSO

7b-24h

7b-48h

(

B)

DMSO

7b-24h

7b-48h

Figure 11. Effect of compound 7b on the cellular and nuclear morphology. MCF-7 cells were seeded on cover slips in a 6- well tissue culture plate, the cells then

were treated with DMSO 0.01% or compound 7b (0.32 mM) for 24 or 48h, the cells were imaged with the light microscope to examine the cellular morphology (A) or

stained with the nuclear stain Dapi to study the nuclear morphology using fluorescence microscope (B).

C- cas-3

DAPI

Merge

DMSO

7

b-48h

Figure 12. Effect of compound 7 b on cleaved caspase-3 protein expression. MCF-7 cells were seeded on cover slips in a 6-well tissue culture plate, the cells then

were treated with DMSO 0.01% or compound 7b at its GI50(0.32 mM) for 48h, the cells were then permiabilised, fixed, blocked and stained with the primary and sec-

ondary antibodies, the nuclear stain Dapi was used as a counter stain. The image were taken using a fluorescence microscope.

their VEGFR-2 inhibitory activity, which revealed the significant significant inhibition of VEGF-stimulated proliferation of human

inhibition of the 4-chlorophthalazines having a biarylurea tail with umbilical vein endothelial cells (HUVEC) with 79.83, 72.58 and

either a terminal 4-chloro (12b) or a terminal 4-chloro-3-trifhuoro- 71.6% inhibition, respectively, at 10 mM concentration. Finally,

methyl substituents (12c and 13c) with IC₅₀of 4.4, 2.7 and molecular docking study of the active biarylurea compounds (6c,

2

.5 mM, respectively. 4-chloropthalazines were found to exhibit 12b, 12c and 13c) in VEGFR-2 kinase active site revealed their

better VEGFR-2 activity than their corresponding 4-methyl or ability to form the essential H bond interactions with Glu 885 or

-unsubstituted analogues. 12b, 12c and 13c also displayed Asp1046 key residues in the VEGFR-2 active site.

4

1

360

S. ELMELIGIE ET AL.

Figure 13. Overlay of the best-scored docking poses of some relevant compounds on the co-crystallised ligand Sorafenib (cyan sticks) in the binding site of VEGFR2

PDB: 4ASD):. (A) The docking pose of 6c as magenta sticks, (B) the docking poses of 12b and 12c as yellow and purple sticks, respectively, and (C) the docking pose

(

of 13c as gold sticks. (D) The docking poses of 12c and 13c at a different perspective of the binding site showing the extended hydrophobic contact of the chloro

and trifluoromethyl phenyl moiety with the side chains of PHE-918 and LEU-840. The yellow dashed-lines represent the polar contacts (H-bonding interactions). Non-

2

8

polar hydrogen atoms were omitted for clarity. All presented docking poses are the best-scored by AutoDock Vina.

Coupling patterns are described as follows: s, singlet; d, doublet,

dd, doubled doublet; t, triplet; m, multiplet. J describes a coupling

Table 5. The score of best-scored poses of some relevant compounds. The score

is expressed as a binding affinity (kcal/mol) as an output of AutoDock Vina.

a

Compound Number

Score (SD)

Average Number of Poses Per Run constant. The coupling constants were rounded off to one decimal

6

1

a

b

c

2a

2b

2c

3a

3b

3c

ꢁ11.4 (0)

ꢁ11.5 (0)

ꢁ12.4 (0.06)

ꢁ11.0 (0)

ꢁ11.1 (0.06)

ꢁ11.5 (0)

ꢁ10.7 (0)

ꢁ10.8 (0)

ꢁ11.0 (0)

4.6

5.3

4.3

5

8.3

9

place. MS spectra mass were recorded on Hewlett Packard 5988

spectrometer (70 eV). Elemental analyses were performed at the

21

Microanalytical Center, Al-Azhar University. Compounds 1a–c ,

3

2

33

34

2a,b

and 3a,b ,

9

and 10

were prepared following

reported procedures.

The shown score is the mean of three consecutive runs. The docking method

was validated by successful pose-retrieval docking experiment of Sorafenib

1

6

.1.1. Synthesis of N -(4-substituted-phthalazin-1-yl)benzene-1,4-

(

score: ꢁ12.3(0)). SD is the standard deviation.

diamine (4a,b)

General procedure:

p-Phenylenediamine (0.33 g, 3.12 mmol, 3equiv) and the

respective chlorophthalazine (2.08 mmol, 2equiv) were treated

ꢀ

with 2-BuOH (7.5 ml) in a tube and heated to 110 C. The reaction

6

. Experimental

quickly became a solid, yellow mass. After four hours, the reaction

was cooled and diluted with water. The resultant slurry was then

6.1. Chemistry

Starting materials and reagents were purchased from Sigma – partitioned between equal volumes of DCM and 1 N NaOH (15 ml).

Aldrich or Acros Organics. Melting points were recorded on Gallen The aqueous layer was extracted into DCM (2x15 ml). The com-

Kamp apparatus and were uncorrected. FT-IR spectra were bined organic layers were dried over anhydrous sodium sulfate, fil-

1

recorded on a Shimadzu IR 435 spectrophotometer. HNMR spec- tered, and concentrated in vacuo. The resulting orange solid was

tra were recorded in d scale given in ppm on a Varian 400 MHz crystallised from (EtOAc/pet ether 1:1) to afford the title com-

spectrophotometer or

a Varian 300 MHz spectrophotometer. pounds (4a,b) as an orange crystals.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1361

1

6

9

–

8

.1.1.1. N -(Phthalazin-1-yl)benzene-1,4-diamine (4a). Yield (0.21 g,

C

21

H

15ClN

): d 9.15 (1H, s, N, 15.22.

2 2

D O exchangeable), d

4

O: C, 67.29; H, 4.03; N, 14.95; Found: C, 67.52; H, 4.08;

ꢀ

1

1%); mp 284–286 C; HNMR (300 MHz, DMSO-d

NH D O exchangeable), 9.04 (2H, s, –NH

6

2

.95–8.92 (2H, d, J ¼ 9 Hz, phthalazine), d 8.18–8.15 (2H,d, J ¼ 9 Hz,

6.1.2.3. N-(4–(4-Methylphthalazin-1-ylamino)phenyl)benzamide(5c).

ꢀ

1

phthalazine), d 7.97 (1H, s, phthalazine), d 7.65–7.62 (2H,d, Yield (0.24 g; 68%), mp 283–285 C; HNMR (400 MHz, DMSO-d ):

6

J ¼ 9 Hz, ArH), d 6.97–6.83 (2H,d, J ¼ 9 Hz, ArH); FT-IR (ύ max, d 10.50 (1H, s, –NH D O exchangeable), d 10.40 (1H, s, –NH D O

2

ꢁ

1

cm ): 3400 (NH

4

2

), 3329 (NH); MS (Mwt.: 236.11): m/z 236.00 (Mþ, exchangeable), d 8.27–8.24 (2H, d, J ¼ 7.6 Hz, phthalazine), d

3.00%), 212.00 (100.00%), 118.00 (54.00%), 56.00 (70.00%); Anal. 8.02–8.00 (2H, m, Ar-H), d 7.99–7.97 (2H,d, J ¼ 7.6 Hz, phthalazine),

Calcd for C14

.17; N, 23.96

12

H N

4

: C, 71.17; H, 5.12; N, 23.71; Found: C, 71.34; H, d 7.77–7.73 (1H, d, J ¼ 9.2 Hz, Ar-H), d 7.63–7.61 (2H,d, J ¼ 7.2 Hz,

5

Ar-H), d 7.59–7.56 (2H,d, J ¼ 7.6 Hz, Ar-H), d 7.54–7.51 (2H, m, Ar-

ꢁ

1

3

H), d 2.91 (3H,s, CH ); FT-IR (ύ max, cm ): 3331, 3257 (2NH), 1649

1

(C¼O); MS (Mwt.: 354.15): m/z 354.95 (Mþ, 1.18%), 315.95

6

.1.1.2. N -(4-Methylphthalazin-1-yl)benzene-1,4-diamine (4b).

ꢀ

1

(15.90%), 105.00 (100.00%); Anal. Calcd for C H N O: C, 74.56; H,

Yield (0.22 g, 88%); mp 240–242 C; HNMR (300 MHz, DMSO-d

d 8.88 (1H, s, –NH D O exchangeable), 8.85 (2H, s, –NH

exchangeable),

8.13–8.08 (2H, d, J ¼ 7 Hz, phthalazine),

6

):

O

d

22 18 4

5

.12; N, 15.81; Found: C, 74.78; H, 5.19; N, 16.04

2

D

2

d

7

.95–7.92 (2H, d, J ¼ 7 Hz, phthalazine), d 7.46–7.43 (2H, d, J ¼ 9 Hz, 6.1.2.4. 4-Chloro-N-(4–(4-methylphthalazin-1-ylamino)phenyl)ben-

ꢀ

1

ArH), d 6.83–6.76 (2H, d, J ¼ 9 Hz, ArH), d 2.78 (3H, s, CH

3

); zamide (5d). Yield (0.20 g; 51%), mp 147–149 C; HNMR

): d 10.65 (1H, s, –NH D O exchangeable), d

13

CNMR (100 MHz, DMSO): d 162.4, 150.5, 135.2, 134.5, 131.2, (400 MHz, DMSO-d

6

2

1

30.7, 128.0, 127.3, 125.0, 122.8, 120.7, 120.2, 115.6, 115.3, 18.2; 10.45 (1H, s, –NH D

2

O exchangeable), d 8.05–8.03 (2H, d, J ¼ 8 Hz,

ꢁ

1

phthalazine), d 7.97–7.95 (2H,d, J ¼ 7.6 Hz, Ar-H), d 7.88–7.86 (2H,

FT-IR (ύ max, cm ): 3385 (NH ), 3321 (NH); MS (Mwt.: 250.12): m/

2

d, J ¼ 8 Hz, phthalazine), d 7.63–7.61 (2H, d, J ¼ 7.6 Hz, Ar-H), d

z 250.00 (Mþ, 50.00%), 249.00 (100.00%), 108.00 (60.92%), 65.00

7

.59–7.57 (2H,d, J ¼ 7.6 Hz, Ar-H), d 7.31–7.28 (2H, m, Ar-H), d 2.91

13

(

77.59%); Anal. Calcd for C H N : C, 71.98; H, 5.64; N, 22.38;

15 14 4

3

(3H, s,CH ); CNMR (100 MHz, DMSO): d 166.9, 165.0, 151.5,

Found: C, 72.09; H, 5.78; N, 22.52.

