Discovery of new quinolines as potent colchicine binding site inhibitors: design, synthesis, docking studies, and anti-proliferative evaluation

Journal of Enzyme Inhibition and Medicinal Chemistry

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

Discovery of new quinolines as potent colchicine

binding site inhibitors: design, synthesis, docking

studies, and anti-proliferative evaluation

Mohamed Hagras, Moshira A. El Deeb, Heba S. A. Elzahabi, Eslam B. Elkaeed,

Ahmed B. M. Mehany & Ibrahim H. Eissa

To cite this article: Mohamed Hagras, Moshira A. El Deeb, Heba S. A. Elzahabi, Eslam B.

Elkaeed, Ahmed B. M. Mehany & Ibrahim H. Eissa (2021) Discovery of new quinolines as

potent colchicine binding site inhibitors: design, synthesis, docking studies, and anti-proliferative

evaluation, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 640-658, DOI:

10.1080/14756366.2021.1883598

To link to this article: https://doi.org/10.1080/14756366.2021.1883598

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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

2021, VOL. 36, NO. 1, 640–658

https://doi.org/10.1080/14756366.2021.1883598

RESEARCH PAPER

Discovery of new quinolines as potent colchicine binding site inhibitors: design,

synthesis, docking studies, and anti-proliferative evaluation

Mohamed Hagras^a, Moshira A. El Deeb^b, Heba S. A. Elzahabi^c, Eslam B. Elkaeed^d,a, Ahmed B. M. Mehany^eand

Ibrahim H. Eissa^f

b

^aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo, Egypt; Department of

c

Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt; Department of Pharmaceutical Medicinal

d

Chemistry & Drug Design, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt; Department of Pharmaceutical Sciences, College of

e

Pharmacy, AlMaarefa University, Ad Diriyah, Riyadh, Saudi Arabia; Department of Zoology, Faculty of Science, Al-Azhar University, Cairo, Egypt;

^fDepartment of Pharmaceutical Medicinal Chemistry & Drug Design, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo, Egypt

ABSTRACT

ARTICLE HISTORY

Received 6 December 2020

Revised 26 December 2020

Accepted 25 January 2021

Discovering of new anticancer agents with potential activity against tubulin polymerisation is still a prom-

ising approach. Colchicine binding site inhibitors are the most relevant anti-tubulin polymerisation agents.

Thus, new quinoline derivatives have been designed and synthesised to possess the same essential phar-

macophoric features of colchicine binding site inhibitors. The synthesised compounds were tested in vitro

against a panel of three human cancer cell lines (HepG-2, HCT-116, and MCF-7) using colchicine as a posi-

tive control. Comparing to colchicine (IC₅₀¼ 7.40, 9.32, and 10.41 mM against HepG-2, HCT-116, and MCF-

7, respectively), compounds 20, 21, 22, 23, 24, 25, 26, and 28 exhibited superior cytotoxic activities with

IC₅₀values ranging from 1.78 to 9.19 mM. In order to sightsee the proposed mechanism of anti-prolifera-

tive activity, the most active members were further evaluated in vitro for their inhibitory activities against

tubulin polymerisation. Compounds 21 and 32 exhibited the highest tubulin polymerisation inhibitory

effect with IC₅₀values of 9.11 and 10.5 nM, respectively. Such members showed activities higher than that

of colchicine (IC₅₀¼ 10.6 nM) and CA-4 (IC₅₀¼ 13.2 nM). The impact of the most promising compound 25

on cell cycle distribution was assessed. The results revealed that compound 25 can arrest the cell cycle at

G2/M phase. Annexin V and PI double staining assay was carried out to explore the apoptotic effect of

the synthesised compounds. Compound 25 induced apoptotic effect on HepG-2 thirteen times more than

the control cells. To examine the binding pattern of the target compounds against the tubulin hetero-

dimers active site, molecular docking studies were carried out.

KEYWORDS

Cancer; colchicine binding

site inhibitors; docking;

quinoline; tubulin

polymerisation

1. Introduction

anti-mitotics–still important counterparts in combination chemo-

therapy for the treatment of several malignancies^9,10

.

Cancer is the uncontrolled growth of abnormal cells¹. There are

about 100 types of cancer which need diagnosis and treatment².

In 2018, according to National Cancer Institute (NIH), approxi-

mately 1,735,350 new cases of cancer have been diagnosed in the

United States and 609,640 people died from the disease. Cancer

of breast, liver, colon, and rectum are common disease with high

rate of incidence³. The current approaches of cancer treatment

include surgery, radiation, chemotherapy; and hormonal

The tubulin heterodimer comprises at least three binding

sites: the paclitaxel, vinblastine, and colchicine binding sites

(Figure 1)¹¹. There are many drugs used in clinical oncology acting

on the paclitaxel and vinblastine binding sites^12,13. These drugs

are highly potent but there are some limitation in clinical use for

many reasons: development of multi-drug resistance, high lipophi-

licity, low water solubility, intravenous administration due to poor

water solubility¹⁴. Aforementioned drawbacks can largely over-

come by use of tubulin inhibitors that bind to the colchicine bind-

ing site. Such inhibitors have therapeutic advantages: high water

solubility so that it can be administered orally and there is no

multi-drug resistance¹⁵.

treatment⁴.

Microtubules are important cytoskeletal structures that have a

crucial role in cell division⁵, which make them attractive targets

for design of new anticancer agents^6,7. Microtubules are com-

posed of two subunits of a- and b-tubulin heterodimers. These

subunits are arranged in slender-shaped filamentous tubes with

many micrometres long⁸. Some natural products that target

Colchicine binding site inhibitors (CBSI) produce their biological

activities by suppressing the vital process of tubulin assembly, and

consequently suppressing microtubule formation¹⁴. Colchicine I,

the most famous inhibitors in this category, binds to tubulin very

the tubulin and the microtubule system- also referred to as tightly, but there is no any compound in this group has a

CONTACT Ibrahim H. Eissa

Azhar University, Cairo 11884, Egypt; Mohamed Hagras

Al-Azhar University, Cairo 11884, Egypt

Ibrahimeissa@azhar.edu.eg

Department of Pharmaceutical Medicinal Chemistry & Drug Design , Faculty of Pharmacy (Boys), Al-

m.hagrs@azhar.edu.eg Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Boys),

Supplemental data for this article can be accessed here.

