Novel Pyrazoloquinolin-2-ones: Design, synthesis, docking studies, and biological evaluation as antiproliferative EGFR-TK inhibitors

Accepted Manuscript

Novel Pyrazoloquinolin-2-ones: Design, Synthesis, Docking Studies, and Bio-

logical Evaluation as Antiproliferative EGFR- TK Inhibitors

Mohammed A.I. Elbastawesy, Ashraf A. Aly, Mohamed Ramadan, Yaseen

A.M.M. Elshaier, Bahaa G.M. Youssif, Alan B. Brown, Gamal El-Din A Abuo-

Rahma

PII:

S0045-2068(19)30497-3

https://doi.org/10.1016/j.bioorg.2019.103045

103045

DOI:

Article Number:

Reference:

YBIOO 103045

Bioorganic Chemistry

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Revised Date:

Accepted Date:

1 April 2019

5 June 2019

Please cite this article as: M.A.I. Elbastawesy, A.A. Aly, M. Ramadan, Y.A.M. Elshaier, B.G.M. Youssif, A.B.

Brown, G. El-Din A Abuo-Rahma, Novel Pyrazoloquinolin-2-ones: Design, Synthesis, Docking Studies, and

Biological Evaluation as Antiproliferative EGFR- TK Inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/

10.1016/j.bioorg.2019.103045

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Novel Pyrazoloquinolin-2-ones: Design, Synthesis, Docking Studies, and Biological

Evaluation as Antiproliferative EGFR- TK Inhibitors

Mohammed A. I. Elbastawesy,^aAshraf A. Aly,^b* Mohamed Ramadan,^aYaseen A. M. M.

Elshaier,^cBahaa G.M. Youssif,^dAlan B. Brown,^eGamal El-Din A Abuo-Rahma^f*

^aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar

University, 71524 Assiut, Egypt.^bDepartment of Chemistry, Faculty of Science, Minia

University, 61519 Minia, Egypt. ^cDepartment of Organic and Medicinal Chemistry, Faculty

of Pharmacy, University of Sadat city, 32958 Menoufia, Egypt .^dDepartment of

Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University, 71526 Assiut,

Egypt. ^eProgram in Chemistry, Florida Institute of Technology, Melbourne, FL 32901, USA,

^fDepartment of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519 Minia,

Egypt

*To whom correspondence should be addressed:

*Ashraf A. Aly_:ashrafaly63@yahoo.com, +201006268742

*G.Abuo-Rahma: gamal.aborahama@mu.edu.eg, +201003069431

1

Abstract

Two new series of diethyl 2-[2-(substituted-2-oxo-1,2-dihydroquinolin-4-yl)hydrazono]-

succinates 6a-g and 1-(2-oxo-1,2-dihydroquinolin-4-yl)-1H-pyrazoles 7a-f have been designed

and synthesized. The structures of the synthesized compounds were proved by IR, mass, NMR

(2D) spectra and elemental analyses. The target compounds were evaluated for their in vitro

cytotoxic activity against 60 cancer cell lines according to NCI protocol. Consequently, seven

compounds were further examined against the most sensitive cell lines, leukemia CCRF-CEM,

and

MOLT-4.

5-Amino-1-(6-bromo-2-oxo-1,2-dihydroquinolin-4-yl)-1H-pyrazole-3,4-

dicarbonitrile (7f) was the most active product, with IC₅₀= 1.35 uM and 2.42 uM against

MOLT-4 and CCRF-CEM, respectively. Also, it showed a remarkable inhibitory activity

compared to erlotinib on the EGFR TK with IC₅₀= 247.14 nM and 208.42 nM, respectively.

Cell cycle analysis of MOLT-4 cells treated with 7f showed cell cycle arrest at G2/M phase

(supported by Caspases, BAX and Bcl-2 studies) with a significant pro-apoptotic activity as

indicated by annexin V-FITC staining. Moreover, the docking study indicated that both the

pyrazole moiety and the quinolin-2-one ring showed good fitting into EGFR (PDB code:

1M17). In order to interpret SAR of the designed compounds, and provide a basis for further

optimization, molecular docking of the synthesized compounds to known EGFR inhibitors was

performed. The study illustrated the effect of several factors on the compounds’ activity.

Keywords: Quinolin-2-one; Pyrazole; NCI; Antiproliferative; Molecular docking, EGFR- TK;

Inhibitors.

2

Introduction

Nitrogen containing heterocycles are indispensable structural units in medicinal chemistry.

Among various heterocyclic compounds, the chemistry and biological applications of quinoline

has been subjected to intense studies from different research groups [1,2]. Recently, quinazoline

derivatives like gefitinib and erlotinib (Figure 1) have been approved by the FDA as EGFR

kinase inhibitors for the treatment of non-small cell lung and breast cancers [3-5]. Moreover, it

was reported that the nitrogen atom at position-3 of the quinazoline core formed a water-

mediated hydrogen bond with Thr⁷⁶⁶(gatekeeper residue) within the EGFR pocket [6,7]. In

contract, bioisosteric replacement of the quinazoline core with a quinoline ring afforded new

quinoline derivatives such neratinib and pelitinib (Figure 1), potent inhibitors of EGFR kinase

[8-12]. This bioisosteric replacement did not require a water molecule to mediate the binding

with the amino acid residue Thr⁷⁶⁶[13]. Moreover, C-3-unsubstituted quinolines can give

similar results; 2-styrylquinoline (SQ-1) derivatives (Figure 1) have been reported as promising

antitumor compounds against several tumor cell lines comparable to gefitinib [14-16].

The pyrazole framework is an exceptionally adaptable drug-like format that is being utilized

broadly in the structure of cancer treatments and cellular apoptosis [17-19]. These compounds

can exhibit anticancer activity by the restraint of various kinds of enzymes, proteins and

receptors which assume basic roles in cell division [20]. Moreover, pyrazolo-fused compounds

have been additionally developed as a significant heterocyclic framework because of their wide

scope of biological activities [21-23], as well as synthetic applications in medicinal chemistry

[24]. Pyrazoles were reported in a variety of potent anticancer active agents as Ruxolitinib

(Figure 1) [25] which were involved in the treatment of intermediate or high-risk myelofibrosis,

a type of myeloproliferative disorder that affects the bone marrow [26, 27]. Crizotinib, another

pyrazole derivative, is an anti-cancer drug acting as an inhibitor of anaplastic lymphoma kinase

3

(ALK) and c-ros oncogene 1 (ROS1), approved for treatment of some non-small cell lung

carcinoma (Figure 1) [28, 29]

Cl

F

HN

O

HN

N

O

N

O

N

O

N

Erlotinib

Gefitinib

Ring replacment

N

F

O

HN

Cl

HN

N

Cl

HN

O

CN

HN

O

CN

N

Pelitinib

Neratinib

Cl

N

HN

N

NH₂

Cl

N

F

N N

Cl

N

N N

O

N

H

HN

Crizotinib

Ruxolitinib

SQ-I

Figure 1. Structure of different quinazoline, quinoline and pyrazole-based-anticancer

agents

In recent years, pyrazolo-quinoline hybrids PQ-1 and PQ-2 (Figure 2) have been reported as

potent antitumor agents [30, 31].