1

38.45, 138.2, 137.5, 137.0, 136.9, 134.6, 133.9, 131.6, 130.9, 130.2,

30.2, 130.1, 129.2, 128.9, 128.1, 125.3, 121.5, 121.2, 18.9; FT-IR (ύ

1

6

.1.2. Synthesis of N-(4–(4-Substitutedphthalazin-1-ylamino)- max, cm- ): 3300, 3192 (2NH), 1678 (C¼O); MS (Mwt.: 388.85): m/z

3

88.90 (Mþ, 7.10%), 279.90 (7.84%), 248.95 (25.65%), 139.00

100%); Anal. Calcd for C22 17ClN O: C, 67.95; H, 4.41; N, 14.41;

Found: C, 68.24; H, 4.48; N, 14.49.

phenyl)-4-substituted-benzamides (5a-d)

General procedure:

To a stirred solution of the respective N1-(4-arylphthalazin-1-

yl)benzene-1,4-diamine (4a,b) (10.0 mmol, 1equiv) and triethyl-

amine (1.1 ml, 10.0 mmol) in acetonitrile (20 ml), the respective

benzoyl chloride (viz.; benzoyl chloride, 4-chlorobenzoyl chloride)

(

H

4

6.1.3. Synthesis of 1-Aryl-3–(4-(4-substitutedphthalazin-1-ylamino)-

phenyl)ureas (6a-f)

General procedure:

To a stirred solution of the respective N1-(4-arylphthalazin-1-

yl)benzene-1,4-diamine (4a,b) (10.0 mmol, 1equiv) in DMF (20 ml),

the respective phenylisocyanate (viz.; phenyl- isocyanate, 4-chloro-

(

10.0 mmol, 1equiv) was added and the mixture was heated under

reflux for 6 h, until the disappearance of starting material as

judged by TLC (CHCl /CH OH 9.5:0.5). The reaction mixture was fil-

3

tered and then the filtrate was concentrated in vacuo, to afford

the crude product which was purified by flash column chromatog-

raphy (EtOAc/pet ether 9:1) to afford 5a–d as yellow crystals.

phenylisocyanate,

10.0 mmol, 1 equiv) was added and the mixture was heated under

reflux for 6 h, after which TLC (CHCl /CH OH 9:1) showed no start-

3-trifluoromethyl-4-chlorophenylisocyanate)

(

3

6

.1.2.1. N-(4-(Phthalazin-1-ylamino)phenyl)benzamide (5a). Yield

ing material. The reaction mixture was poured over ice water, the

formed precipitate was allowed to settle, then filtered off and

dried to afford the crude products(6a-f) which was further crystal-

lised from EtOAc.

ꢀ

1

(

9

0.17 g; 50%), mp 142–144 C; HNMR (400 MHz, DMSO-d

.66 (1H, s, –NH D O exchangeable), d 9.12 (1H, s, –NH D

exchangeable), d 8.62 (1H, s, J ¼ 8 Hz, phthalazine), d 8.11–8.08

6

): d

2

O

(

7

2H, d, J ¼ 7.6 Hz, Ar-H), d 7.98–7.96 (2H, d, J ¼ 8 Hz, phthalazine), d

.93–7.88 (2H, m, phthalazine), d 7.64–7.62 (1H, m, Ar-H), d

.62–7.60 (2H, d, J ¼ 7.6 Hz, Ar-H), d 7.59–7.57 (2H, d, J ¼ 6.8 Hz, Ar-

6.1.3.1. 1-Phenyl-3–(4-(phthalazin-1-ylamino)phenyl)urea (6a).

ꢀ

1

Yield (0.10 g; 30%), mp 145– 147 C; HNMR (400 MHz, DMSO-d ):

d 9.22 (1H, s,–NH D O exchangeable), d 9.16 (1H,s, –NH D O

2 2

exchangeable), d 9.09 (1H, s, –NH D O exchangeable), d 8.74 (1H,

s, phthalazine), d 8.68–8.66 (2H, d, J ¼ 8 Hz, phthalazine), d

6

ꢁ

1

H), d 7.50–7.48 (2H, m, Ar-H); FT-IR (ύ max, cm ): 3550, 3510

1

(

1

2NH), 1649 (C¼O); MS (Mwt.: 340.38): m/z 340.15 [M , 1.58%),

2

38.05 (2.70%), 86.10 (100%); Anal. Calcd for C H N O: C, 74.10;

21

16

4

H, 4.74; N, 16.46; Found: C, 74.34; H, 4.80; N, 16.58.

8

7

.02–8.00 (2H, d, J ¼ 8 Hz, phthalazine), d 7.58–7.56 (2H, m, Ar-H), d

.48–7.46 (2H, d, J ¼ 7.6 Hz, Ar-H), d 7.31–7.29 (2H,d, J ¼ 7.2 Hz, Ar-

6

.1.2.2.

4-Chloro-N-(4-(phthalazin-1-ylamino)phenyl)benzamide H), d 7.19–7.17 (1H, m, Ar-H), d 6.98–6.96 (2H, d, J ¼ 7.2 Hz, Ar-H)

1

ꢁ1

ꢀ

(

5b). Yield (0.20 g; 54%), mp 156–158 C; HNMR (400 MHz, FT-IR (ύ max, cm ): 3300, 3047 (3NH), 1700 (C¼O); MS (Mwt.:

DMSO-d ): d 10.50 (1H, s, –NH D

NH D O exchangeable), d 8.99 (1H, s, phthalazine), d 8.11–8.08

2H, d, J ¼ 9 Hz, Ar-H), d 7.94–7.92 (2H,d, J ¼ 7.6 Hz, phthalazine), d

.91–7.90 (2H, d, J ¼ 7.6 Hz phthalazine), 7.56–7.54 (2H,d,

J ¼ 9 Hz, Ar-H), d 7.53–7.51 (2H,d, J ¼ 7.5 Hz, Ar-H), d 7.31–7.28 (2H, 6.1.3.2.

O exchangeable), d 9.15 (1H, s, 355.14): m/z 355.10 (Mþ, 8.88%), 289.10 (9.51%), 123.10 (15.43%),

6

2

6

1

9.00(100.00%); Anal. Calcd for C H N O: C, 70.97; H, 4.82; N,

9.71; Found: C, 71.21; H, 4.89; N, 19.88

–

(

2

21 17

5

7

d

1–(4-Chlorophenyl)-3–(4-(phthalazin-1-ylamino)pheny-

ꢁ

1

ꢀ

1

m, Ar-H); FT-IR (ύ max, cm ): 3355, 3310 (2NH), 1640 (C¼O); MS l)urea (6b). Yield (0.20 g; 53%), mp 168–171 C; HNMR (400 MHz,

(

Mwt.: 374.82): m/z 375.00 (Mþ, 1.57%), 245.00 (2.41%), 139.05 DMSO-d ): d 10.33 (1H, s, –NH D O exchangeable), d 10.07 (1H, s,

6

2

2 2

46.44%), 111.10 (25.30), 64.00 (100.00%); Anal. Calcd for –NH D O exchangeable), d 9.16 (1H, s, –NH D O exchangeable), d

1

362

S. ELMELIGIE ET AL.

8

.96 (1H, s, phthalazine), d 8.09–8.07 (2H, d, J ¼ 9.2 Hz, phthala- 125.6, 124.8, 123.9, 122.1, 121.8, 121.4, 119.8, 119.5, 118.8, 118.6,

ꢁ1

zine), d 7.63–7.61 (2H, d, J ¼ 9.2 Hz, phthalazine), d 7.49–7.47 (2H, 117.6, 115.4, 19.6; FT-IR (ύ max, cm ): 3363, 3255, 3153 (3NH),

d, J ¼ 8.8 Hz, Ar-H), d 7.44–7.42 (2H, d, J ¼ 8 Hz, Ar-H), d 7.33–7.31 1681(C¼O); MS (Mwt.: 471.86): m/z 472.10(Mþ, 0.30%), 223.00

13

(

d, J ¼ 8.8 Hz, 2H, Ar-H), d 6.56–6.54 (2H,d, J ¼ 8 Hz, Ar-H); CNMR (43.56%), 195.00 (70.63%), 52.05 (100%); Anal. Calcd for

100 MHz, DMSO): d 160.2, 153.0, 145.4, 139.4, 137.6, 135.8, 134.8, C H ClF N O: C, 58.54; H, 3.63; N, 14.84; Found: C, 58.61; H, 3.61;

23 17 3 5

1

33.2, 132.6, 129.7, 129.2, 128.9, 127.7, 122.6, 122.4, 121.8, 121.5, N, 15.03

1

20.3, 119.9, 118.6, 115.6; FT-IR (ύ max, cmꢁ ): 3396, 3341 (3NH),

672 (C¼O); MS (Mwt.: 389.10): m/z 389.00 (Mþ, 1.51%), 281.95

6.1.4.

1-Aryl-3–(4-(4-substituted-phthalazin-1-yloxy)phenyl)ureas

(19.41%), 253.95(100.00%), 152.95 (13.00%); Anal. Calcd for

C H ClN O: C, 64.70; H, 4.14; N, 17.96; Found: C, 64.89; H, 4.21; (7a-f)

2

1

16

5

N, 18.17.