ß 2021 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.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

641

Figure 1. Reported colchicine binding site inhibitors.

significant use in treatment of cancer¹⁶. combretastatin A-1 (CA-1) with a lower side effects²⁴. The antitumor activity of indibulin is

II and combretastatin A- 4 (CA-4) III are combretastatin analogs believed to be related to its effects on microtubules²⁵.

having microtubule inhibitory activity with limited value due to

E7010 VII is an orally bioavailable tubulin-binding agent that

their low water solubility¹⁷. To improve their water solubility, such was developed for cancer treatment. It is a sulphonamide deriva-

analogs were designed as prodrugs of monosodium phosphate tives having an antimitotic effect. It binds to the colchicine site on

salt. These prodrugs can be metabolised in vivo into CA-1 and b-tubulin subunit, leading to cell cycle arrest at the G2/M phase,

CA-4 as active components^18,19. CA-4P showed no bone marrow resulting in cellular apoptosis²⁶. It exhibited a broad spectrum of

toxicity, stomatitis, and hair loss in phase II clinical trial²⁰. One of antitumor activity in vitro and in vivo²⁶. Its clinical trial indicated

CA-4 analogs is ombrabulin IV which has better water solubility, that it has dose-limiting toxicities included abdominal pain, consti-

oral activity, enhanced anti-cancer activity and decreased adverse pation, and fatigue¹⁴.

effects¹⁴.

Podophyllotoxin VIII, a naturally occurring anticancer agent,

Plinabulin V is an active molecule against non-small cell lung binds to the colchicine site of tubulin leading to inhibition of the

cancer²¹. It restricts tubulin polymerisation with immune-enhanc- tubulin assembly into microtubules²⁷. It has severe toxicity which

ing effects^22,23. Indibulin VI has an effective antitumor activity limits its clinical application as

a cancer therapy. However,

642

M. HAGRAS ET AL.

podophyllotoxin is still considered an attractive lead compound Ala316b, Met259b, Ala317b, Val238b, Thr353b, and Ala354b resi-

dues (Figure 2)^39,43

.

for the generation of new antitumor agents²⁸.

Moreover, several derivatives (e.g. compounds IX²⁹, X³⁰, XI³¹,

and XII³²) has been synthesised and evaluated as tubulin inhibi-

tors targeting the colchicine binding site. These compounds were

modified and tested to find highly potent agents for treatment of

cancer.

It was reported that the colchicine binding site inhibitors pos-

sess seven essential pharmacophoric groups; one hydrogen bond

donor (D1), three hydrogen bond acceptors (A1, A2, and A3), two

hydrophobic centres (H1 and H2), and one planar group (R1)^14,44

(Figure 3(A)). The different pharmacophoric features are arranged

in two planes. The Features A1, D1, H1, and R1 lie in plane A, and

features A2, A3, and H2 lie in plane B. The angle between the two

planes is about 45^39,44(Figure 3(B)).

Colchicine as a prototype of CBSIs is formed of three rings (A,

B (linker), and C). some reports revealed that A- and C-ring

encompasses the least pharmacophoric features required for bind-

ing to tubulin⁴⁵. In addition, it was found that changes in the ring

B (linker) could affect the antiproliferative activity the CBSIs³¹.

Figure 4 shows the pharmacophoric points on colchicine and

podophyllotoxin as representative examples of CBSIs.

In continuation to our previous efforts of design and synthesis

of new anticancer agents^33–42, a new series of quinoline deriva-

tives were designed and synthesised. The synthesised derivatives

have the same pharmacophoric features of CBSI to examine their

effect as anticancer agents with potential tubulin inhibitory activ-

ities targeting the colchicine binding site.

1.1. Rational of molecular design

The colchicine binding site is a funnel shaped with a volume of

about 600 Å. the cavity of the active site is surrounded by

Ala180a, Asn101a, Thr179a, Val181a, Thr314b, Asn349b, Lys352b,

Asn350b, Tyr202b, Thr239b, Asp251b, Cys241b, Asn258b, Leu242b,

The main target of this work was the synthesis of new quin-

oline derivatives having the same essential pharmacophoric fea-

tures of the reported CBSIs (Figure 5). The core of our molecular

design rational comprised bioisosteric modification strategies of

Leu248b, Leu252b, Ile378b, Leu255b, Val318b, Ala250b, Lys254b, CBSIs at three different positions (Figure 6).

The first position was the C-ring, where the 2-chloro-6-methox-

yquinoline moiety used as a bioisosteric ring equivalent. The

second position was the B-ring (linker region), where different het-

ero-rings were used including five-membered rings as 4,5-dihydro-

1H-pyrazole (compounds 8 2 10 and 14 2 25) and 4,5-dihydroi-

soxazol (compounds 11 2 13), or six-membered rings as pyrimi-

dine-2(1H)-thione (compounds 26 2 28), 2-aminopyrimidine

(compounds 29 2 31), and pyrimidin-2(1H)-one (compounds

32 2 34). The third position was the A-ring, where different substi-

tuted phenyl rings were used including 4-chlorophenyl, 4-amino-

phenyl, and 3,4,5-trimethoxyphenyl.

Figure 2. The colchicine binding site having a funnel shape.³⁹

Figure 4. Pharmacophoric points of colchicine and podophyllotoxin as CBSIs.