4

OH

S

HN

N N

N

HO

N

O

N

OH

PQ-2

PQ-1

COOEt

NH

NC

H₂N

CN

O

N

O

N

O

N

HN

R₁

N

R

O

N

R

O

R₁

N

Y

R

X

( Not Observed)

Obtained

Figure 2: Structure of different anticancer pyrazolo-quinoline hybrids and the target

compounds.

In our previous studies, we synthesized ethyl pyrano[3,2-c]quinoline-4-carboxylates [32],

spiro(indoline-3,4'-pyrano[3,2-c]-quinoline)-3'-carbonitriles [33], 2,3-bis-(4-hydroxy-2-oxo-1,2-

dihydroquinolin-3-yl) succinates, and arylmethylene-bis-3,3’-quinoline-2-ones [34]. Besides,

two series of N-2,3-bis(6-substituted-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)naphthalene-

1,4-diones and substituted N-(methyl/ethyl)bis-quinolinone triethyl-ammonium salts were

successfully synthesized. The synthesized compounds were targeted as candidates to

extracellular signal-regulated kinases 1/2 (ERK1/2) with considerable antineoplastic activity

[35]. Moreover, we reported on the design and synthesis of novel series of fused naphthofuro[3,2-

c]quinoline-6,7,12-triones and pyrano[3,2-c]quinoline-6,7,8,13-tetraones as potential ERK

inhibitors. The newly prepared inhibitors were evaluated for their ability to inhibit ERK1/2 in an

in vitro radioactive kinase assay. Some of these compounds were found to inhibit ERK1 with

IC₅₀of 0.5 and 0.19 µM, and inhibited ERK2 with IC₅₀of 0.6 and 0.16 µM respectively. Kinetic

5

mechanism studies revealed that the inhibitors are ATP-competitive inhibitors where other

inhibited ERK2 with a K_iof 0.09 µM [36].

In view of the above mentioned findings and our previous success in screening the antitumor

activities of quinolones [35,36], the aim of this research became the synthesis of new quinoline

bio-conjugates of potential EGFR inhibitory and anticancer properties. Our drug design strategy

was based upon optimizing the bio-active hits by manipulation of the lead compound PQ-1

(Figure 2) through the following:

a) Molecular hybridization of quinolin-2-one with pyrazole to form designed

structures 7a-f and Y that included pyrazole nucleus which is an important

pharmacophore;

b) Replacing the lateral phenyl group in PQ-1 by carbonitrile and amino

functional groups able to form hydrogen bonds with EGFR receptors, and test

their antitumor activity against different tumor cell lines.

Accordingly, two new series of 1-(2-oxo-1,2-dihydroquinolin-4-yl)-1H-pyrazoles

(7a-f) and 5-Oxo-1-(2-oxo-quinolin-4-yl)-1H-pyrazole-3-carboxylic acid ethyl

ester (Y) have been designed. Detailed biological studies were carried out on the

newly synthesized pyrazole-quinoline conjugates to test their activities as an

EGFR inhibitor and as antitumor active agents. Attempted synthesis of Y led to

by-products 6a-g, which were tested as open-chain analogues of pyrazoles 7.

2. Results and discussion

2.1. Chemistry

The target compounds 6a-g and 7a-f were prepared according to Scheme 1. Compounds 1-5

were synthesized according to the literature [37-39], and their structures were confirmed by

matching their spectral data with those reported [39]. The key intermediates, 4-hydrazino-

quinolin-2-ones 5a-g, were prepared by heating at reflux compounds 4a-g with hydrazine

6

hydrate for 12 h [39]. To our surprise, refluxing 5a-g with diethyl acetylene dicarboxylate

(DEAD) in DMF furnished diethyl 2-(2-(subsituted-2-oxo-1,2-dihydroquinolin-4-yl)hydrazono)

succinates 6a-g instead of pyrazoles Y. The spectral and elemental data results revealed that all

derivatives 5a-g underwent the reaction smoothly to give the respective open structures 6a-g in

74-90% yield.

Cl

b

R₁

c

R₁

R = H

N

Cl

N

H

O

R

OH

HN

3a-f

4a-f

a

R₁

N

O

5a: R = R₁=H

Cl

5b: R= H; R₁= 5-CH₃

5c: R = H ; R₁= 6-CH₃

5d: R = H ; R₁= 6-OCH₃

5e: R = H ; R₁= 6-Cl

5f: R = H ; R₁= 6-Br

5g: R = CH₃; R₁=H

R

1a-g

2a-g

b

R = Me

N

O

CH₃

4g

O

N

CO₂Et

O

NHNH₂

d

NH

O

N

R₁

4a-g

e

HN

N

R

O

R₁

N

R

O

R₁

5a-g

N

R

f

7a: R= R₁=H

6a-g

Y: not observed

7b: R= H; R₁= 5-CH₃

7c: R = H ; R₁= 6-CH₃

NC

CN

6a: R = R₁=H

7d: R = H ; R₁= 6-OCH₃

7e: R = H ; R₁= 6-Cl

7f: R = H ; R₁= 6-Br

6b: R= H; R₁= 5-CH₃

6c: R = H ; R₁= 6-CH₃

6d: R = H ; R₁= 6-OCH₃

6e: R = H ; R₁= 6-Cl

6f: R = H ; R₁= 6-Br

6g: R = CH₃; R₁=H

N

H₂N

N

R₁

N

R

O

7a-f

Scheme 1. Synthesis of target compounds 6a-g and 7a-f

Reagent and reaction conditions: a) Diethyl malonate, PPA, reflux 3 h; b) POCl₃, reflux 2 h;

c) AcOH, reflux 10 h; d) NH₂NH₂·H₂O (85%), EtOH, reflux 8 h; e) DEAD, DMF, Et₃N, reflux

10-16 h; f) TCNE, EtOH, reflux 20-24 h.