General procedure:

To a stirred solution of the respective chlorophthalazine deriva-

tive (3a,b) (10.0 mmol, 1equiv), and cesium carbonate (6.5 g,

20.0 mmol, 2equiv) in acetonitrile (20 ml), the appropriate 1-aryl-

3–(4-hydroxyphenyl)urea (1a-c) (10.0 mmol, 1equiv) was added

and the mixture was heated under reflux for 6 h, after which TLC

6

.1.3.3. 1–(4-Chloro-3-(trifluoromethyl)phenyl)-3–(4-(phthalazin-1-

ꢀ

ylamino)phenyl)urea (6c). Yield (0.22 g; 49%), mp 288–291 C;

1

HNMR (400 MHz, DMSO-d ): d 10.08 (1H, s, –NH D O exchange-

6

2

able), d 9.36 (1H, s, –NH D O exchangeable), d 9.11 (1H, s, –NH

D O exchangeable), d 8.22 (1H, s, Ar-H), 8.09–8.07 (2H, d,

(

CHCl /CH OH 9:1) showed no starting material. The filtrate was

3 3

2

evaporated in-vacuo to afford the crude product (7a-f) which was

further purified by column chromatography (using gradient elu-

tion starting from EtOAc then 1% MeOH/EtOAc).

J ¼ 8.8 Hz, phthalazine), d 7.97–7.95 (2H, d, J ¼ 8.8 Hz, phthalazine),

d 7.79 (1H, s, Ar-H), d 7.69–7.67 (2H, d, J ¼ 9.6 Hz, Ar-H), d

7

.52–7.50 (2H, d, J ¼ 8.4 Hz, Ar-H), d 7.39–7.37 (2H, d, J ¼ 8.4 Hz, Ar-

ꢁ1

H); FT-IR (ύ max, cm ): 3321, 3263, 3126 (3NH), 1649 (C¼O); MS

(

Mwt.: 457.09): m/z 457.90 (Mþ, 1.06%), 415.90 (7.68%), 6.1.4.1. 1-Phenyl-3–(4-(phthalazin-1-yloxy)phenyl)urea (7a). Yield

ꢀ

1

94.95(100.00%); Anal. Calcd for C22

H15ClF

3

N

5

O: C, 57.71; H, 3.30; (0.22 g; 64%), mp 180–182 C; HNMR(300 MHz, DMSO-d

): d 9.57

6

N, 15.30; Found: C, 57.93; H, 3.28; N, 15.48

(1H, s, –NH D O exchangeable), d 9.37 (1H, s, –NH D O exchange-

2

able), d 8.36 (1H, s, phthalazine), d 8.10–8.00 (2H, m, phthalazine),

d 7.93–7.91 (2H, d, J ¼ 7 Hz, phthalazine), d 7.91–7.90 (2H, m, Ar-H),

d 7.49–7.47 (2H, d, J ¼ 7.8 Hz, Ar-H), d 7.19–7.25 (m, 3H, Ar-H), d

6

.1.3.4.

rea(6d). Yield (0.06 g; 20%), mp 284–286 C; HNMR (300 MHz,

DMSO-d ): d 9.20(1H, s, –NH D O exchangeable), d 9.14 (1H, s,–NH

D O exchangeable), d 9.06 (1H, s,–NH D O exchangeable), d

1–(4-(4-Metyhlphthalazin-1-ylamino)phenyl)3-phenylu-

ꢀ

1

6

.89–6.86 (1H, d, J ¼ 7.5 Hz, Ar-H), d 6.67–6.65 (1H, d, J ¼ 7.8 Hz, Ar-

ꢁ1

6

2

H); FT-IR (ύ max, cm ): 3304, 3235 (2NH), 1641 (C¼O); MS (Mwt.:

2

þ

3

56.38): m/z 355.00 (M-1 , 0.11%), 146.05 (23.71%), 118.05

8

.93–8.91 (1H, d, J ¼ 7 Hz, phthalazine), d 8.77–8.75 (1H, d, J ¼ 7 Hz,

(

8.47%), 79.95 (100.00%); Anal. Calcd for C21

H

16 4

N O

2

: C, 70.77; H,

phthalazine), d 8.22–8.20 (2H, d, J ¼ 7 Hz, phthalazine), d 7.85–7.83

4

.53; N, 15.72; Found: C,71.02; H, 4.61; N, 15.89

(

7

6

2H, d, J ¼ 8.4 Hz, Ar-H), d 7.52–7.49 (2H, d, J ¼ 8.4 Hz, Ar-H), d

.43–7.40 (2H, d, J ¼ 7.2 Hz, Ar-H), d 7.25–7.23 (1H, m, Ar-H), d

.95–6.93 (2H, d, J ¼ 7.2 Hz, Ar-H), d 2.81 (3H, s, CH

);) ; FT-IR (ύ 6.1.4.2.

1–(4-Chlorophenyl)-3–(4-(phthalazin-1-yloxy)phenyl)urea

3

ꢁ

1

ꢀ

1

max, cm ): 3292, 3194, 3132 (3NH), 1651 (C¼O); MS (Mwt.: (7b). Yield (0.19 g; 50%), mp 166–168 C; HNMR (400 MHz,

69.16): m/z 369.00 (Mþ, 2.99%), 212.00 (6.90%), 93.00 DMSO-d ): d 9.72 (1H, s, –NH D O exchangeable), d 9.44 (1H, s,

100.00%);Anal. Calcd for C H N O: C, 71.53; H, 5.18; N, 18.96; –NH D O exchangeable), d 8.37 (1H, s, phthalazine), d 8.28–8.26

3

(

6

2

22 19 5

2

Found: C, 71.69; H, 5.27; N, 19.21

(2H, d, J ¼ 8 Hz, phthalazine), d 8.18–8.16 (2H, d, J ¼ 8 Hz, phthala-

zine), d 7.58–7.56 (2H, d, J ¼ 8.8 Hz, Ar-H), d 7.56–7.53 (2H, d,

J ¼ 8.8 Hz, Ar-H), d 7.36–7.34 (2H, d, J ¼ 7.2 Hz, Ar-H), d 7.14–7.12

6

.1.3.5. 1–(4-Chlorophenyl)-3–(4-(4-methylphthalazin-1-ylamino)-

ꢁ

1

ꢀ

1

(2H, d, J ¼ 7.2 Hz, Ar-H); FT-IR (ύ max, cm ): 3278, 3161 (2NH),

phenyl)urea (6e). Yield (0.20 g; 50%), mp 178–180 C; HNMR

400 MHz, DMSO-d ): d 9.20(1H, s, –NH D O exchangeable), d

.17 (1H, s, –NH D O exchangeable), d 9.03 (1H, s, –NH D O

1

701 (C¼O); MS (Mwt.: 390.82): m/z 390.10 (4.47%), 365.10

24.02%), 75.00 (100.00%);Anal. Calcd for C21 15ClN : C, 64.54;

H, 3.87; N, 14.34; Found: C, 64.69; H, 4.01; N, 14.62

(

6

2

(

H

4 2

O

9

2

exchangeable), d 8.18–8.16 (2H, d, J ¼ 7.6 Hz, phthalazine), d

8

.09–8.05 (2H, d, J ¼ 7.6 Hz, phthalazine), d 7.86–7.84 (2H, d,

J ¼ 8.8 Hz, Ar-H), d 7.73–7.71 (2H, d, J ¼ 8.8 Hz, Ar-H), d 7.49–7.47 6.1.4.3. 1–(4-Chloro-3-(trifluoromethyl)phenyl)-3–(4-(phthalazin-1-

ꢀ

1

(

2H, d, J ¼ 8.8 Hz, Ar-H), d 7.33–7.30 (2H, d, J ¼ 8.8 Hz, Ar-H), d 2.80 yloxy)phenyl)urea (7c). Yield (0.22 g; 49%), mp 158–160 C; HNMR

ꢁ1

3

6 2

3H, s,CH ); FT-IR (ύ max, cm ): 3294, 3188, 3168 (3NH), 1681 (400 MHz, DMSO-d ): d 9.91 (1H, s, –NH D O exchangeable), d

C¼O); MS (Mwt.: 403.86): m/z 403.00 (Mþ, 1.51%), 276.95 9.08 (s, 1H, –NH D O exchangeable), d 8.39 (s, 1H, phthalazine), d

2

17.01%), 127.00 (100.00%); Anal. Calcd for C22

H

18ClN

5

O: C, 65.43; 8.24–8.21 (d, J ¼ 8 Hz, 2H, phthalazine), d 8.17–8.15 (d, J ¼ 8 Hz, 2H,

phthalazine), d 7.99 (s, 1H, Ar-H), d 7.85–7.83 (d, J ¼ 7.6 Hz, 1H, Ar-

H), d 7.76–7.74 (d, J ¼ 7.6 Hz, 1H, Ar-H), d 7.28–7.26 (d, J ¼ 8.4 Hz,

H, 4.49; N, 17.34; Found: C, 65.62; H, 4.52; N, 17.52.

1

3

2

H, Ar-H), d 6.76–6.74 (d, J ¼ 8.4 Hz, 1H, Ar-H); CNMR (100 MHz,

6

.1.3.6.