(A)

(B)

Figure 3. (A) Seven pharmacophoric features of colchicine binding site inhibitors: three hydrogen bond acceptors (A1, A2 & A3), one hydrogen bond donor (D1), two

hydrophobic centres (H1 & H2), and one planar group (R1) (based on Ref.^14,44). (B) The pharmacophoric model with two planes: plane A (green) points A1, D1, H1 and

R1, Plane B (turquoise) consists of points A2, A3, and H2, and (based on Ref.^39,44).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

643

Figure 5. Representative examples of the newly synthesised compounds having the same essential pharmacophoric features of the CBSIs.

strong absorption bands around 1655 cm^ꢀ1due to carbonyl

ketone stretching, which appeared at low absorption value

because of extended conjugation with the double bond. The

absolute geometry of the a,b-unsaturated carbonyl linker was

assigned to be in trans form based on the coupling constant

1

alkene protons. In more details, the H NMR spectrum of chalcone

5 showed two doublets, each equivalent to one proton, at d

8.1 ppm due to CH alkene (b-proton) and at d 7.9 ppm due to CH

alkene (a-proton). Both protons have the same coupling constant

value of 15.2 Hz, which confirms the E-configuration. Additionally,

the ¹H NMR spectrum of compound 5 displayed two more dou-

blets, each of two protons, at d 8.2 and 7.1 ppm, with coupling

constant value of 8.4 Hz which is attributed to the p-disubstituted

phenyl moiety (Scheme 1, Supplementary data).

The reactivity of chalcone can attributed to the fact that their

molecules have two electron poor centres at C-1 and C-3 in add-

ition to one electron rich centre at C-2, so the double bond in

chalcone can be looked on as an electron rich bond that may

enter in 1,3-dipolar cycloaddition reactions. Chalcone are heavily

utilised synthons in 1,3-cycloadditon reactions to build a wide var-

iety of heterocyclic systems such as pyrrolidines, oxazolines and

pyrimidines. In this work, nine different 1,3-dipolar cyclo-addition

reactions have been achieved. all reactions proceeded smoothly

and final products were obtained in relatively good yields as

detailed in the experimental part. The subsequent paragraphs

point out those reactions in depth (Scheme 2).

Figure 6. Summary for all chemical modifications.

The wide variety of modifications enabled us to study the SAR

of these compounds as effective anti-cancer agents with potential

tubulin inhibitory activity which is considered as a crucial object-

ive of our work. All modification pathways and molecular design

rationale were illustrated and summarised in Figure 7.

2. Results and discussion

2.1. Chemistry

The synthetic pathways employed to prepare the target com-

First, to build a dihydroprazole ring system, a mixture of the

pounds are outlined in Schemes 1–3. Firstly, p-aminoacetophe- chalcone (5, 6, and 7) with excess hydrazine hydrate in absolute

none 1 was refluxed in acetic anhydride/sodium acetate mixture ethanol was heated up at reflux temperature to finally give the

to produce N-(4-methoxyphenyl) acetamide 2. Boiling compound desired corresponding compounds 8, 9, and 10. The structure of

2 with POCl₃in the presence of catalytic amount of DMF, pro- compound 8 was established on the basis of its elemental and

duced aldehyde derivative 3. Chalcone derivatives 5, 6, and 7 spectral data. IR spectrum of compound 8 exhibited an important

were prepared through the reaction of compound 3 with the cor- band at 3295 cm^ꢀ1due to NH stretching of the newly formed

responding acetophenones 4 in the presence of alcoholic NaOH. dihydropyrazole ring. The later NH was also appeared on the ¹H

The reaction occurred by ratio (1:1) to form chalcone which crys- NMR spectrum as a broad deuterium-exchangeable singlet within

tallised from ethanol, the structure of compounds 5, 6, and 7 the aromatic region at d 7.1 ppm. The hydrocarbon backbone of

were established on the basis of its elemental and spectral data. the newly built dihydropyrazole ring revealed three more signals;

The IR spectra of compounds 5, 6, and 7 were characterised by i.e. a triplet, of one proton at d 5.1 ppm due to pyrazole-H5 that

644

M. HAGRAS ET AL.

Figure 7. Rational of molecular design of the new proposed CBSIs.

appeared downfield, as expected, because of the neighbouring and NaOH in ethanol was heated up till reflux to give the corre-

nitrogen atom; on the other hand, there were two doublet of sponding target compounds 11,12, and 13. The IR spectra of

doublets, each of one proton at d 3.7 and 2.8 ppm with coupling compound 11 characterised by a band at 1590 cm^ꢀ1due to C¼N,

constants of 5.6 and 16.8 Hz assignable for pyrazole-H4 axial pro- ¹H NMR spectra of compound 11 showed, a doublet of doublet of

ton, 9.6 and 16.4 Hz of pyrazol-H4 equatorial proton. In addition, one proton at d 6 ppm due to isoxazole-H-5 which appeared

mass spectrum of compound 8 showed a strong base peak with downfield as expected because C-5 of isoxazole attached to oxy-

the proper chlorine-isotope distribution pattern (see experimental gen atom, a two doublet of doublet, each equivalent for one pro-

part). Formation of dihydropyrazole derivatives were postulated to ton at d 4.0 and 3.5 ppm which attributed to isoxazole-H4 axial

pass through two steps. The first step involves Michael-type add- proton and isoxazole-H4 equatorial proton with coupling constant

ition on carbonyl b-carbon, followed by protonation to afford (11.2, 16.8 Hz) of axial proton and (6.8, 17.2 Hz) of equatorial pro-

b-hydrazinylpropanone intermediate. The terminal primary amine ton, respectively, the most characteristic feature of ¹H NMR spec-

attacks the carbonyl group to finally give the dihydropyrazole final trum of compound 11 is the disappearance of the olefinic protons

product after loss

(Supplementary data).

a

molecule of water as detailed below (Supplementary data).