The IR spectra of 6a-g showed an absorption band at 3328-3215 cm^-1corresponding to the NH

group, and bands at 1743-1731 cm^-1characteristic of the ester C=O. In addition, absorptions at

1

2992-2975 and 1675-1630 cm^-1are characteristic of C=CH and C=N, respectively. The H

7

NMR spectra of 6a, for example, showed two sets of ethoxy signals, immediately excluding the

cyclic pyrazole structure, which would have only one. All 19 Hs showed HSQC correlation with

either a C or an N, confirming that they belong to the sample under analysis, not a solvent or

water. In addition to the ethoxy groups, there are two NH protons, a four-spin aromatic system,

a 1H vinylic singlet at δ_H6.34 and a 2H singlet at δ_H3.97. The latter singlet is assigned as H-4e

(Table 1). These two protons correlated with both ester carbonyls at δ_C168.28 and 163.94,

assigned as C-4f and C-4i respectively; with a carbon at δ_C135.30, assigned as C-4d; and with

an sp²nitrogen at δ_N342.4, assigned as N-4c. The assignments of H-4e, C-4e, C-4d, and N-4c

require that the oxaloacetate subunit exist as an imino tautomer not an enamine (Table 1).

Table 1: NMR spectroscopic assignments of compound 6a (italicized correlations are weak).

¹H NMR:

11.38 (s; 1H)

COSY:

Assignment:

H-1

10.07 (s; 1H)

H-4b

H-5

H-7

H-8

H-6

H-3

H-4j

H-4g

H-4e

H-4k

H-4h

4i

4j 4k

8.14 (d, J = 8.0; 1H)

7.54 (“t”, J = 7.3; 1H)

7.33 (d, J = 8.0; 1H)

7.21 (dd, J = 7.8, 7.3; 1H) 8.14, 7.54

6.34 (s; 1H)

4.25 (q, J = 7.1; 2H)

4.12 (q, J = 7.1; 2H)

3.97 (s; 2H)

1.29 (t, J = 7.1; 3H)

1.20 (t, J = 7.1; 3H)

¹³C NMR: HSQC:

168.28

7.54, 7.21

8.14, 7.33, 7.21

7.54

4c

4b

4d

N

CO₂CH₂CH₃

4e

CO₂CH₂CH₃

4a

HN

5

4

6

7

3

4f

4g

4h

N

O

2

1

8a

1.29

1.20

8

H

6a

4.25

4.12

HMBC:

4.12, 3.97

4.25, 3.97

Assignment:

C-4f

C-4i

C-2

163.94

162.33

148.13

139.36

135.30

130.80

122.95

120.76

115.62

111.93

99.13

10.07, 8.14, 7.33, 6.34

8.14, 7.54

10.07, 3.97

8.14, 7.21

7.54

C-4

C-8a

C-4d

C-7

C-5

C-6

C-8

C-4a

C-3

C-4j

C-4g

C-4e

7.54

8.14

7.21

7.33

7.54, 7.21

11.38, 10.07, 8.14, 7.54, 7.33, 7.21, 6.34

11.38, 10.07

1.29

6.34

4.25

4.12

3.97

61.04

60.73

32.12

1.20

8

14.08

1.29

1.20

HSQC:

4.25

4.12

HMBC:

10.07, 3.97

7.33

C-4k

C-4h

13.98

¹⁵N NMR

342.4

Assignment:

N-4c

N-1

145.1

141.6

11.38

10.07

6.34

N-4b

The mass spectra and elemental analyses indicated that the molecular weight is consistent with

the molecular formula of the open chain analogue. Scheme 2 suggests the mechanism of

formation of such compounds as due to nucleophilic attack of the lone pair of electrons of NH₂-

hydrazine to the acetylenic bond to give intermediate A (Scheme 2). Neutralization of

intermediate A would give B, which on proton transfer would give 6a-g (Scheme 2). Similar

mechanisms have been reported [33,36,40,41].

DEAD

CO₂Et

H

CO₂Et

EtO₂C

..

NH₂

EtO₂C

N

NH

H

O

H

O

HN

R₁

6a-g

R₁

N

R

N

R

N

R

O

5a-g

A

B

Scheme 2. Mechanism for the formation of compounds 6a-g

On other hand, heating at reflux compounds 5a-f with ethene-1,1,2,2-tetracarbonitrile (TCNE)

in ethanol furnishes the corresponding 1-(2-oxo-1,2-dihydroquinolin-4-yl)-1H-pyrazoles 7a-f in

moderate yields. Elemental analyses, IR and NMR in addition to mass spectra were in good

agreement with the assigned product structures. As a representative example for this series, the

IR spectrum of compound 7d showed frequencies corresponding to amino, nitrile and C=N

1

groups at  3347, 3196 (NH₂), 2227 (CN), 1674 (C=N) cm^-1, respectively. Moreover, the H

NMR spectrum of 7d showed a broad singlet signal at δ_H7.56 (disappeared upon deuteration)

representing NH₂protons; their attached nitrogen appears at δ_N62.8, and gives HMBC

correlation with two carbons in the pyrazole ring not with the quinolone. In addition, there is a

9

13

characteristic singlet signal at δ_H3.38 related to OCH₃group. The C NMR spectrum of 7d

showed two peaks at δ_C111.95 and 111.91 assigned to the cyano groups, a signal at δ_C126.90

for C=N, and a peak at δ_C160.70 assigned to the C=O of the quinolone moiety (Table 2).

Table 2: NMR spectroscopic assignments of compound 7d (italicized correlations are weak)

¹H NMR

12.13 (s; 1H)

7.56 (bs; 2H)

7.39 (d, J = 9.7; 1H) 7.31, 6.58

7.31 (d, J = 8.8; 1H) 7.39, 6.58

6.81 (s; 1H)

6.58 (s; 1H)

3.38 (s; 3H)

¹H-¹H COSY:

6.81

Assignment:

H-1

H-5a’

H-7

H-8

H-3

4a'

5a'

NC

5'

CN

3'

2'

3a'

4'

N

H₂N

N

1'

6a

5

4

4a

8a

O

6

3

12.13, 6.58

7.39, 7.31, 6.81

12.13

H-5

H-6a

7

N

O

2

8

H

1

7d

¹³C NMR:

160.77

154.50

152.99

142.25

134.16

126.90

122.72

120.60

117.21

116.38

111.95, 111.91

105.38

75.86

HSQC

HMBC

Assignment:

C-2

7.39, 7.31, 6.81, 6.58, 3.72 C-6

7.56

C-5’

C-4

C-8a

C-3’

C-3

7.39, 7.31, 6.81, 6.58

7.39, 7.31, 6.58

6.81

7.31

7.39

12.13

6.58

C-7

C-8

12.13, 7.39, 7.31, 6.81

C-4a

C-3a’, 4a’

C-5

C-4’

C-6a

6.58

3.72

7.39, 7.31, 6.81

7.56

55.62

¹⁵N NMR: HSQC:

189.0

HMBC:

7.56, 6.81

7.39, 6.81

Assignment:

N-1’

N-1

151.6

62.8

12.13

7.56

N-5a’

Mass spectra and elemental analyses data confirmed the suggested structural formula.