1–(4-Chloro-3-(trifluoromethyl)phenyl)-3–(4-(4-methyl-

DMSO): d171.0, 153.7, 153.2, 141.1, 138.7, 133.8, 132.7, 132.0,

phthalazin-1-ylamino)phenyl)-urea (6f). Yield (0.30 g; 63%), mp

ꢀ

1

131.7, 129.5, 129.3, 129.1, 128.5, 127.0, 125.8, 124.7, 121.4, 121.2,

2

58–260 C; HNMR (400 MHz, DMSO-d

6 2

): d 9.43(1H, s, –NH D O

ꢁ

1

(

21.0, 119.6, 118.7, 115.6; FT-IR (ύ max, cm ): 3300, 3153 (2NH),

679 (C¼O); MS (Mwt.: 458.82): m/z 458.10 (2.63%), 271.05

exchangeable), d 9.14 (1H, s, –NH D O exchangeable), d 9.02 (1H,

2

s, –NH D O exchangeable), d 8.88–8.86 (2H, d, J ¼ 8 Hz, phthala-

2

7.73%), 195.00 (100.00%); Anal. Calcd for C H ClF N O : C,

zine), d 8.24–8.22 (2H, d, J ¼ 8 Hz, phthalazine), d 8.13 (1H, s,Ar-H),

d 7.97–7.95 (1H, d, J ¼ 9.2 Hz, Ar-H), d 7.88–7.86 (1H, d, J ¼ 9.2 Hz,

Ar-H), d 7.63–7.61 (2H, d, J ¼ 10 Hz, Ar-H), d 6.78–6.76 (2H, d,

22 14 3 4 2

5

7.59; H, 3.08; N, 12.21; Found: C, 57.82; H, 3.06; N, 12.57

1

3

J ¼ 10 Hz, Ar-H), d 2.80 (3H, s, CH ); CNMR (100 MHz, DMSO): d 6.1.4.4. 1–(4-(4-Methylphthalazin-1-yloxy)-3-phenylurea (7d). Yield

3

ꢀ

1

60.8, 153.0, 140.7, 139.4, 134.0, 132.7, 131.9, 127.7, 127.3, 127.1, (0.20 g; 54%), mp 174–176 C; HNMR (400 MHz, DMSO-d ): d

6

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1363

8

.76 (1H, s, –NH D

2

O exchangeable), d 8.69 (1H, s, –NH D

2

O

d

DMSO): d 179.9, 154.9, 149.2, 134.1, 132.3, 129.6, 127.2, 123.1,

ꢁ

1

exchangeable),

d

8.39–8.37 (1H, d, J ¼ 8 Hz, phthalazine),

122.7, 61.6, 43.1, 41.2, 30.1, 30.0, 14.6; FT-IR (ύ max, cm ): 1672

þ

8

.23–8.21 (1H, d, J ¼ 8 Hz, phthalazine), d 8.11–8.09 (1H, d, J ¼ 8 Hz, (C¼O); MS (Mwt.: 286.33): m/z 286.05 (M , 0.95%), 270.00 (3.77%),

phthalazine), d 8.08–8.06 (1H, d, J ¼ 8 Hz, phthalazine), d 7.55–7.52 56.00 (100.00%); Anal. Calcd for C15

18 4 2

H N O : C, 62.92; H, 6.34; N,

(

2H, d, J ¼ 8.8 Hz, Ar-H), d 7.46–7.43 (2H, d, J ¼ 8.8 Hz, Ar-H), d 19.57; Found: C, 63.23; H, 6.38; N, 19.71

7

.33–7.30 (2H, d, J ¼ 7.6 Hz, Ar-H), d 7.22–7.20 (2H, d, J ¼ 8.8 Hz, Ar-

H), d 6.69–6.67 (1H, d, J ¼ 7.6 Hz, Ar-H), d 2.96 (3H, s,CH ); FT-IR (ύ

max, cm ): 3302, 3141 (2NH), 1643 (C¼O); MS (Mwt.: 370.40): m/z

3

6

.1.5.2. 1–(4-Phenylpiperazin-1-yl)phthalazine (8b). Yield (0.20 g;

ꢁ

1

ꢀ

1

6

9%), mp 242–244 C; HNMR (300 MHz, DMSO): d 9.72–7.96 (1H,

1

3

70.00 [M , 2.28%), 277.95 (19.33%), 251.00 (100.00%); Anal. Calcd

for C22 : C, 71.34; H, 4.90; N, 15.13; Found: C, 71.49; H, 4.97;

N, 15.29.

s, phthalazine), d 8.17–8.15 (1H, d, J ¼ 8 Hz, phthalazine), d

18 4 2

H N O

8

.12–8.10 (1H, d, J ¼ 8 Hz, phthalazine), d 8.00–7.95 (2H, d, J ¼ 8 Hz,

phthalazine), d 7.28–7.24 (2H, d, J ¼ 7.6 Hz, Ar-H), d 7.05–7.03 (2H,

d, J ¼ 7.6 Hz, Ar-H), d 6.84–6.81 (1H, d, J ¼ 7.6 Hz, Ar-H), d 3.58–3.56

6

.1.4.5. 1–(4-Chlorophenyl)-3–(4-(4-methylphthalazin-1-yloxy)phe-

(

4H, m, piperazine ring), d 3.44–3.42 (4H, m, piperazine ring);

ꢀ

1

13

nyl)urea (7e). Yield (0.17 g; 42%), mp 169–171 C; HNMR

400 MHz, DMSO-d ): d 10.06 (1H, s, –NH D O exchangeable), d

0.05 (1H, s, –NH D O exchangeable), d 8.38–8.36 (1H, d,

CNMR (100 MHz, DMSO): d151.5, 148.2, 129.5, 128.6, 127.3,

ꢁ

1

(

1

6

2

1

24.3, 119.7, 116.0, 51.2, 48.7; FT-IR (ύ max, cm ): 3428 (CH aro-

2

matic), 2991, 2919 (CH aliphatic); MS (Mwt.: 290.36): m/z 290.10

þ

J ¼ 7.6 Hz, phthalazine), d 8.20–8.18 (1H, d, J ¼ 7.6 Hz, phthalazine),

d 8.07–8.04 (2H, d, J ¼ 7.6 Hz, phthalazine), d 7.58–7.56 (2H, d,

J ¼ 8.4 Hz, Ar-H), d 7.29–7.22 (2H, d, J ¼ 8.4 Hz, Ar-H), d 7.20–7.18

(

M , 0.41%), 162.10 (8.10%), 120.10 (100.00%), Anal. Calcd for

18 18 4

C H N : C, 74.46; H, 6.25; N, 19.30; Found: C, 74.62; H, 6.37;

N, 19.54.

(

2H, d, J ¼ 9.2 Hz, Ar-H), d 6.66–6.63 (2H, d, J ¼ 9.2 Hz, Ar-H), d 2.81

ꢁ1

3H, s, CH ); FT-IR (ύ max, cm ): 3300, 3153 (2NH), 1643 (C¼O);

3

6

.1.5.3. 1–(4-(2-Fluorophenyl)piperazin-1-yl)phthalazine (8c). Yield

MS (Mwt.: 404.85): m/z 404.90(Mþ, 2.04%), 385.85 (1.29%), 267.90

4.75), 250.95 (28.16), 127.00 (100.00%); Anal. Calcd for

C H ClN O : C, 65.27; H, 4.23; N, 13.84; Found: C, 65.52; H, 4.28;

ꢀ

1

(

0.21 g; 70%), mp 88–90 C; HNMR (300 MHz, DMSO): d 8.61 (1H,

s, phthalazine), d 8.27–8.24 (2H, d, J ¼ 9 Hz, phthalazine), d

.14–8.11 (2H, d, J ¼ 9 Hz, phthalazine), d 7.19–7.17 (1H, d,

J ¼ 7.6 Hz, Ar-H), d 7.15–7.13 (1H, d, J ¼ 7.6 Hz, Ar-H), d 7.08–7.06

1H, d, J ¼ 7.6 Hz, Ar-H), d 6.99–6.97 (1H, d, J ¼ 7.6 Hz, Ar-H), d

.70–3.69 (2H, m, piperazine ring), d 3.31–3.30 (2H, m, piperazine

(

2

17

4 2

8

N, 13.97.

(

3

6

.1.4.6. 1–(4-Chloro-3-(trifluoromethyl)phenyl)-3–(4-(4-metyhlph-

thalazin-1-yloxy)phenyl)urea (7f). Yield (0.20 g; 42%), mp

13

ring), d 3.23–3.21 (4H, m, piperazine ring); CNMR (100 MHz,

DMSO): d 175.9, 159.2,145.3, 138.9, 138.3, 134.8, 134.0, 130.0,

ꢀ

50–152 C; H NMR (400 MHz, DMSO-d ): d 10.31 (1H, s, –NH

6

1

2 2

D O exchangeable), d 10.30 (1H, s, –NH D O exchangeable), d 8.37

1

27.6, 125.6, 123.3, 122.8, 122.7, 116.0, 50.8, 49.9, 47.0, 46.9; FT-IR

(

7

1H, s, Ar-H), d 8.19–8.16 (2H, d, J ¼ 8 Hz, phthalazine), d 8.07–8.05

ꢁ1

(ύ max, cm ): 3039 (CH aromatic), 2924 (CH aliphatic); MS (Mwt.:

1H, d, J ¼ 7.6 Hz, Ar-H), d 7.78–8.75 (1H,d, J ¼ 8 Hz, phthalazine), d

þ

3

08.35): m/z 308.05 (M , 1.81%), 180.05 (19.88%), 138.10

.61–7.59 (2H, d, J ¼ 7.6 Hz, Ar-H), d 7.56–7.54 (1H, d, J ¼ 8.8 Hz, Ar-

(

100.00%); Anal. Calcd for C H FN : C, 70.11; H, 5.56; N, 18.17;

18 17 4

H), d 7.24–7.21(2H, d, J ¼ 8.8 Hz, Ar-H), d 2.80 (3H, s, CH

3

); FT-IR (ύ

max, cm ): 3300, 3153 (2NH), 1679 (C¼O); MS (Mwt.: 472.85): m/z

72.80 (Mþ, 2.07%), 391.95 (10.86), 335.85 (25.53), 250.95

86.76%), 194.90 (100%); Anal. Calcd for C23 16ClF : C, 58.42;

H, 3.41; N, 11.85; Found: C, 58.49; H, 3.39; N, 12.01

Found: C, 70.29; H, 5.60; N, 18.28.