Next, to prepare pyrimidine-2(1H)-thione derivatives 26,27, and

Next, to synthesise dihydroisoxazole derivatives 11, 12, and 28, a mixture of chalcone, thiourea, and NaOH in ethanol was

13, a mixture of chalcone (5,6,7), hydroxylamine hydrochloride, heated to reflux to give the corresponding target derivatives.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

645

Scheme 1. Synthesis of the target compounds 5–7.

Taking compound 26 as an example, the IR spectrum was charac- mainly base on the absence its molecular ion peak from the MS.

terised by a band at 3308 cm^ꢀ1assignable to one NH stretching,

Other spectral and elemental data confirm this assumption and

confirm the structure of compounds 32, 33, and 34

(Supplementary data).

1

the H NMR spectrum shows, a singlet of one proton at 14.1 ppm

due to NH which is D₂O exchangeable, a singlet for one proton at

d 8.6 ppm due to quinoline-H4, a doublet for one proton at d

8.3 ppm due to quinoline-H-8 with coupling constant 8.8 Hz, a

doublet of two protons at d 8.1 ppm due to phenyl-H2,H6 with

coupling constant 8.4 Hz, a doublet for one proton at 7.8 ppm due

to quinoline-H7 with coupling constant 8.8 Hz, a doublet of two

protons at d 7.5 ppm due to phenyl-H3,H5 with coupling constant

8.4 Hz, a singlet for one proton at 7.4 ppm due pyrimidine-H5, a

singlet of one proton at d 7.3 ppm due to quinoline-H5, a singlet

2.2. Biological evaluation

2.2.1. In vitro anti-proliferative activity

In vitro cytotoxic activities of the synthesised compounds were

assessed using standard MTT method^46–48. a panel of human can-

cer cell lines namely; hepatocellular carcinoma (HepG-2), colorectal

carcinoma (HCT-116), and breast cancer (MCF-7) were used in this

test utilising colchicine as a reference standard. The antiprolifera-

tive activity was expressed in IC₅₀values as reported in Table 1.

From the cytotoxic screening, the tested compounds showed

different degrees of activities against the tested cells. In general,

some compounds were proven to be efficient anticancer candi-

dates with promising activities against the tested cells. Comparing

to colchicine (IC₅₀¼ 7.40, 9.32, and 10.41 mM against HepG-2, HCT-

116, and MCF-7, respectively), compounds 20, 21, 22, 23, 24, 25,

26, and 28 exhibited superior cytotoxic activities with IC₅₀values

ranging from 1.78 to 9.19 mM. Taking compound 25 as demonstra-

tive example, it showed 3.91, 6.51 and 2.47 times of colchicine

cytotoxic activity against HepG-2, HCT-116, and MCF-7, respect-

ively. Also, compound 20 exhibited 2.84, 5.15 and 2.30 times of

colchicine activity against HCT-116, HepG-2, and MCF-7, respect-

ively. In addition, Compounds 14, 19, 32, and 33 exhibited super-

ior activities against HCT-116 and MCF-7 with IC₅₀values ranging

from 6.48 to 10.27 mM.

Additionally, compounds 15, 17, 18, 29, and 34 displayed

moderate anti-proliferative activities against all the tested cell

lines with IC₅₀values ranging from 10.18 to 17.62 mM. On the

other hand, compounds 5, 6, 8, 12, and 30 showed weak anti-pro-

liferative activities against all cell lines with IC₅₀values ranging

from 22.43 to 45.53 mM.

Finally, compound 16 showed strong activity against HCT-116,

HepG-2 cell lines (IC₅₀¼ 9.30 and 8.87 mM, respectively) and

moderated activity against MCF-7 cells (IC₅₀¼ 13.89 mM).

of three protons at

(Scheme 3).

d 3.8 ppm due to OCH₃of quinoline

Similarly, pyrimidin-2-amine was synthesised. Briefly, a mixture

of chalcone, guanidine hydrochloride, and NaOH in ethanol was

heated to reflux to give the corresponding compounds 29, 30,

and 31. The structure of the isolated product was confirmed by

spectral data, taking compound 29 as a representative example,

the IR spectrum characterised by a band at 3320 and 3284 cm^ꢀ1

due to NH₂stretching, the ¹H NMR spectrum revealed a singlet

for one proton at d 8.4 ppm due to quinoline-H4, a doublet of

one proton at d 8.3 ppm due to quinoline-H8 with coupling con-

stant 8.8 Hz, a doublet for two protons at d 7.9 ppm due to phe-

nyl-H2,H6 with coupling constant 8.4 Hz, a singlet of one proton

at d 7.5 ppm due to pyrimidine-H5, a singlet of one proton at

7.1 ppm due to quinoline-H5, a doublet of two protons at 7 ppm

due to phenyl-H3,H5, a doublet for one proton at d 6.7 ppm due

to quinoline-H7 with coupling constant 8.8 Hz, a singlet for two

protons at 5.4 ppm which attributed to NH₂, D₂O exchangable, a

singlet of three protons at 3.7 ppm due to OCH₃of quinoline.

Unlike other reactions in this series, reaction of chalcone with

urea was preceded first under basic conditions, but the yield was

poor, therefore we shifted towards the acidic medium. In brief,

compounds 32, 33, and 34 were prepared, in a good yield, by

allowing the corresponding chalcone to react with urea in the

presence of conc. hydrochloric acid at a reflux temperature. Under

strong acidic media, an alternative product was also anticipated,

where the chlorine atom was expected to be hydrolysed to afford

the amide-containing structure. Later structure was excluded Compound 13 showed strong activity against HepG-2 cell line

646

M. HAGRAS ET AL.

Scheme 2. Synthesis of the target compounds 8–25.