The formation of the pyrazole structure can be explained as due to nucleophilic attack of the

lone pair of electrons of NH₂-hydrazine to the ethylene bond to give intermediate C (Scheme 3).

Neutralization and elimination of a molecule of HCN from intermediate C would give D.

Subsequently, nucleophilic attack of the lone pair of electrons of the NH-hydrazine atom to the

10

carbonitrile carbon would give Zwitter-ion E. Ultimately; neutralization of E would give F,

which on aromatization would give 7a-f (Scheme 3).

NC

CN

NC

HN

NC

CN

NH

NC

TCNE

CN

H

CN

..

N

..

HN

N

NH₂

H

HN

- HCN

R₁

N

H

O

N

H

O

N

H

O

C

D

5a-f

NC

CN

NC

CN

NH

N

H

HN

N

H

7a-f

R₁

N

O

N

H

O

H

E

F

Scheme 3. Mechanism for the formation of compounds 7a-f

2.2. Biology

2.2.1 Evaluation of in vitro antiproliferative activity for compounds 6a-g and 7a-f.

The target compounds 6a-g and 7a-f were selected by the National Cancer Institute [NCI,

Bethesda, MD, USA (http://www.dtp.nci.nih.gov.)] for evaluation at a single concentration of

10 µM towards a panel of sixty cancer cell lines of nine diverse tissues according to NCI

protocol. The screening results were reported (Table 3) as the percent growth of treated cells

compared to untreated control cells. The cyclized 1-(2-oxo-1,2-dihydroquinolin-4-yl)-1H-

pyrazole compounds 7a-f are more active than the open chain analogues containing hydrazino-

succinic acid diethyl ester backbone 6a-g, which reflects a positive influence of cyclization on

the antiproliferative activity. 1-(2-Oxo-1,2-dihydroquinolin-4-yl)-1H-pyrazole scaffold

compounds 7a-f showed promising activities against a majority of the tested cancer cell lines.

Substituents attached to the phenyl moiety affected the activity: it was found that compounds 7e

and 7f, possessing 6-chloro and 6-bromo substituents respectively, were more active against all

11

tested leukemia cancer cell lines than compound 7d with a 6-methoxy substituent, which

showed no inhibitory effect against any of the tested leukemia cell lines. A similar pattern was

also observed to PC-3 prostatic cancer cell line and HOP-92 non-small cell lung cancer. Here,

the halogen bearing compound 7e showed excellent cell growth inhibition activity against HOP-

92 non-small cell lung cancer and PC-3 prostatic cancer cell line with a high GI values of -

79.6% and -87.3%, respectively; 7f resembled 7e, with GI values of -90.8% and -86.9%, but the

methoxy-bearing 7d gave GI values of 35.29 and 10.65%, respectively (Table 3). On the other

hand, compounds 6a-g showed moderate cytotoxic activities against majority of cancer cell

lines. Among them, compounds 6d, 6e and 6f have good anticancer activity against all the

tested leukemia cell lines.

It is obvious that the electronic effect and the position of substituent on the tested scaffolds had

a pronounced effect on their cytotoxic activity. Compounds 6d-f with electron withdrawing

groups on the pendant phenyl group showed higher antiproliferative activity with GI values

73.91 % (6d) and lethal effect -0.31% (6e) and -15.7% (6f) against MOLT-4 leukemia cell line

than compounds 6b and 6c with electron donating groups (GI = 0 and 81.67 %, respectively)

(Table 3). Compared with 6-substituted 6c, the 5-substituted 6b showed no inhibitory effect

against all the tested leukemia cell lines, indicating the importance of the 6-substitution for

optimizing activity where a similar trend was also observed in case of PC-3 prostatic cancer cell

line. From the results, it is clear that substitution on the phenyl group of quinolone is important

for the antiproliferative activity where the most active compounds against all of the tested

leukemia cell line are substituted at position 6, compared to the unsubstituted analogues (6d-f vs

6a).

12

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

concentration for compounds 6a-g and 7a-f.

Subpanel

cancer cell

Lines

% Growth Inhibition (GI %)^a

6a

6b

6c

6d

6e

6f

6g

7a

7b

7c

7d

7e

7f

Leukemia

CCRF-CEM

HL-60(TB)

K-562

-

-57.19

-

-47.5

-

-51.5

-

-36.7

-7.48

75.85

-15.7

16.79

59.20

-

11.44

28.73

-

-15.8

99.03

-43.9

-3.9

-32.0

-11.2

-12.4

-16.1

-29.7

-14.2

13.77

28.23

12.41

10.89

25.37

-

10.56

81.67

-

10.69

73.91

31.45

33.01

56.05

-0.31

42.82

53.04

24.05

10.71

-

MOLT-4

RPMI-8226

SR

-24.6

99.29

53.64

Non-small cell lung cancer

-

A549/ATCC

EKVX

-

12.78

-

32.15

31.86

15.70

21.32

11.09

33.20

18.93

22.97

-

12.53

-

17.29

13.67

12.81

16.81

HOP-62

20.17

12.82

12.52

23.85

10.55

-

35.29

-79.6

-90.8

HOP-92

-

13.13

12.62

14.59

14.56

-

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

Colon cancer

COLO 205

HCC-2998

HCT-116

HCT-15

-

11.45

18.59

22.18

10.98

12.90

21.63

-

20.04

37.15

-

21.53

-

24.02

13.52

12.95

15.61

14.27

12.13

-

HT29

KM12

16.47

SW-620

CNS cancer

SF-268

-

SF-295

-

10.20

11.39

-

SF-539

-

10.30

-

11.77

-

17.55

-

12.74

-

SNB-19

SNB-75

13.56

U251

Melanoma

LOX IMVI

-

11.06

-

20.47

-

18.98

21.79

15.11

-

15.69

-

10.57

-

MALME-3M

M14

-

13

-

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

-

12.12

19.75

11.73

13.97

13.03

18.06

12.64

22.08

12.96

13.41

Ovarian cancer

-

17.41

-

24.71

35.93

11.26

14.50

-

IGROV1

-

10.8

-

OVCAR-3

-

12.63

-

10.97

-

18.37

OVCAR-4

-

OVCAR-5

-

14.09

-

10.59

-

OVCAR-8

-

NCI/ADR-RE

-

12.84

25.54

21.67

21.72

25.42

30.92

SK-OV-3

Renal cancer

-

14.75

-

10.39

-

786-0

-

A498

-

13.40

16.87

17.42

-

ACHN

15.27

12.94

-

16.86

10.36

-

CAKI-1

RXF 393

SN12C

TK-10

-

23.63

-

12.75

-

19.52

27.67

13.32

21.87

16.29

28.62

10.49

38.76

54.77

23.63

28.38

24.15

34.26

UO-31

Prostate cancer

-

11.73

-

12.59

-

28.88

-

13.89

-

10.65

-

-87.3

-

-86.9

-

PC-3

-

DU-145

Breast cancer

-

12.13

-

14.91

14.51

-

MCF7

MDA-MB-

231/ATCC

-

15.58

14.72

-

11.41

14.89

43.91

-

HS 578T

-

14.41

-

13.11

22.95

-

13.47

11.25

-

12.07

32.76

-

BT-549

-

21.08

13.91

T-47D

-

10.94

MDA-MB-468

(–): Weak activity GI < 10%.