ꢁ

1

4

(

6

.1.5.4. 1–(4-(Pyridin-2-yl)piperazin-1-yl)phthalazine (8d). Yield

H

3 4 2

N O

ꢀ

1

(

8

0.20 g; 69%), mp 286–288 C; HNMR (300 MHz, DMSO): d

.14–8.12 (1H, d, J ¼ 7 Hz, pyridine ring), d 7.93–7.91 (3H, m,

phthalazine), d 7.59–7.56 (2H, d, J ¼ 9 Hz, phthalazine), d 7.56–7.54

1H, d, J ¼ 7 Hz, pyridine ring), d 6.69–6.67 (2H, d, J ¼ 7 Hz, pyridine

ring), d 3.67–3.65 (4H, m, piperazine ring), d 3.06–3.04 (4H, m,

(

6

.1.5. Synthesis of ethyl 4–(4-substitutedphthalazine-1-yl)pipera-

zine-1-carboxylate and 1-Substituted-4-(arylpiperazine-1-yl)phtha-

lazine (8a–j)

General procedure:

ꢁ

1

piperazine ring); FT-IR (ύ max, cm ): 3030 (CH aromatic), 2926

(

CH aliphatic); MS (Mwt.: 291.35): m/z 291.10 (Mþ, 0.19%), 279.05

0.28%), 160.10 (27.24%), 95.05 (100.00); Anal. Calcd for C H N :

1

7 17 5

To a stirred mixture of the respective 1-chlorophthalazine

C, 70.08; H, 5.88; N, 24.04; Found: C, 70.32; H, 5.64; N, 24.31

(

2

3a,b) (10.0 mmol, 1equiv), potassium carbonate (0.27 g,

0.0 mmol, 2equiv), potassium iodide (0.01 g, 0.01 mmol, 0.1equiv)

in absolute ethanol (20 ml), the respective piperazine (viz.; ethyl

piperazinecarboxylate, phenylpiperazine, 2-flourophenylpiperazine, (8e). Yield (0.23 g; 77%), mp 90–92 C; HNMR (300 MHz, DMSO):

-pyridylpiperazine, 2-furoylpiperazine) (10.0 mmol, 1equiv) was d 8.36 (1H, s, phthalazine), d 8.12–8.10 (1H, d, J ¼ 7 Hz, furoyl ring),

added and the mixture was heated under reflux for 2 h, after d 8.03–8.01 (2H, d, J ¼ 7.8 Hz, phthalazine), d 7.98–7.96 (2H, d,

which TLC (CHCl /CH

OH 9:1) showed no starting material. The J ¼ 7.8 Hz, phthalazine), d 6.99–6.98 (2H, d, J ¼ 7 Hz, furoylring), d

mixture was then concentrated in vacuo, the residue was washed 3.69–3.67 (4H, m,piperazine ring), d 2.91–2.90 (4H, m, piperazine

6.1.5.5.

Furan-2-yl-(4-(phthalazin-1-yl)piperazin-1-yl)methanone

ꢀ

1

2

3

13

with water, extracted with EtOAc, dried over anhydrous Na SO . ring); CNMR (100 MHz, DMSO): d 175.9, 159.8, 154.6, 154.2,

2

4

The crude product was recrystallised from EtOAc.

142.3, 135.4, 133.9, 128.5, 127.8, 125.7, 123.2, 122.2, 117.2, 111.7,

ꢁ

1

5

3

0.2, 42.8, 40.2, FT-IR (ύ max, cm ): 1679 (C¼O); MS (Mwt.:

08.33): m/z 308.05 (Mþ, 4.64%), 274.00 (6.66%), 158.05 (57.70%),

6

.1.5.1. Ethyl 4-(phthalazin-1-yl)piperazine-1-carboxylate (8a).

ꢀ

1

16 4 2

95.00 (100.00%) Anal. Calcd for C17H N O : C, 66.22; H, 5.23; N,

Yield (0.19 g; 68%), mp 271–273 C; HNMR (300 MHz, DMSO): d

.10 (1H, s, phthalazine), d 7.98–7.96 (2H, d, J ¼ 7 Hz, phthalazine),

d 7.93–7.91 (2H, d, J ¼ 7 Hz, phthalazine), d 4.07 (2H, q,CH -CH ), d

.63–3.62 (2H, m, piperazine ring), d 2.86–2.85 (2H, m, piperazine 6.1.5.6. Ethyl 4–(4-methylphthalazin-1-yl)piperazine-1-carboxylate

1

8.17; Found: C, 66.41; H, 5.32; N, 18.40

8

2

3

ꢀ

1

ring), d 2.55–2.54 (2H, m, piperazine ring), d 2.27–2.26 (2H, m, (8f). Yield (0.22 g; 73%), mp 95–97 C; HNMR (300 MHz, CDCl

3

): d

1

3

2 3

piperazine ring), d 1.14 (3H, t, CH -CH );

CNMR (100 MHz, 8.43–8.41 (2H, d, J ¼ 8.1 Hz, phthalazine), d 8.36–8.34 (2H, d,

1

364

S. ELMELIGIE ET AL.

ꢀ

2 3

-CH ), d 3.75–3.74 (4H, 2-BuOH (7.5 ml) in a tube and heated to 110 C. The reaction

J ¼ 8.1 Hz, phthalazine), d 4.19 (2H, q, CH

m, piperazine ring), d 3.65–3.64 (4H, m, piperazine ring), d 3.37 quickly became a solid, yellow mass. After four hours, the reaction

ꢁ

1

(

1

3H, s, CH ), d 1.33 (3H, t,CH -CH ); FT-IR (ύ max, cm ): 1693 was cooled and diluted with water. The resultant slurry was then

3 3 2

C¼O); MS (Mwt.: 300.36): m/z 300.05(Mþ, 5.71%), 244.05 (3.15%), partitioned between equal volumes of DCM and 1 N NaOH (15 ml).

84.55 (13.85%), 172.30 (48.47%), 55.65 (100%); Anal. Calcd for The aqueous layer was extracted into DCM (2 ꢂ 15 ml). The com-

C H N O : C, 63.98; H, 6.71; N, 18.65; Found: C, 64.17; H, 6.83; bined organic layers were dried over anhydrous sodium sulfate, fil-

1

6 20 4 2

N, 19.01

tered, and concentrated in vacuo. The resulting orange solid was

crystallised from (EtOAc/pet ether 1:1) to afford the title com-

6

(

7

.1.5.7. 1-Methyl-4–(4-phenylpiperazin-1-yl)phthalazine (8 g). Yield pound (11) as an orange crystals.

ꢀ

1

ꢀ

1

0.15 g; 56%), mp 229–231 C; HNMR (300 MHz, DMSO): d

Yield 0.22 g, (88%); mp 170–172 C; HNMR (300 MHz, DMSO-

): d 13.21 (s, 1H, –NH D O exchangeable), 9.27 (s, 2H, 2H, –NH

O exchangeable), d 8.30–8.28 (d, J ¼ 7 Hz, 2H, phthalazine), d

.88–6.86 (1H, m, Ar-H), d 6.85–6.83 (2H, d, J ¼ 7.8 Hz, Ar-H), d 8.07–8.04 (d, J ¼ 7 Hz, 2H, phthalazine), d 7.46–7.43 (d, J ¼ 9 Hz, 2H,

); FT-IR (t

), 3321(NH); MS (Mwt.: 270.72): m/z 250.00

.28–7.26(2H, d, J ¼ 7.2 Hz, phthalazine), d 7.25–7.23 (2H, d,

d

D

2

6

2

J ¼ 7.2 Hz, phthalazine), d 6.99–6.97 (2H, d, J ¼ 7.8 Hz, Ar-H), d

6

3

.34–3.32 (4H, m, piperazine ring), d 3.22–3.20 (4H, m, piperazine ArH), d 6.83–6.76 (d, J ¼ 9 Hz, 2H, ArH), d 2.48 (s, 3H, CH

3

ꢁ

1

ꢁ1

ring), d 2.48 (3H, s, CH

3 2

); FT-IR (ύ max, cm ): 3070 (CH aromatic), max, cm ): 3385 (NH

2

954 (CH aliphatic); MS (Mwt.: 304.39): m/z 304.00(Mþ, 1.73%), (Mþ, 50.00%), 249.00 (100.00%), 108.00 (60.92%), 65.00 (77.59%);

2

84.60 (2.19%), 192.35 (5.24%), 105.20 (34.33%), 55.65 (100.00%); Anal. Calcd for C H ClN : C, 62.11; H, 4.10; N, 20.70; Found: C,

14

11

4

20 4

Anal. Calcd for C19H N : C, 74.97; H, 6.62; N, 18.41; Found: C, 62.37; H, 4.18; N, 20.94.

8

0.32; H, 6.70; N, 18.48.

6

.1.7. Synthesis of 1-aryl-3–(4-(4-substituted phthalazin-1-ylamino)

6

.1.5.8. 1–(4-(2-Fluorophenyl)piperazin-1-yl)-4-methylphthalazine

ꢀ

1

phenyl)urea (12a-c)

General procedure:

To a stirred solution of the N1-(4-chlorophthalazin-1-yl)ben-

zene-1,4-diamine (11) (10.0 mmol, 1equiv) in DMF (20 ml), the

respective phenyl isocyanate (viz.; phenyl isocyanate, 4-chloro

phenyl isocyanate, 3-trifluromethyl-4-chlorophenyl isocyanate)

(8 h). Yield (0.12 g; 71%), mp 149–151 C; HNMR (300 MHz,

CDCl ): d 8.15–8.13 (1H, d, J ¼ 9.6 Hz, phthalazine), d 8.05–8.01

3

(

1H, d, J ¼ 9.6 Hz, phthalazine), d 7.87–7.83 (2H, d, J ¼ 9.6 Hz,

phthalazine), d 7.07–7.04 (2H, d, J ¼ 8.7 Hz, Ar-H), d 7.01–7.00 (2H,

d, J ¼ 8.7 Hz, Ar-H), d 3.69–3.67 (4H, m, piperazine ring), d

13

3

.39–3.36 (4H, m, piperazine ring), d 2.92 (3H, s, CH ); CNMR

3

(10.0 mmol, 1equiv) was added and the mixture was heated under

(

100 MHz, DMSO): d 173.5, 158. 9, 153.1, 137.4, 131.8, 131.5,

reflux for 8–10 h, after which TLC (CH Cl /CH OH 9:1) showed no

1

5

27.2, 125.2, 123.2, 123.1, 122.3, 120.4, 119.3, 116.1, 115.8, 50.9,

0.0, 47.0, 19.0, FT-IR (ύ max, cm ): 3035 (CH aromatic), 2951 (CH

2

3

ꢁ

1

starting material. The reaction mixture was poured over ice-water;

the formed precipitate was allowed to settle, then filtered off and

dried to afford the crude product (12a-c) which was further crys-

tallised from EtOAc.