(IC₅₀¼ 9.96 mM) and moderated activity against HCT-116 and MCF-

7 cells (IC₅₀¼ 12.87 and 17.04 mM, respectively). Compounds 9 and

10 showed moderate activity against HCT-116 (IC₅₀¼ 16.64 and

15.38 mM, respectively), HepG-2 (IC₅₀¼ 17.40 and 17.41 mM,

respectively) cell lines, and showed weak activity against MCF-7

cells (IC₅₀¼ 20.49 and 25.12 mM, respectively).

Compounds 21 and 32 exhibited the highest tubulin polymer-

isation inhibitory effect with IC₅₀values of 9.11 and 10.5 nM,

respectively. Such members showed activities higher than that of

colchicine (IC₅₀¼ 10.6 nM) and CA-4 (IC₅₀¼ 13.2 nM). Moreover,

compounds 20, 22, 25, 26, 28, and 33 showed promising activ-

ities with IC₅₀values of 36.8, 19.1, 41.3, 34.5, 37.4 and 47.2 nM,

respectively. Compounds 14 showed moderate activity with IC₅₀

value of 50.1 nM. Finally, Compound 24 exhibited weak tubulin

polymerisation inhibitory activity with IC₅₀value of 146.2 nM.

2.2.2. Tubulin polymerisation assay

The most active antiproliferative members were further evaluated

for their inhibitory effect against tubulin polymerisation. The

inhibition assay on microtubule polymerisation was evaluated tur-

bidimetrically using a fluorescent plate reader⁴⁹. Colchicine and

CA-4 were used as positive controls (Table 2).

2.2.3. Cell cycle analysis

The impact of the most promising compound 25 on cell cycle dis-

tribution was assessed. Such analysis gave a better insight into

the effect of the synthesised compounds on cancer cell growth

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

647

Scheme 3. Synthesis of the target compounds 26–34.

Table 1. In vitro anti-proliferative activities of the tested compounds and

The results revelled that compound 25 induced an increase in

the percentage of HepG-2 cells from 24.83% to 28.95% at S phase.

It exhibited marked increase in the percentage of HepG-2 cells

from 13.8% to 30.17% at G2/M phase. Also, it increased the per-

centage of HepG-2 cells from 2.57% to 21.46% at Pre-G1 phase.

On the other hand, it decreased the percentage of HepG-2 cells

from 61.37% to 40.88% at G0-G1phase. These findings indicated

that compound 25 can arrest the cell cycle at G2/M phase, and

can cause apoptosis at pre-G1 phase (Table 3 and Figure 8).

colchicine

IC₅₀( mM )^a

Comp.

HepG-2

HCT-116

MCF-7

5

6

7

8

29.17 1.9

32.91 2.1

23.63 1.3

30.92 1.2

16.64 0.8

15.38 0.9

20.52 1.0

35.44 1.8

12.87 0.6

8.92 0.4

10.25 0.4

9.30 0.3

12.73 0.5

10.18 0.4

10.94 0.5

2.60 0.1

7.32 0.2

4.76 0.2

2.64 0.1

3.14 0.1

1.89 0.2

3.62 0.1

16.26 0.4

5.47 0.2

16.49 0.8

28.53 1.3

9.45 0.5

8.16 0.2

9.37 0.4

15.27 0.4

7.40 0.2

28.92 1.7

28.63 1.7

25.08 1.2

22.43 1.1

17.40 1.0

17.41 1.1

19.72 1.3

40.47 2.4

9.96 0.3

7.62 0.4

11.56 0.9

8.87 0.3

11.42 0.4

10.41 0.3

7.38 0.4

1.81 0.1

8.91 0.3

3.04 0.3

1.78 0.1

4.18 0.2

1.43 0.1

2.24 0.2

22.36 0.5

5.85 0.3

13.04 0.6

32.74 0.9

10.49 0.4

6.48 0.2

8.50 0.2

11.92 0.4

9.32 0.2

32.19 2.0

37.31 2.1

16.87 1.2

33.53 2.4

20.49 1.1

25.12 1.2

22.77 1.4

45.53 2.8

17.04 1.2

10.04 0.4

13.60 0.7

13.89 0.3

15.94 0.6

13.70 0.8

9.04 0.5

4.51 0.2

9.19 0.3

6.45 0.2

4.48 0.1

4.97 0.1

4.21 0.1 control (1.39%).

5.55 0.2

24.34 0.9

7.34 0.2

17.62 0.8

33.75 1.5

15.19 0.7

9.89 0.4

10.27 0.5

15.44 0.8

10.41 0.3

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

Colchicine

2.2.4. Annexin V-FITC apoptosis assay

Annexin V and PI double staining assay⁵¹was carried out to

explore the proposed apoptotic effect of the synthesised com-

pounds. Compound 25 as a representative example was tested

against HepG-2 cells. In this test, HepG-2 cells were incubated

with compound 25 at a concentration of 1.89 mM for 24 h.

As shown in Table 4 and Figure 9, compound 25 induced

apoptotic effect equal 19.05% (7.66% and 11.39 at early and late

apoptosis, respectively), which was thirteen times more than the

2.3. Docking studies

The synthesised compounds were docked against tubulin hetero-

dimers using MOE2014 software to determine the binding free

energy and binding mode (Table 5). Molecular docking studies

gives perception about the binding mode and the degree of affin-

ity between the docked compounds and the prospective target.

good biological effect is shown by lower binding free energy and

similar binding mode to that of the reference co-crystallised

ligand.⁵²

^aIC₅₀values are the mean SD of three separate experiments. Bold figures indi-

cate superior potency than colchicine and CA-4.

A docking study of the co-crystallised ligand, DAMA-colchicine

provides a binding energy value of ꢀ12.88 Kcal/mol with four

hydrogen bonds and six hydrophobic interactions. The trimethoxy

phenyl (A-ring) ring occupied the first pocket of the colchicine

inhibition. HepG-2 cells were utilised in this test. The reported

method described by Wand et al.⁵⁰was followed. HepG-2 cells

were treated with compound 25 at a concentration equals its IC₅₀

value (1.89 mM) for 24 h.