2.2.2 In vitro five dose full NCI 60 cell panel assay

Compounds 7e and 7f were selected for five dose testing against the full panel of 60 human

tumor cell lines according to NCI protocol (http://www.dtp.nci.nih.gov.), Appendix A.

14

Compound 7e exhibited noteworthy antiproliferative activity against ovarian cancer cell lines

with GI₅₀= 8.63 µM (Table 4) and selectivity ratio = 4.99, and against non-small cell lung cancer

with GI₅₀= 11.35 and selectivity ratio = 3.79. Compound 7f showed antiproliferative activity

against ovarian cancer cell lines with GI₅₀= 37.90 µM and selectivity ratio = 1.29, and against

non-small cell lung cancer with GI₅₀= 22.27 and selectivity ratio = 2.21.

Selective index .

Table 4. Results of in vitro five dose testing of nine human cancer types and selectivity for

compounds 7e and 7f.

7e

7f

GI₅₀

Conc.

per cell

line

Conc.

TGI LC₅₀

Panel

Cell line

TGI LC₅₀

Selectivity

ratio

Selectivity

ratio

per cell MID^b

MID^b

line

CCRF-

CEM

> 100

> 100 > 100

HL-60(TB)

K-562

> 100

74.2

> 100 > 100

Leukemia

-

74.2

0.66

MOLT-4

RPMI-8226

SR

> 100

A549/ATCC

EKVX

> 100

11.5

> 100

4.60

HOP-62

> 100

11.2

> 100

51.5

HOP-92

Non-small cell lung

cancer

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

> 100

59.3

11. 3.79

22.27 2.21 > 100 > 100

> 100 > 100

> 100

22.7

> 100 > 100

86.4 > 100

COLO 205

HCC-2998

HCT-116

HCT-15

HT29

> 100

> 100 > 100

> 100

> 100 > 100

Colon Cancer

59. 0.73

-

> 100 > 100

KM12

15

SW-620

> 100

67.0

> > 100

> 100 > 100

SF-268

SF-295

SF-539

SNB-19

SNB-75

U251

> 100

51.7

CNS Cancer

-

65.97 0.74

> 100

57.1

88.1

LOX IMVI

> 100

MALME-

3M

> 100

> 100 > 100

M14

-

MDA-MB-

435

Melanoma

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

IGROV1

> 100

8.63

> 100

37.9

> 100 > 100

-

OVCAR-3

OVCAR-4

> 100

37.9

1.29

OVCAR-5

OVCAR-8

> 100

Ovarian Cancer

8.6 4.99

NCI/ADR-

RES

> 100

> 100 > 100

SK-OV-3

> 100

82.3

> 100

64.1

> 100 > 100

786-0

ACHN

CAKI-1

RXF 393

SN12C

UO-31

Renal Cancer

-

PC-3

Prostate Cancer

DU-145

MCF7

MDA-MB-

231/ATCC

89.8

> 100

> 100 > 100

HS 578T

BT-549

T-47D

> 100

38.9

> 100

64.9

> 100 > 100

45.15 1.09

Breast Cancer

70. 0.61

> 100

6.46

MDA-MB-

468

> 100

> 100 > 100

16

MID^afor 7e= 43.09, MID^afor 7f= 49.19

Figure 3: Dose-antiproliferative response of compound 7e against nine different cancer

cell lines.

17

Figure 4. Dose-antiproliferative response of compound 7f against nine different cancer

cell lines

2.2.3. Effect of compounds 6c-f, 7b, 7e and 7f on MOLT-4 and CCRF-CEM leukemia

cancer cells growth

Seven compounds 6c-f, 7b, 7e and 7f were evaluated for their anticancer activity against

MOLT-4 and CCRF-CEM leukemia cancer cell lines by MTT assay (Appendix A) [42]: the

results are shown in Table 5. Compounds 7b, 7e and 7f, with the 1-(2-oxo-1,2-

dihydroquinolin-4-yl)-1H-pyrazole scaffold, showed better anticancer activity than open chain

analogues 6c-f. Compounds 7e and 7f with 6-chloro and 6-bromo substitution on the

quinoline core were found the most potent against tested cancer cell lines showing IC₅₀of

4.34 ± 0.14 µM and 1.35 ± 0.08 µM, respectively against MOLT-4 where the standard

erlotinib showed IC₅₀= 07.38 ± 0.56 µM. The same pattern was observed against the CCRF-

CEM leukemia cell line (Table 5). Compounds 7e and 7f substituted at 6-position showed

better activity against the MOLT-4 leukemia cell line (IC₅₀4.34 ± 0.14 µM for 7e, and 1.35 ±

0.08 µM for 7f) than compounds 6e and 6f bearing the same substitution at 6-position but

with hydrazino-succinic acid diethyl ester backbone (IC₅₀= 11.34 ± 0.22 µM for 6e and 7.57

± 0.16 µM for 6f). Finally, it is noteworthy that compound 6c, with 6-methyl substitution on

the quinoline core, was found to be the least potent among the tested compounds against the

MOLT-4 line, with IC₅₀43.19 ± 0.71µM.

In short, the presence of cyclized pyrazole at position 4 of quinolone gave much better activity

than the open scaffold at the same position against the majority of the tested cancer cell lines.

Moreover, substitution at the quinolone 6 position with electron withdrawing groups like Br

or Cl enhances greatly the anticancer activity, while introduction of an electron-donating 6-

methyl group reduces activity greatly. Similarly, shift of the substituent into position 5 of the

quinolone moiety abolished activity (cf. 6b and 6c).

18

Table 5. Antiproliferative activities of compounds 6c-f, 7b, 7e and 7f

Compound Antiproliferative activity IC₅₀± SEM (µM)

MOLT-4

43.19 ±0.71

14.75 ±0.28

11.34 ±0.22

7.57 ±0.16

7.05 ±0.11

4.34 ±0.14

1.35 ±0.08

7.38 ±0.56

CCRF-CEM

6c

6d

6e

6f

7b

7e

7f

12.55 ±0.06

10.32 ±0.07

11.38 ±0.02

16.39 ±0.32

5.42 ±0.05

5.07 ±0.10

2.42 ±0.04

4.92 ±0.24

Erlotinib

2.2.4. EGFR TK Inhibitory Activity

The mechanism at molecular level of the most active antiproliferative compounds 6e, 6f, 7b,

7e and 7f were investigated on EGFR TK kinase (Table 6) [43]. The findings of the cancer

cell-based assays have been powerfully complemented by the results from the EGFR assay.