þ

aliphatic); MS (Mwt.: 322.38): m/z 323.50 [M þ 1H] (9.74%), 172.15

4

(48.77%,), 122.05 (100.00%); Anal. Calcd for C19H19FN : C, 70.79; H,

5

.94; N, 17.38; Found: C, 71.03; H, 5.99; N, 18.48.

6

.1.7.1. 1–(4-(4Chlorophthalazin-1-ylamino)phenyl)-3-phenylurea

6

.1.5.9. 1-Methyl-4–(4-(pyridin-2-yl)piperazin-1-yl)phthalazine (8i).

ꢀ

1

ꢀ

1

Yield (0.10 g; 59%), mp 226–228 C; HNMR (300 MHz, DMSO): d (12a). Yield 0.12 g; (68%), mp 225–227 C; HNMR (400 MHz,

): d 12.85 (s, 3H, –NH D O exchangeable), d 8.63 (d,

8

.16–8.13(1H, d, J ¼ 8.7 Hz, pyridyl ring), d 7.62–7.60 (2H, d, DMSO-d

6

2

J ¼ 8.4 Hz, phthalazine), d 7.58–7.56 (2H, d, J ¼ 8.4 Hz, phthalazine), J ¼ 10 Hz 1H, phthalazine), d 8.30–8.27 (d, J ¼ 10 Hz, 2H, phthala-

d 6.91–6.89 (1H, d, J ¼ 8.7 Hz, pyridyl ring), d 6.74–6.72 (2H, d, zine), d 8.08–8.05 (d, J ¼ 10 Hz, 1H, phthalazine), d 8.03–7.02 (d,

J ¼ 8.7 Hz, pyridyl ring), d 3.75–3.73 (4H, m, piperazine ring), d J ¼ 8 Hz, 2H, Ar-H), d 7.99–7.97 (d, J ¼ 8 Hz, 2H, Ar-H), d 7.95–7.94

3

.12–3.10 (4H, m, piperazine ring), d 2.08 (3H, s, CH ); FT-IR (ύ (d, J ¼ 8 Hz, 1H, Ar-H), d 7.46–7.43 (d, J ¼ 9.2 Hz, 2H, Ar-H), d

3

ꢁ

1

max, cm ) 3132 (CH aromatic), 2954 (CH aliphatic); MS (Mwt.: 7.29–7.24 (d, J ¼ 9.2 Hz, 1H, Ar-H), d 6.98–6.94 (d, J ¼ 9.2 Hz, 1H, Ar-

13

3

(

05.38): m/z 305.15 (Mþ, 1.16%), 185.65 (16.50%), 172.35 H); CNMR (100 MHz, DMSO): d 159.0, 152.5, 146.1, 139.6, 137.0,

41.23%,), 55.55 (100.00%); Anal. Calcd for C H N : C, 70.80; H, 134.4, 132.9, 128.7, 128.3, 126.4, 125,4, 121.7, 120.5, 119.5, 118.1;

18 19 5

ꢁ

1

6

.27; N, 22.93; Found: C, 71.04; H, 6.38; N, 23.18.

FT-IR (t max, cm ): 3296, 3286, 3155 (3NH), 1668 (C¼O); MS

þ

(Mwt.: 389.84): m/z 389.28 (M , 2.84%), 367.27 (6.23%), 322.10

6

.1.5.10. Furan-2-yl-(4–(4-methylphthalazin-1-yl)piperazin-1-yl)

(11.84%), 128.10(100.00%); Anal. Calcd for C H ClN O: C, 64.70;

2

1

16

5

ꢀ

1

methanone (8j). Yield (0.11 g; 60%), mp 90–92 C; HNMR H, 4.14; N, 14.96; Found: C, 64.71; H, 3.89; N, 14.52.

300 MHz, DMSO): d 8.32–8.30 (2H, d, J ¼ 7 Hz, phthalazine), d

.29–8.27 (2H, d, J ¼ 7 Hz, phthalazine), d 7.82–7.81 (1H, d,

J ¼ 7.2 Hz, furoyl ring), d 6.97–6.96(1H, d, J ¼ 7.2 Hz, furoyl ring), d

(

8

6.1.7.2.

1–(4-Chlorophenyl)-3–(4-(4-chlorophthalazin-1-ylamino)-

ꢀ

1

phenyl)urea (12b). Yield 0.20 g; (53%), mp 236–238 C; HNMR

6

.62–6.60 (1H, d, J ¼ 7.2 Hz, furoyl ring), d 3.64–3.62 (4H, m, pipera-

(

400 MHz, DMSO-d

.81 (s, 1H, –NH D

O exchangeable), d 8.29–8.27 (d, J ¼ 8.8 Hz, 1H,

phthalazine),

8.07–8.05 (d, J ¼ 8.8 Hz, 1H, phthalazine),

.02–8.00 (d, J ¼ 8.8 Hz, 1H, phthalazine), d 7.98–7.96 (d, J ¼ 8.8 Hz,

H, phthalazine), d 7.95–7.94 (d, J ¼ 6.4 Hz, 2H, Ar-H), d 7.57–7.56

6 2

): d 12.84 (s, 2H, –NH D O exchangeable), d

zine ring), d 3.41–3.39 (4H, m, piperazine ring), d 2.92 (3H, s, CH );

3

8

2

1

FT-IR (ύ max, cm- ): 1610 (C¼O); MS (Mwt.: 322.36): m/z 322.00

d

(

Mþ, 6.60%), 184.00 (31.70%), 172.00 (100.00%); Anal. Calcd for

8

1

C H N O : C, 67.07; H, 5.63; N, 17.38; Found: C, 67.21; H, 5.69;

1

8 18 4 2

N, 17.52.

(

7

d, J ¼ 6.4 Hz, 2H, Ar-H), d 7.48–7.47 (d, J ¼ 6.4 Hz, 2H, Ar-H), d

ꢁ

1

.31–7.30 (m, 2H, Ar-H); FT-IR (t max, cm ): 3446, 3300, 3157

1

þ

6

.1.6. Synthesis of N -(4-chlorophthalazin-1-yl)benzene-1,4- (3NH), 1670 (C¼O); MS (Mwt.: 423.07): m/z 424.88 [M þ 1H]

þ

diamine (11)

(3.74%), 423.50 (M , 9.84%), 328.27 (52.58%), 252.97 (20.15%),

General procedure:p-Phenylenediamine (0.33 g, 3.12 mmol, 3equiv) 119.04(100.00%); Anal. Calcd for C21H15Cl N O: C, 59.45; H, 3.56; N,

2 5

and the dichlorophthalazine (2.08 mmol, 2equiv) were treated with 16.51; Found: C, 59.49; H, 3.37; N, 16.29.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1365

ꢁ

1

6

.1.7.3. 1–(4-Chloro-3-(trifluoromethyl)phenyl)-3–(4-(4-chlorophtha- Ar-H), d 6.69–6.66 (d, J ¼ 8.4 Hz, 2H, Ar-H); FT-IR (t max, cm ):

þ

lazin-1-ylamino)phenyl)-urea (12c). Yield 0.22 g; (49%), mp 3294, 3155(2NH), 1662 (C¼O); MS (Mwt.: 493.27): 493.46 (M ,

ꢀ

87–189 C; HNMR (400 MHz, DMSO-d ): d 12.84 (s, 2H, –NH 1.24%), 281.00 (2.26%), 194.97(100.00%); Anal. Calcd for

6

1

D

2 2

O exchangeable), d 9.36 (s, 1H, –NH D O exchangeable), d

22 2 3 4 2

C H13Cl F N O : C, 53.57; H, 2.66; N, 11.36; Found: C, 53.81; H,

8

2

.29–8.27 (d, J ¼ 8.8 Hz, 2H, phthalazine), d 8.27–8.25 (d, J ¼ 8.8 Hz, 2.90; N, 11.3.

H, phthalazine), d 8.09 (s, 1H, Ar-H), 8.05–8.03 (d, J ¼ 9.6 Hz, 1H,

Ar-H), d 8.01–7.97 (d, J ¼ 9.6 Hz, 1H, Ar-H), d 7.59–7.50 (d,

6

.1.9. Synthesis of N-(4-chlorophenyl)-4-methylphthalazin-1-

J ¼ 8.4 Hz, 2H, Ar-H), d 7.39–7.37 (d, J ¼ 8.4 Hz, 2H, Ar-H); FT-IR (t

ꢁ

1

amine (14)

General procedure:

max, cm ): 3387, 3344, 3290 (3NH), 1664(C¼O); MS (Mwt.:

þ

4

92.28): m/z 493.06 [M þ 1H] (2.63%), 395.08 (32.95%), 372.90

To a stirred mixture of the 1-chloro-4-methyl-phthalazine (3b)

(

5

2 3 5

35.24%), 137.07 (100.00%); Anal. Calcd for C22H14Cl F N O: C,

(

1

(

1.78, 10.0 mmol, 1 equiv) in butanol (50 ml), triethylamine (1 ml,

0.0 mmol, 1 equiv) the respective amine (viz.; aniline, p-Cl aniline)

10.0 mmol, 1equiv) was added and the mixture was heated under

reflux for 2 h, after which TLC (CHCl /CH OH 9:1) showed no start-

3.68; H, 2.87; N, 14.23; Found: C, 53.72; H, 2.68; N, 14.44.

6

.1.8. Synthesis of 1-aryl-3–(4-(4-chlorophthalazin-1-yloxy)pheny-

3

ing material. The mixture was then concentrated in vacuo, the

crude product was recrystallised from methanol.