648

M. HAGRAS ET AL.

binding site forming one hydrogen bond with Cys241. Other inter- (1–(2,4-dinitrophenyl)-4,5-dihydro-1H-pyrazole)

occupied

the

second cavity of the colchicine binding site forming five hydrogen

bonds with Lys352, Ser178, and Asn258. Also, it formed three

hydrophobic interactions with Lys352, Ala250 and Leu248.

Furthermore, the 2-chloro-6-methoxyquinoline moiety (C-ring)

occupied the third pocket of the colchicine binding site forming

eight hydrophobic interactions with Leu255, Cys241, and Leu248

(Figure 12).

The binding mode of compound 28 exhibited an affinity value

of ꢀ12.74 kcal/mol. The 3,4,5-trimethoxyphenyl moiety (A-ring)

occupied the first cavity of the colchicine binding site forming

four hydrophobic interactions with Ala316, Ala250, Cys241, and

Leu255. The pyrimidine-2(1H)-thione (B-ring) occupied the second

cavity of the colchicine binding site forming one hydrogen one

hydrophobic bonds with Lys352. Furthermore, the 2-chloro-6-

methoxyquinoline moiety (C-ring) occupied the third pocket of

the colchicine binding site forming three hydrophobic interactions

with Lys254 and Ala180. The methoxy group formed one hydro-

gen bond with Gln11 (Figure 13).

actions were observed between the ring-A and different essential

amino acid residues as Cys241, Leu255, and Ala250. Additionally,

the side chain of the B-ring (2-mercaptoacetamide moiety) occu-

pied the second cavity of the colchicine binding site forming two

hydrogen bonds with Ser178 and Leu248. Furthermore, the

methoxytropone moiety (C-ring) occupied the third pocket of the

receptor forming one hydrogen and one hydrophobic bonds with

Lys352 (Figure 10).

Compound 19 as a representative example showed a binding

mode like that of DAMA-colchicine, with affinity value of

ꢀ16.51 kcal/mol. The 3,4,5-trimethoxyphenyl moiety (A-ring) occu-

pied the first cavity of the colchicine binding site forming three

hydrophobic interactions with Ala250, Leu255, and Leu248.

Additionally, the 2-acetyl-4,5-dihydro-1H-pyrazol moiety (B-ring)

occupied the second cavity of the colchicine binding site forming

one hydrogen bond with Asn258 and one hydrophobic interaction

with Leu248. Furthermore, the 2-chloro-6-methoxyquinoline moi-

ety (C-ring) occupied the third pocket of the colchicine binding

site (Figure 11).

The binding mode of compound 25 exhibited an affinity value

of ꢀ17.12 kcal/mol. The 3,4,5-trimethoxyphenyl moiety (A-ring)

occupied the first cavity of the colchicine binding site forming

one hydrophobic interaction with Leu248. The linker moiety

2.4. Structure-activity relationship (SAR)

SAR as an aim of our work was based on the results of in vitro

cytotoxic activities of the synthesised compounds. Initially, the

effect of the A-ring on the activity was explored. Comparing the

cytotoxic activity of compounds 22, 25, 28, 31, and 34 incorpo-

rating 4-methoxyphenyl as an A-ring with compounds 20, 23, 26,

29, and 32 incorporating 4-chlorophenyl as an A-ring, and com-

pound 21, 24, 27, 30, and 32 incorporating 4-aminophenyl as an

A-ring indicated that the cytotoxic activities decreased in the

order of 4-methoxyphenyl > 4-chlorophenyl > 4-aminophenyl.

Then, the impact of the B-ring (linker region) was investigated.

The increased IC₅₀values of compounds 5, 6, and 7 incorporated

open structure at the linker region than those of their correspond-

ing members incorporating cyclic structure, indicated that

Table 2. In vitro tubulin polymerisation inhibition.

IC₅₀(nM)^a

Tubulin polymerisation

inhibition

IC₅₀(nM)^a

Tubulin polymerisation

inhibition

Comp.

14

20

21

22

24

25

50.1

36.8

9.11

19.1

146.2

41.3

26

28

32

34.5

37.4

10.5

47.2

10.6

13.2

33

Colchicine

CA-4

^aIC₅₀values are the mean SD of three separate experiments.

Table 4. Apoptosis and necrosis percent induced by compound 25 in HepG-2

cells.

Table 3. Effect of compound 25 on cell cycle progression in HepG-2 cells.

Cell cycle distribution (%)

Apoptosis

Sample

%G0-G1

%S

%G2-M

%Pre-G1

Sample

Total

Early

Late

Necrosis

25 / HepG-2

Cont. HepG-2

40.88

61.37

28.95

24.83

30.17

13.8

21.46

2.57

25 / HepG-2

Cont. HepG-2

21.46

2.57

7.66

0.67

11.39

0.72

2.41

1.18

Figure 8. HepG-2 cells distribution upon treatment with compound 25.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

649

Figure 9. Induced apoptosis on HepG-2 cells by compound 25.

cancer cell lines including; colorectal carcinoma (HCT-116), hepato-

cellular carcinoma (HepG-2), and breast cancer (MCF-7).

Compounds 20, 21, 22, 23, 24, 25, 26, and 28 exhibited superior

cytotoxic activities with IC₅₀values ranging from 1.78 to 9.19 mM.

Additionally, the most promising members were tested for their

tubulin polymerisation inhibitory effect. Compounds 21 and 32

exhibited the highest tubulin polymerisation inhibitory effect with

IC₅₀values of 9.11 and 10.5 nM, respectively. Structure-activity

relationship of the synthesised compounds revealed that 4-

methoxyphenyl > 4-chlorophenyl > 4-aminophenyl as an A-ring.