Compounds 7e and 7f showed potent EGFR inhibition with IC₅₀298.27 and 247.14 nM,

respectively while that of erlotinib was 208.42 nM. Compound 6e displayed the least potent

activity (IC₅₀= 703.84 nM) among all the tested compounds. The inhibitory effect against

EGFR TK kinase is consistent with their antiproliferative effects, where the cyclized 5-

amino-1H-pyrazole 3,4-dicarbonitrile compounds are superior in activity when compared

with the open chain analogues. This study demonstrates that compounds 7e and 7f are strong

inhibitors of EGFR and can probably be promising anticancer agents.

Table 6. Effects of compounds 6e, 6f, 7b, 7e and 7f on EGFR TK kinase

Compound

EGFR IC₅₀( nM)

6e

6f

703.84

472.41

7b

7e

345.19

298.27

247.14

208.42

7f

Erlotinib

19

2.2.5. Activation of caspases cascade.

Activation of caspases plays a key role in the initiation and effecting of the apoptotic process

[44]. Among the most common caspases, caspase-3 is an essential player that cleaves multiple

proteins in the cells, leading to apoptotic cell death [45]. The effect of compounds 7e and 7f on

caspase 3 were evaluated and compared to erlotinib as a reference drug. Compounds 7e and 7f

showed an increase in the level of active caspase 3 by 5.14 and 5.82 fold versus control,

respectively, compared to erlotinib (6.49 fold) as shown in Table 7. Moreover, the effects of

compounds 7e and 7f on caspases 8 and 9 was also evaluated; compound 7e increases the

levels of caspases 8 and 9 by 2.82 and 10.10 fold respectively, while compound 7f increases

their level by 3.52 and 9.63 fold, respectively, compared to erlotinib (6.52 and 11.15 fold

respectively: Table 7). Thus, both 7e and 7f are potential activators of both intrinsic and

extrinsic caspase pathways with more prominent effect on the intrinsic pathway, since caspase

9 levels are higher.

Table 7. Effect of compounds 7e, 7f and erlotinib on the active caspases-3, 8 and 9 in

MOLT-4 cell line.

Compound Number

Caspase-3

Caspase-8

Conc Fold

(ng/ml) change (ng/ml) change

Caspase-9

Conc Fold

Conc

(pg/ml)

319

Fold

change

5.14

7e

7f

0.48

0.60

1.11

2.82

3.52

6.52

24.05

22.94

26.56

10.10

361

405

5.82

6.53

9.63

Erlotinib

11.15

Control

62

1

0.17

1

02.38

1

2.2.6. Effects on BAX and Bcl-2 proteins.

Bcl-2 (B-cell lymphoma 2) family members are significant regulators of apoptosis that can be

classified into three groups. The first group protects against apoptosis (Bcl-2 itself). The

second group of proteins is represented by Bax (Bcl-2 associated protein X) and Bak (Bcl-2

20

antagonist/killer), which are the key activators of the apoptosis machinery in response to

cellular stress stimuli. The third group is comprised of different categories of proteins such as

Bid (BH3 interacting death domain), Bim (Bcl-2 interacting mediator) and others [46].

Overexpression of the antiapoptotic Bcl-2 members leading to antagonizing apoptosis and

development of resistance of tumor cells to common chemotherapeutic agents. Thus, the

development of new drugs that can inhibit the action of antiapoptotic Bcl-2 members is a very

attractive strategy in the design of anticancer agents [47]. The effect of compounds7e and 7f on

the expression levels of Bcl2 and BAX was determined after treatment of MOLT-4 cells with

the IC₅₀of compounds7e and 7f (Table 8). Compound 7e caused up-regulation in the level of

the proapoptotic protein BAX approximately five-fold while it markedly down-regulated the

levels of the antiapoptotic proteins Bcl2 up to four-fold compared to the control untreated cells.

Compound 7f caused up-regulation in the level of the BAX over four-fold whiles it markedly

down-regulated the levels of the antiapoptotic proteins Bcl2 three-fold compared to the control.

These findings proved the proapoptotic effect of compounds 7e and 7f.

Table 8: Bax and Bcl-2 levels for compounds 7e, 7f, and erlotinib on MOLT-4 cells

Bax

Bcl-2

Compound

Number

Conc

Fold

Conc

Fold

(pg/ml) change (ng/ml) change

7e

7f

249.10

221.80

04.89

04.35

1.36

1.70

03.83

03.06

Erlotinib

251.40

50.88

0.99

5.21

05.26

1

04.94

1

Control

2.2.6. Cell cycle analysis and apoptosis assay

2.2.6.1. Cell cycle analysis

The cell cycle includes four phases: The G1 phase where cell enlargement and preparation of

DNA duplication occurs; the S phase (synthesis) is the stage of DNA replication and

chromatid duplication; in G2 Phase, repairing of new DNA and more growth occurs while in

21

the M stage nuclear division takes place. Studies on the effect of compound 7f on cell cycle

development and induction of apoptosis in the MOLT-4 were done. MOLT-4 was incubated

with IC₅₀concentration of compound 7f for 24 h. Consequently, the cell line was stained with

PI/Annexin V and analyzed by flow cytometry using BD FASCC alibur [48]. Investigation of

the results in Figure 5, exposed that percentage of pre G1 apoptosis induced by 7f on MOLT-

4 was 22.42%. A high percent of cell accumulation was observed in G2-M phase in MOLT-4

treated with compound 7f after 24 h incubation, indicating arrest of cell cycle at G2-M phase.

Figure 5: Cell cycle analysis results for compound 7f

2.2.6.2. Apoptosis assay

Cell cycle analysis of MOLT-4 after treatment with compound 7f showed presence of pre-G1

peak which is an indication of apoptosis. To confirm the ability of 7f to induce apoptosis, cells

were stained with Annexin V/PI, incubated for 24 h and analyzed. Analysis of early and late

apoptosis showed that compound 7f was able to induce significant levels of apoptosis with

necrosis percent 4.61 and 15.32, respectively (Figures 6 and 7) and that % of necrosis was

2.49.

22

Figure 6. Apoptosis induction analysis using Annexin V/PI for compound7f

Figure 7: Apoptosis induction analysis using Annexin V/PI for compound 7f.