Yield 1.56 g; (88%), mp 213–215 C; HNMR (400 MHz, DMSO-

d ): d 10.47 (s, 1H, –NH D O exchangeable), d 9.10–9.07 (d,

l)urea (13a-c)

General procedure:

ꢀ

1

To a stirred solution of 1,4 dichlorophthalazine derivative (10)

6

2

(

2

10.0 mmol, 1equiv), and cesium carbonate (6.5 g, 20.0 mmol,

equiv) in acetonitrile (20 ml), the appropriate 1-aryl-3–(4-hydroxy-

phenyl)urea (1a-c) (10.0 mmol, 1equiv) was added and the mixture

was heated under reflux for 6 h, after which TLC (CHCl /CH OH

:1) showed no starting material. The solvent was evaporated

J ¼ 9 Hz, 1H, phthalazine), d 8.45 –8.44 (d, J ¼ 9 Hz, 1H, phthala-

zine), d 8.31–8.29 (d, J ¼ 9 Hz, 1H, phthalazine), d 8.23–8.20 (d,

J ¼ 9 Hz, 1H, phthalazine), d 7.85–7.82 (d, J ¼ 8 Hz, 1H, Ar-H) , d

3

7

.51–7.49 (d, J ¼ 8 Hz, 1H, Ar-H), d 7.10–7.07 (d, J ¼ 8 Hz, 1H, Ar-H),

9

d 6.70–6.68 (d, J ¼ 8 Hz, 1H, Ar-H) , d 2.97 (s, 3H, CH

3

); FT-IR

under vacuum and the resultant solid was stirred with cold 1 M

NaOH solution (20 ml), filtered off, dried and recrytallised from

acetonitrile to afford compounds (13a-c).

ꢁ

1

(t max, cm ) 3360 (NH), 3050 (CH aromatic); MS (Mwt.: 269.73):

þ

m/z 269.28 (M , 8.26%), 239.18 (15.43%), 125.22 (24.70%),

9

0.97(100.00%); Anal. Calcd for C15

3

H12ClN : C, 66.79; H, 4.48; N,

1

5.58; Found: C, 67.03; H, 4.57; N, 15.75.

6

(

.1.8.1.

13a). Yield 0.12 g; (46%), mp 191–193 C; HNMR (300 MHz,

DMSO-d ): d 10.48 (s, 1H, –NH D O exchangeable), d 10.31 (s, 1H, 6.1.10. Synthesis of 4-(bromomethyl)-N-(4-chlorophenyl)phthala-

1–(4-(4-Chlorophthalazin-1-yloxy)phenyl)-3-phenylurea

ꢀ

1

6

2

–

NH D

d 8.29–8.27 (d, J ¼ 8 Hz, 2H, phthalazine), d 7.90–7.88 (d, J ¼ 8.6 Hz, General procedure:

H, Ar-H), d 7.69–7.66 (d, J ¼ 8.6 Hz, 2H, Ar-H), d 7.40–7.37 (d,

mixture of the N-aryl-4-methylphthalazin-1-amine (14b)

J ¼ 8 Hz, 1H, Ar-H), d 7.27–7.25 (d, J ¼ 8 Hz, 2H, Ar-H), d 6.94–6.92 (0.26 mmol, 1 equiv), N-bromo-succinimide (4.6 g, 0.26 mmol, 1

2

O exchangeable), d 8.46–8.44 (d, J ¼ 8 Hz, 2H, phthalazine), zin-1-amine (15)

2

A

1

3

(

d, J ¼ 8 Hz, 2H, Ar-H); CNMR (100 MHz, DMSO): d 164.7, 158.8, equiv) and dibenzoyl peroxide (0.44 g, 0.018 mmol, 0.07 equiv) in

1

3

2

53. 8, 152.4, 141.3, 136.6, 135.3, 132.3, 128.4, 128.0, 127.3, 125. 7, carbon tetrachloride (150 ml) was refluxed for 24 h. The reaction

ꢁ

1

ꢀ

23.8, 121.6, 120.6, 120.2, 118.1, 115.1; FT-IR (t max, cm ): 3325,

mixture was cooled to 40 C, and then filtered. The filtrate was

þ

295 (2NH), 1662 (C¼O); MS (Mwt.: 390.82): m/z 390.97.00 (M ,

concentrated under vacuum to afford the crude product, which

was triturated with ether to give a yellowish solid product.

Yield 0.10 g; (42%), mp 172.-174 C; HNMR (400 MHz, DMSO-

): d 11.06 (s, 1H, –NH D O exchangeable), d 8.07–8.05 (d,

.24%), 346.01 (5.06%), 268.11 (18.11%), 179.11 (100.00%); Anal.

ꢀ

1

Calcd for C21

H, 4.21; N, 14.12.

4 2

H15ClN O : C, 64.54; H, 3.87; N, 14.43; Found: C, 64.97;

d

6

2

J ¼ 7.2 Hz, 2H, phthalazine), d 7.99 –7.97 (d, J ¼ 7.2 Hz, 2H, phthala-

zine), d 7.62–7.60 (d, J ¼ 7.6 Hz, 2H, Ar-H), d 7.45–7.44 (d,

6

.1.8.2. 1–(4-Chlorophenyl)-3–(4-(4-chlorophthalazin-1-yloxy)phe-

ꢁ

1

ꢀ

1

J ¼ 7.6 Hz, 2H, Ar-H), d 4.02 (s, 2H, CH

2

); FT-IR (t max, cm ) 3450

nyl)urea (13b). Yield 0.19 g; (50%), mp 172–174 C; HNMR

400 MHz, DMSO-d ): d 9.06 (s, 1H, –NH D O exchangeable), d

þ2

(

NH), 3053 (CH aromatic); MS (Mwt.: 348.62): m/z 350.00 (M

,

(

6

2

þ

2

.86%), 348.00 (M , 9.89%), 268.04 (65.94), 102.03 (100.00%); Anal.

11BrClN : C, 51.68; H, 3.18; N, 12.05; Found: C, 51.90;

H, 3.16; N, 12.18.

8

.54 (s, 1H,–NH D O exchangeable), d 8.34–8.32 (d, J ¼ 8.4 Hz, 2H,

2

Calcd for C15

H

3

phthalazine),

d

8.18–8.16 (d, J ¼ 8.4 Hz, 2H, phthalazine),

d

7

.44–7.42 (d, J ¼ 8.8 Hz, 2H, Ar-H), d 7.35–7.33 (d, J ¼ 8.8 Hz, 2H, Ar-

H), d 7.27–7.25 (d, J ¼ 7.2 Hz, 2H, Ar-H), d 6.78–6.76 (d, J ¼ 7.2 Hz,

ꢁ

1

2

H, Ar-H); FT-IR (t max, cm ): 3278, 3155 (2NH), 1666 (C¼O); MS

6.1.11. Synthesis of N-(4-chlorophenyl)-4–(3-substitutedphenylami-

þ

(Mwt.: 425.27): m/z 425.28 (M , 5.29%), 305.13 (7.74%), 201.08

no)methyl)phthalazin-1-amines (16a-d)

General procedure:

2 4 2

(11.73%), 63.03 (100.00%); Anal. Calcd for C21H14Cl N O : C, 59.31;

H, 3.32; N, 13.17; Found: C, 59.62; H, 3.60; N, 13.65.

To a stirred solution of 4-(bromomethyl)-N-(4-chlorophenyl)ph-

thalazin-1-amine (15) (10.0 mmol, 1 equiv), potassium carbonate

6

.1.8.3. 1–(4-Chloro-3-(trifluoromethyl)phenyl)-3–(4-(phthalazin-1- (0.27 g, 20.0 mmol, 2equiv), potassium iodide (0.01 g, 0.01 mmol,

ꢀ

yloxy)phenyl)urea (13c). Yield 0.22 g; (49%), mp 210–212 C; 0.1equiv) in acetone (20 ml), the respective amine (viz.; aniline, m-

1

HNMR (400 MHz, DMSO-d ): d 10.20 (s, 1H, –NH D O exchange- Chloroaniline, m-touilidine, m-anisidine) (10.0 mmol, 1equiv) was

6

2

able), d 10.13 (s, 1H, –NH D

O exchangeable), d 8.28–8.26 (d, added and the mixture was heated under reflux for 4–6 h, after

2

J ¼ 9 Hz, 2H, phthalazine), d 8.25–8.23 (d, J ¼ 9 Hz, 2H, phthalazine), which TLC (CH

d 8.09 (s, 1H, Ar-H), d 7.93–7.92 (d, J ¼ 7.6 Hz, 1H, Ar-H), d mixture was then filtered and the filtrate concentrated in vacuo,

.76–7.74 (d, J ¼ 7.6 Hz, 1H, Ar-H), d 7.54–7.51 (d, J ¼ 8.4 Hz, 2H, the residue was crystallised from acetone.