In the same time. The it was found that substituted five-mem-

bered ring > non-substituted six-membered ring > non-substituted

five-membered ring as a B-ring. Moreover, compound 25 was

approved to arrested the cell cycle at the G2/M phase and

induced apoptosis in HepG-2 cells. Docking experiments assisted

these findings by anticipating potential binding interactions

between the target compounds and the active sites of tubulin

heterodimers. The most effective candidates in the quest for

strong and selective antineoplastic agents will serve as valuable

lead compounds and merit further investigations.

Table 5. The docking scores of the synthesised compounds, colchicine, and co-

crystallised ligand (DAMA-colchicine) against tubulin

Binding free energy

(kcal/mol)

Binding free energy

(kcal/mol)

Comp.

5

6

7

8

– 9.95

–10.04

–13.48

–10.23

–10.71

–14.02

–10.20

–10.47

–13.73

–13.28

–11.63

–14.62

–12.07

–11.89

–16.51

–12.35

21

22

23

24

25

26

27

28

29

30

31

32

33

34

–11.67

–14.72

–13.42

–14.35

–17.12

–11.23

–11.25

–12.74

–11.18

–11.11

–14.65

–11.05

–11.02

–13.14

–12.88

–13.45

9

10

11

12

13

14

15

16

17

18

19

20

DAMA–colchicine

Colchicine

cyclisation of the linker region (B-ring) is advantageous. For the B-

ring, the cytotoxic activities decreased in the order of substituted

five-membered ring (compounds 14–25)

> non-substituted

six-membered ring (compounds 26–34) > non-substituted five-

membered ring (compounds 8–13). With regard to the substituted

five-membered ring, it was found that the cytotoxicity decrease in

the order of 2,4-dinitrophenyl (compounds 23–25) > phenyl

(compounds 20–22) > ethane-1-thione (compounds 14–16) >

ethan-1-one (compounds 17–19).

For the non-substituted five-membered ring, it was found 4,5-

dihydro-1H-pyrazole derivatives (compounds 8–9) are almost

equipotent with 4,5-dihydroisoxazole ones (compounds 11–13).

For the non-substituted six-membered ring, it was found pyrimi-

dine-2(1H)-thione derivatives (compounds 26–28) are more active

than 2-amino pyrimidine derivatives (compounds 29–31), which

were more active than pyrimidin-2(1H)-one derivatives (com-

pounds 32–34) (Figure 14).

4. Experimental

4.1. Chemistry

¹H NMR spectra were run at 400 MHz and ¹³C spectra were deter-

mined at 100 MHz in deuterated chloroform (CDCl₃), or dimethyl

sulfoxide (DMSO-d₆) on a Varian Mercury VX-400 NMR spectrom-

eter. Chemical shifts are given in parts per million (ppm) on the

delta (d) scale. Chemical shifts were calibrated relative to those of

the solvents. Flash chromatography was performed on 230–400

mesh silica. The progress of reactions was monitored with Merck

silica gel IB2-F plates (0.25 mm thickness). The infra-red spectra

were recorded in potassium bromide discs on pye Unicam SP

3300 and Shimadzu FT IR 8101 PC infra-red spectrophotometer.

Mass spectra were recorded at 70 eV. High resolution mass spectra

for all ionisation techniques were obtained from a Finnigan MAT

3. Conclusion

In summary, twenty-nine of quinoline derivatives were designed XL95. Melting points were determined using capillary tubes with a

and synthesised. The synthesised compounds were evaluated for Stuart SMP30 apparatus and are uncorrected. All yields reported

their anti-proliferative activities against a group of three human refer to isolated yields.

650

M. HAGRAS ET AL.

Figure 11. (A) 3D structure of compound 19 docked into the colchicine binding

site (B) 2D structure of compound 19 docked into the colchicine binding site.

Figure 10. (A) 3D structure of co-crystallised ligand, DAMA-colchicine, docked

into the colchicine binding site (B) 2D structure of co-crystallised ligand, DAMA-

colchicine, docked into the colchicine binding site.

1H, quinoline-H8), 7.50 (d, J ¼ 9.2 Hz, 1H, quinoline-H7), 7.39 (s, 1H,

quinoline-H5), 7.14 (d, J ¼ 8.4, 2H, phenyl-H3, H5 protons), 3.87 (s,

3H, quinoline-OCH₃); MS (m/z) 361 (0.8%, M þ 4), 359 (4.3%,

M þ 2), 357 (6.7%, M^þ), 246 (61%), 138 (100%); Anal. Calc. for:

(C₁₉H₁₃Cl₂NO₂): C, 63.71; H, 3.66; N, 3.91%; Found: C, 63.74; H,

3.71; N, 3.97%.

4.1.1. General procedure for synthesis of compounds (5, 6, and 7)

To a stirred and ice-cooled aqueous solution of sodium hydroxide

(10 mmole, 50% w/w) and absolute ethanol (12.5 mL), substituted

acetophenone (10 mmole) namely, 4-chloroacetophenoe, 4-aminoace-

tophenone, and 3,4,5-trimethoxyacetophenone was added followed

by 2-chloro-6-methoxyquinoline-3-carbadehyde 4 (10 mmole). The

reaction mixture was vigorously stirred for 3–6h while

temperature was maintained below 25 ^ꢁC till the reaction mixture

became thick. The reaction mixture was left in the refrigerator over-

night. The formed precipitate was filtered off under vaccum and

washed with copious amount of water until the filtrates became neu-

tral to litmus paper, washed with ice-cold ethanol (20 mL), and then

recrystallized from ethanol to give the titled compound 5, 6, and 7,

respectively.

4.1.1.2.