2.3. Docking study

A library of the target compounds, structurally related compounds (Figures 1 and 2), and

known EGFR inhibitors (Erlotinib and Gefitinib) was designed and energy minimized using

MMFF94 force field calculations. The catalytic domain of EGFR (PDB code: 1M17) [49] was

prepared for docking using Open Eye® software. Docking was conducted using the Fred

application and the data was visualized by the Veda application. This software package

generates consensus scoring which is a filtering processes to obtain virtual binding affinity: a

lower consensus score denotes better binding affinity of the ligands towards the receptor. The

23

standard EGFR inhibitors PQ-1[30, 48], Erlotinib, and Gefitinib docked in the receptor with

high consensus score (0, 8, 13 respectively). The docking results showed that compounds 7e

and 7f have high consensus score (50 and 49 respectively) in comparison to other synthesized

derivatives. This evidence emphasizes our design strategy about incorporating the pyrazole

ring together with the quinolin-2-one scaffold.

Erlotinib interacts in the EGFR receptor with formation of a hydrogen bond (HB; 1.71 Å)

towards the ATP-binding site of EGFR coming from pyrimidine-N2 with Met:769:A. This

docking mode is similar to the cocrystallized docking pose with the receptor [50].

The lead compound PQ-1 [32, 50] showed good interaction with the EGFR receptor through

formation of two HBs with Met:769:A coming from its lactam group (donor and acceptor).

Moreover, it participates in HB with Lys:721:A through its phenolic OH group (Figure 8A).

The most potent derivative (compound 7f) interacts with the EGFR receptor by formation of

HB with Met:769:A through the carbonyl of its quinolinone ring. Moreover, it formed HB with

Lys:721:A through the nitrile group (adjacent to the amino functionality) of pyrazole ring

(Figure 8B). This docking mode demonstrated that both scaffolds, quinolone and pyrazole,

participate in essential HB interactions. Interestingly, compound 7e showed good overlay with

the standard Erlotinib (Figure 8C). Compound 7e forms HB with Asp:831:A through the amino

group of the pyrazole moiety. Also, it formed two HBs with Thr:830:A through the nitrile

functionality which is adjacent to the amino group (Figure 8D). This interaction of the amino

and ortho nitrile parts with the peptide Thr:830-Asp:831:A looks like a cyclic system.

24

A

B

C

D

Figure 8.A): Visual representation of compounds docked with 1M17: (A) PQ-1 (B) Visual

representation of 7f docked with 1M17, showed HB with Met:769:A and Lys:721:A; (C)

Visual representation of 7f overlaid with standard erlotinib, showed HB interactions with the

same amino acid Met:769:A; (D) Visual representation of 7e with formation of HB with

Thr:830:A and Asp:831:A.

2.4. Structure activity relationship

The presence of cyclized pyrazole at position 4 of quinolone gave much better activity than the

open scaffold at the same position on the majority of the tested cancer cell lines. This is due to

rigidification of pyrazole part to occupy the pocket occupied by the aryl part as shown by the

25

docking pose (Figure 9). Moreover, it is clear that substitution at quinolone 6-position with

electron withdrawing groups like Br or Cl enhances greatly the anticancer activity, while

introduction of a 6-methyl group reduces activity greatly. Similarly, shift of the substituent into

position 5 of the quinolone moiety abolished activity (cf. 6b and 6c) . This is due to increased

strength of HB interaction with the amino acids and also separation of the exocyclic pyrazole

part from the quinolinone ring. The most potent compound 7f interacts with the EGFR receptor

by formation of HB with Met:769:A amino acid through the 2-carbonyl of its quinolone ring.

Moreover, it formed HB with Lys:721:A amino acids through the nitrile group (adjacent to

amino functionality) of pyrazole ring.

According to compounds pose and mode shown from docking studies (Figures 7-8), we found

that our designed compounds have a resemblance to the reported compounds erlotinib and PQ-

1, as illustrated from the graphical representation of the SAR of the most potent series in this

work (Figure 9). The anticancer and activity against EGFR of the target compounds depends

on both the two parts constituting the hybrid, the 2-quinolone and pyrazole scaffolds, and also

on the nature of the substituent.

HO

Aryl group

that characterize

the tail

N

occupied the

cleft in

polar part

N

that characterize the

head occupied the

cleft in the receptor

HN

O

the receptor

N

OH

HO

O

PQ-1

O

Erlotinib

O

NC

CN

N

CN

NC

CN

N

H₂N

N

H₂N

Br

N

X

Cl

N

H

O

N

H

O

N

H

O

7f

7e

7f: IC₅₀= 1.35 ±0.08 and 2.42 ±0.04 µM against

MOLT-4 CCRF-CEM leukemia cell lines, respectively

IC₅₀= 247.14nM against EGFR TK

7e: IC₅₀

= 4.34 ±0.14and 5.07 ±0.10 µM against

MOLT-4 CCRF-CEM leukemia cell lines, respectively

IC₅₀= 298.27nM against EGFR TK

Figure 9. Graphical representation of the SAR of the most potent of the designed compounds,

and standards erlotinib and PQ-1

26

3. Conclusion

A new series of 4-pyrazolyl-quinolin-2-ones 7a-f and the open chain analogues 6a-g were

1

13

characterized by different spectroscopic and elemental techniques including IR, H NMR, C

NMR, 2D NMR, MS and elemental microanalyses. In vitro one dose anticancer screening

results revealed that the cyclized 5-amino-1H-pyrazole-3,4-dicarbonitrile derivatives 7a-f are

more potent than the open chain analogues 6a-g. Compounds 7e and 7f were selected for five

dose testing against the full panel of 60 human tumor cell lines and compound 7e showed

moderate selectivity against ovarian, small cell lung, leukemia and cancer cell lines. Testing of

the most active compounds 7a-f showed that 6-halogen substituted derivatives 7e and 7f are

the most active against leukemia MOLT-4 and CCRF-CEM cell lines with IC₅₀= 4.34 ± 0.14

M and 5.07 ± 0.10 M (7e); 1.35 ± 0.08 M and 2.42 ± 0.04 M (7f) compared to erlotinib

with IC₅₀= 7.38 ± 0.56 M and 4.92 ± 0.24 M, respectively.

Compounds 7e and 7f experienced an outstanding inhibitory activity comparable to erlotinib

on the EGFR TK with IC₅₀= 247.14 nM and 208.42 nM, respectively. Cell cycle analysis

indicated cell cycle arrest at G2/M phase (supported by Caspases, BAX and Bcl-2 studies) with

a significant pro-apoptotic activity as indicated by annexin V-FITC staining. Moreover, the

docking study mode showed that both pyrazole moiety and quinolone ring showed good fitting

into EGFR receptor (PDB code: 1M17). The 2-quinolone-pyrazoles 7e and 7f are considered

promising lead antiproliferative EGFR TK inhibitors.