Cl2/CH OH 99:1) showed no starting material. The

2

3

7

1

366

S. ELMELIGIE ET AL.

6

.1.11.1. N-(4-Chlorophenyl)-4-((phenylamino)methyl)phthalazin-1- 6.1.12. Synthesis of N-(4-chlorophenyl)-4-(aryloxymethyl)phthala-

ꢀ

1

amine (16a). Yield 0.10 g; (42%), mp 120–122 C; HNMR zin-1-amine (17a,b)

400 MHz, DMSO-d ): d 11.03 (s, 1H, –NH D

O exchangeable), d General procedure:

.65 (s, 1H, –NH D O exchangeable), d 8.75–8.72 (d, J ¼ 9.2 Hz, 2H,

To a stirred solution of 4-(bromomethyl)-N-(4-chlorophenyl)ph-

phthalazine), d 8.28 –8.25 (d, J ¼ 9.2 Hz, 2H, phthalazine), d thalazin-1-amine (15) (10.0 mmol, 1 equiv), sodium hydride (0.27 g,

.90–7.88 (d, J ¼ 8.6 Hz, 2H, Ar-H), d 7.85–7.87 (d, J ¼ 8.6 Hz, 2H, 20.0 mmol, 2equiv) in acetone (20 ml), the respective phenol (viz.;

Ar-H), d 7.46–7.43 (d, J ¼ 7.6 Hz, 1H, Ar-H), d 7.05–7.03 (d, phenol, m-cresol) (10.0 mmol, 1equiv) was added and the mixture

was heated under reflux for 6–8 h, after which TLC (CH Cl /CH OH

9:1) showed no starting material. The mixture was then filtered

(

9

6

2

7

J ¼ 7.6 Hz, 2H, Ar-H), d 6.83–6.81 (d, J ¼ 7.6 Hz, 2H, Ar-H), d 3.89 (s,

2

3

ꢁ

1

9

2

H, CH

2

); FT-IR (t max, cm ) 3446, 3383 (2NH), 3068 (CH aro-

þ,

and the filtrate concentrated in vacuo, the residue was crystallised

from acetone.

matic), 2976 (CH aliphatic); MS (Mwt.: 360.84): m/z 360.02 (M

2

.87%), 345.88 (13.64%), 268.02 (49.93%), 75.01 (100%); Anal.

Calcd for C21 17 ClN : C, 69.90; H, 4.75; N, 15.53; Found: C, 70.12;

H, 4.82; N, 15.69.

H

4

6.1.12.1. N-(4-Chlorophenyl)-4-(phenoxymethyl)phthalazin-1-amine

ꢀ

1

(

17a). Yield 0.10 g; (42%), mp 242–244 C; HNMR (400 MHz,

DMSO-d ): d 9.33 (s, 1H,–NH D O exchangeable), d 8.74–8.75 (d,

6

2

6

.1.11.2. N-(4-Chlorophenyl)-4-((3-chlorophenylamino)methyl)ph-

J ¼ 9.2 Hz, 2H, phthalazine), d 8.30 –8.28 (d, J ¼ 9.2 Hz, 2H, phthala-

zine), d 8.11–8.09 (d, J ¼ 8.6 Hz, 2H, Ar-H), d 7.89–7.87 (d,

J ¼ 8.6 Hz, 2H, Ar-H), d 7.49–7.47 (d, J ¼ 7.6 Hz, 2H, Ar-H), d

ꢀ

thalazin-1-amine (16b). Yield 0.10 g; (42%), mp 164–166 C;

1

HNMR (400 MHz, DMSO-d ): d 9.90 (s, 2H, –NH D O exchange-

6

2

able), d 8.86–8.84 (d, J ¼ 9.2 Hz, 2H, phthalazine), d 8.34 –8.32 (d,

7

.17–7.15 (d, J ¼ 7.6 Hz, 1H, Ar-H), d 6.76–6.74 (d, J ¼ 7.6 Hz, 2H, Ar-

ꢁ

1

J ¼ 9.2 Hz, 2H, phthalazine), d 8.20–8.18 (d, J ¼ 8.6 Hz, 2H, Ar-H), d H), d 3.89 (s, 2H, CH ); FT-IR (t max, cm ) 3379 (NH), 3066 (CH

2

þ

8

.12–8.09 (d, J ¼ 8.6 Hz, 2H, Ar-H), d 7.88–7.86 (d, J ¼ 7.6 Hz, 1H, Ar- aromatic), 2978 (CH aliphatic); MS (Mwt.: 361.82): m/z 361.00 (M ,

H), d 7.53–7.51 (d, J ¼ 7.6 Hz, 1H, Ar-H), d 7.44–7.42 (d, J ¼ 7.6 Hz, 5.29%), 349.01 (17.15%), 310.24 (12.19%), 268.11 (64.63%), 133.44

ꢁ

1

2

3

H, Ar-H), d 3.79 (s, 2H, CH

2

); FT-IR (t max, cm ) 3379 (2NH), (100.00%); Anal. Calcd for C H ClN O: C, 69.71; H, 4.46; N, 11.61;

2

1

16

3

080 (CH aromatic), 2954 (CH aliphatic); MS (Mwt.: 395.28) m/z Found: C, 69.88; H, 4.39; N, 11.69.

þ,

95.99 (M 2.17%), 271.01 (16.80%), 268.01 (100%); Anal. Calcd

2 4

for C21H16Cl N : C, 63.81; H, 4.08; N, 14.17; Found: C, 63.94; H,

6

.1.12.2.

N-(4-Chlorophenyl)-4-(m-tolyloxymethyl)phthalazin-1-

4

.12; N, 14.28.

ꢀ

1

amine (17b). Yield 0.10 g; (42%), mp 193–195 C; HNMR

(

400 MHz, DMSO-d ): d 9.33 (s, 1H,–NH D O exchangeable), d

6

2

8

2

.74–8.72 (d, J ¼ 8.8 Hz, 2H, phthalazine), d 8.31–8.28 (d, J ¼ 8.8 Hz,

H, phthalazine), d 8.14–8.11 (d, J ¼ 8.6 Hz, 2H, Ar-H), d 8.09–8.06

6

.1.11.3. N-(4-Chlorophenyl)-4-((m-tolylamino)methyl)phthalazin-1-

ꢀ

1

amine (16c). Yield 0.10 g; (42%), mp 130–132 C; HNMR (400 MHz,

DMSO-d ): d 9.70 (s, 2H, –NH D O exchangeable), d 8.86–8.84 (d,

(

7

d, J ¼ 8.6 Hz, 2H, Ar-H), d 7.49–7.47 (d, J ¼ 7.6 Hz, 1H, Ar-H), d

6

2

.47–7.44 (d, J ¼ 8.6 Hz, 2H, Ar-H), d 6.55–6.53 (d, J ¼ 8.6 Hz, 1H, Ar-

13

J ¼ 9.2 Hz, 2H, phthalazine), d 8.34 –8.32 (d, J ¼ 9.2 Hz, 2H, phthala-

zine), d 8.20–8.18 (d, J ¼ 8.6 Hz, 2H, Ar-H), d 8.12–8.09 (d,

J ¼ 8.6 Hz, 2H, Ar-H), d 7.88–7.86 (d, J ¼ 7.6 Hz, 1H, Ar-H), d

H), d 3.89 (s, 2H, CH

DMSO): d 173.4, 162.4, 151.0, 139.4, 138.4, 133.1, 132.1, 128.9,

2 3

), d 2.19 (s, 3H, CH ); CNMR (100 MHz,

1

28.6, 128.4, 126.9, 126.9, 125.9, 123.3, 123.15, 121.9, 119.4, 115.7,

12.2, 75.4, 20.9, FT-IR (t max, cm ) 3414 (NH), 3034 (CH aro-

7

.53–7.51 (d, J ¼ 7.6 Hz, 1H, Ar-H), d 7.44–7.42 (d, J ¼ 7.6 Hz, 2H, Ar-

ꢁ1

1

3

H), d 3.79 (s, 2H, CH

2

), d 2.69 (s, 3H, CH

3

);): CNMR (100 MHz,

þ

matic), 2976 (CH aliphatic); MS (Mwt.: 375.85): m/z 375.94 (M ,

1

C H ClN O: C, 70.30; H, 4.83; N, 11.18; Found: C, 70.43; H, 4.88;

N, 11.32.

DMSO): d 162.2, 152.9, 151.1, 138.9, 135.0, 133.8, 131.7, 127.3,

.09%), 349.94 (4.65%), 267.15 (100.00%); Anal. Calcd for

1

3

27.1, 126.8, 126.4, 126.3, 124.7, 124.3, 124.1, 120.5, 120.2, 117.4,

13.2, 110.5, 45.5, 16.7, FT-IR (t max, cm ) 3446, 32992 (2NH),

050 (CH aromatic), 2922 (CH aliphatic); MS (Mwt.: 374.13): m/z

74.75 (M

2

18

3

ꢁ

1

þ,

2.65%), 268.06 (21.45%), 189.95 (18.36%), 77.06

(

100%); Anal. Calcd for C H ClN : C,70.49; H, 5.11; N, 14.95; Acknowledgements

22 19 4

Found: C, 70.73; H, 5.19; N, 15.08.

The authors are grateful to The National Cancer Institute,

Maryland, USA for performing anticancer activity. TMI would like

to acknowledge Prof. Frank Boeckler (University of Tuebingen,

Germany) for granting access to XBScore and to some functions

of MOE.

6

.1.11.4. N-(4-Chlorophenyl)-4-((3-methoxyphenylamino)methyl)ph-

ꢀ

thalazin-1-amine (16d). Yield 0.10 g; (42%), mp 175–177 C;

1

6 2

HNMR (400 MHz, DMSO-d ): d 11.34 (s, 1H, –NH D O exchange-

able), d 9.78 (s, 1H, –NH D O exchangeable), d 8.73–8.70 (d,

2

J ¼ 9.6 Hz, 2H, phthalazine), d 8.48 –8.46 (d, J ¼ 9.6 Hz, 2H, phthala-

zine), d 8.06–8.04 (d, J ¼ 8.6 Hz, 2H, Ar-H), d 7.87–84 (d, J ¼ 8.6 Hz,

Disclosure statement

No potential conflict of interest was reported by the authors.

2

H, Ar-H), d 7.68–7.66 (d, J ¼ 7.5 Hz, 1H, Ar-H), d 7.46–7.44 (d,

J ¼ 7.5 Hz, 2H, Ar-H), d 7.14–7.12 (d, J ¼ 7.5 Hz, 1H, Ar-H), d 4.29 (s,

1

3

2

1

5

2 3

H, CH ), d 3.63 (s, 3H, OCH ); CNMR (100 MHz, DMSO): d

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Article Doi

Design and synthesis of phthalazine-based compounds as potent anticancer agents with potential antiangiogenic activity via VEGFR-2 inhibition

DOI: 10.1080/14756366.2019.1642883

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Article abstract of DOI:10.1080/14756366.2019.1642883

Full text of DOI:10.1080/14756366.2019.1642883

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