4. Experimental

4.1. Chemistry

Melting points were determined using open glass capillaries on a Gallenkamp melting point

apparatus (Weiss-Gallenkamp, Loughborough, UK), and are uncorrected. The IR spectra were

recorded from potassium bromide disks with a FT device, Minia University. NMR spectra

were measured in DMSO-d₆on a Bruker AV-400 spectrometer (400 MHz for ¹H, 100 MHz for

27

¹³C, and 40.55 MHz for ¹⁵N); chemical shifts are expressed in  (ppm), versus internal

tetramethylsilane (TMS) = 0 for ¹H and ¹³C, and external liquid ammonia = 0 for ¹⁵N. Coupling

1

constants are stated in Hz. Correlations were established using H-¹H COSY, and H-¹³C and

¹H-¹⁵N HSQC and HMBC experiments. ¹⁵N signals were observed indirectly, through the

proton channel in HSQC or HMBC experiments. Mass spectra were recorded on a Finnigan

Fab 70 eV, Institute of Organic Chemistry, Karlsruhe University, Karlsruhe, Germany. TLC

was performed on analytical Merck 9385 silica aluminum sheets (Kieselgel 60) with Pf₂₅₄

indicator; TLC's were viewed at _max= 254 nm. Elemental analyses were carried out at the

Microanalytical Center, Cairo University, Egypt.

General Details: See Appendix A

4.1.1 General method for the Synthesis of compounds 6a-g

To a solution of hydrazino quinolone 5 (1mmol) in dry DMF (30 ml), diethyl acetylene

dicarboxylate (0.17 gm, 1mmol) was added. The reaction mixture was heated under reflux for

10-16 h (monitored by TLC). After reaction completion, the reaction mixture was poured onto

a mixture of crushed ice and water with vigorous stirring. The precipitated solid was filtered,

washed with water, dried and crystallized from methanol to afford compounds 6a-g.

4.1.1.1 Diethyl 2-(2-(2-oxo-1,2-dihydroquinolin-4-yl)hydrazono)succinate (6a)

Yield: 0.29 g (84%); mp 190–192 °C, IR (KBr) υ_max3291, 2983, 1742, 1655. NMR: Table 1.

LCMS: m/z calcd: 345.13, found [M-H]^-: 344. Anal. Calcd for C₁₇H₁₉N₃O₅(345.13): C, 59.12;

H, 5.55 ; N, 12.7. Found: C, 58.89; H, 5.37; N, 12.41.

4.1.1.2 Diethyl 2-(2-(5-methyl-2-oxo-1,2-dihydroquinolin-4-yl)hydrazono)succinate(6b)

1

Yield: 0.28 g (80%); mp 198–200 °C, IR (KBr) υ_max3299, 2975, 1743, 1647. H NMR _H

12.37 (s, 1H: NH-4b), 11.35 (s, 1H; NH-1), 7.37 (t, J = 7.6, 1H; H-7), 7.21 (d, J = 7.8, 1H; H-

8), 6.89 (d, J = 7.0, 1H; H-6), 6.47 (s, 1H; H-3), 4.28 (q, J = 6.8, 2H; H-4j), 4.14 (q, J = 6.7,

28

2H; H-4g), 3.63 (s, 2H; H-4e), 2.88 (s, 3H; H-5a), 1.25 (t, J = 6.9, 3H; H-4k), 1.22 (t, J = 7.0,

3H; H-4h). ¹³C NMR _C169.85 (C-4f), 161.70, 161.65 (C-2, 4i), 149.36 (C-4), 140.97 (C-8a),

133.30 (C-5), 130.27, 130.10 (C-4d, 7), 125.45 (C-6), 114.73 (C-8), 111.59 (C-4a), 98.73 (C-

3), 61.55 (C-4j), 60.55 (C-4g), 39.16 (C-4e), 23.82 (C-5a), 14.04 (C-4k), 13.72 (C-4h). ¹⁵N

NMR _N343.6 (N-4c), 151.2 (N-4b), 147.0 (N-1). LCMS: m/z calcd: 359.15, found [M+H]⁺:

359.9. Anal. Calcd for C₁₈H₂₁N₃O₅(359.15):C, 60.16; H, 5.89 ; N, 11.69. Found: C, 60.42; H,

6.08; N, 11.75.

4.1.1.3 Diethyl2-(2-(6-methyl-2-oxo-1,2-dihydroquinolin-4-yl)hydrazono)succinate (6c)

1

Yield: 0.32 g (90%); mp 221–223 °C, IR (KBr) υ_max3307, 2975, 1740, 1647. H NMR _H

11.31 (s, 1H; NH-1), 10.02 (s, 1H; NH-4b), 7.93 (s, 1H; H-5), 7.37 (d, J = 8.3, 1H; H-7), 7.23

(d, J = 8.4, 1H; H-8), 6.32 (s, 1H; H-3), 4.25 (q, J = 7.1, 2H; H-4j), 4.13 (q, J = 7.1, 2H; H-4g),

3.97 (s, 2H; H-4e), 2.39 (s, 3H; H-6a), 1.29 (t, J = 7.1, 3H; H-4k), 1.20 (t, J = 7.1, 3H; H-4h).

¹³C NMR _C168.30 (C-4f), 163.95 (C-4i), 162.24 (C-2), 147.91 (C-4), 137.39 (C-8a), 135.11

(C-4d), 131.96 (C-7), 129.80 (C-6), 122.34 (C-5), 115.56 (C-8), 111.84 (C-4a), 99.22 (C-3),

15

61.02 (C-4j), 60.72 (C-4g), 32.13 (C-4e), 20.61 (C-6a), 14.09 (C-4k), 13.98 (C-4h). N NMR

_N342.6 (N-4c), 144.3 (N-1), 141.8 (N-4b). LCMS: m/z Calcd: 359.15, found [M+H]⁺: 359.9.

Anal. Calcd for C₁₈H₂₁N₃O₅(359.15):C, 60.16; H, 5.89; N, 11.69. Found: C, 60.04; H, 6.13; N,

11.88.

4.1.1.4. Diethyl 2-(2-(6-methoxy-2-oxo-1,2-dihydroquinolin-4-yl)hydrazono) succinate

(6d)

Obtained as an equally-populated mixture of C=N stereoisomers. Plain numbers denote one

isomer, primed numbers the other. Yield 0.28 g (77%), mp 188–189 °C, IR (KBr) υ_max3215,

1

2991, 1738, 1651. H NMR _H12.40 (s, 1H; NH-4b’), 11.30 (s, 1H; NH-1, 1’), 10.04 (s, 1H;

H-4b), 7.64 (d, J = 2.4, 1H; H-5), 7.32 (d, J = 9.0, 1H; H-8’), 7.29 (d, J = 8.9, 1H; H-8), 7.28

29

Article Doi

Novel Pyrazoloquinolin-2-ones: Design, synthesis, docking studies, and biological evaluation as antiproliferative EGFR-TK inhibitors

DOI: 10.1016/j.bioorg.2019.103045

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