(E)-1–(4-Aminophenyl)-3–(2-chloro-6-methoxyquinolin-3-

yl)prop-2-en-1-one (6). Orange solid (89%) mp ¼ 120–121 ^ꢁC; IR

(KBr) cm^ꢀ1: 3310, 3295 (NH₂), 3054 (CH aromatic), 2962 (CH ali-

phatic), 1653 (C¼O); ¹H NMR (DMSO-d₆) d: 9.08 (s, 1H, quinoline-

H4), 8.07 (d, J ¼ 15.2 Hz, 1H, CH alkene b proton), 7.96 (d, J ¼ 8.8,

2H, phenyl-H2, H6 protons), 7.92 (d, J ¼ 15.2 Hz, 1H, CH alkene a

proton), 7.88 (d, J ¼ 9.2, 1H, quinoline-H8), 7.49 (d, J ¼ 9.2 Hz, 1H,

quinoline-H7), 7.38 (s, 1H quinoline-H5), 6.64 (d, J ¼ 8.8, 2H, phe-

nyl-H3, H5 protons), 6.25 (brs, 2H, NH₂, D₂O-exchangeable), 3.91

(s, 3H, quinoline-OCH₃); MS (m/z) 340 (1.96%, M^þþ2), 338 (5.8%,

M^þ), 246 (66%), 120 (100%); Anal. Calc. for: (C₁₉H₁₅ClN₂O₂): C,

67.36; H, 4.46; N, 8.27%; Found: C, 67.41; H, 4.49; N, 8.31%.

4.1.1.1.

(E)-3–(2-Chloro-6-methoxyquinolin-3-yl)-1–(4-chlorophe-

nyl)prop-2-en-1-one (5). Yellow solid (93%) mp ¼ 135–136 ^ꢁC; IR

(KBr) cm^ꢀ1: 3080 (CH aromatic), 2900 (CH aliphatic), 1655 (C¼O);

¹H NMR (DMSO-d₆) d: 9.10 (s, 1H, quinoline-H4), 8.20 (d, J ¼ 8.4 Hz,

2H, phenyl-H2, H6 protons), 8.13 (d, J ¼ 15.2, 1H, CH alkene b pro-

4.1.1.3. (E)-3–(2-Chloro-6-methoxyquinolin-3-yl)-1–(3,4,5-trimethox-

yphenyl)prop-2-en-1-one (7). Yellow solid (91%) mp ¼ 127–128 ^ꢁC;

IR (KBr) cm^ꢀ1: 3064 (CH aromatic), 2910 (CH aliphatic), 1655

ton), 7.96 (d, J ¼ 15.2 Hz, 1H, CH alkene a proton), 7.92 (d, J ¼ 9.2, (C¼O); ¹H NMR (DMSO-d₆) d: 9.12 (s, 1H, quinoline-H4), 8.16 (d,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

651

Figure 13. (A) 3D structure of compound 28 docked into the colchicine binding

site (B) 2D structure of compound 28 docked into the colchicine binding site.

recrystallized from ethanol to afford the corresponding com-

Figure 12. (A) 3D structure of compound 25 docked into the colchicine binding pounds 8–25.

site (B) 2D structure of compound 25 docked into the colchicine binding site.

4.1.2.1. 2-Chloro-3-[3–(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-5-

yl]-6-methoxyquinoline (8). White solid (76%) mp ¼ 118–119 ^ꢁC; IR

1

(KBr) cm^ꢀ1: 3295 (NH), 3071 (CH aromatic), 2945 (CH aliphatic); H

NMR (DMSO-d₆) d: 8.40 (s, 1H, quinoline-H4), 7.84 (d, J ¼ 8.8 Hz,

2H, phenyl-H2, H6 protons), 7.62 (d, J ¼ 9.6, 1H, quinoline-H8), 7.46

(s, 1H, quinoline-H5), 7.41 (d, J ¼ 9.6 Hz, 1H, quinoline-H7), 7.01

(brs, 1H, NH, D₂O-exchangeable), 6.94 (d, J ¼ 8.8, 2H phenyl-H3, H5

protons), 5.13 (t, J ¼ 10 Hz, 1H, pyrazole-H5), 3.75 (s, 3H, quinoline-

OCH₃), 3.70 (dd, J ¼ 5.6, J ¼ 16.8, 1H, pyrazole- H4 axial proton),

2.89 (dd, J ¼ 9.6, J ¼ 16.4, 1H, pyrazole-H4 equatorial proton); ¹³C

NMR (DMSO-d₆) d: 159.4, 153.4, 147.9, 144.4, 142.3, 134.2, 133.2,

130.5, 129.3, 129.1, 128.1, 126.4, 123.8, 106.5, 55.9, 50.6, 46.1; MS

(m/z) 375 (0.8%, M^þþ4), 373 (4.3%, M þ 2), 371 (6.7%, M^þ), 179

(61%), 192 (100%); Anal. Calc. for: (C₁₉H₁₅Cl₂N₃O): C, 61.31; H, 4.06;

N, 11.29%; Found: C, 61.35; H, 4.11; N, 11.35%.

J ¼ 15.3 Hz, 1H, CH alkene b proton), 8.02 (d, J ¼ 15.3, 1H, CH

alkene a proton), 7.91 (d, J ¼ 9.3 Hz, 1H, quinoline-H8), 7.52 (d,

J ¼ 9.3 Hz, 1H, quinoline-H7), 7.41 (s, 1H, quinoline-H5), 7.14 (s, 2H,

phenyl-H2, H6 protons), 3.94 (s, 6H, 2OCH₃), 3.89 (s, 6H, 2OCH₃);

MS (m/z) 415 (2.7%, M^þþ2), 413 (7.8%, M^þ), 246 (76%), 195

(100%); Anal. Calc. for: (C₂₂H₂₀ClNO₅): C, 63.85; H, 4.87; N, 3.38%;

Found: C, 63.92; H, 4.92; N, 3.40%.

4.1.2. General procedure for synthesis of compounds (8–25)

A mixture of chalcone 5–7 (10 mmole) and the appropriate amine

derivatives namely, hydrazine hydrate, hydroxylamine hydrochlor-

ide, thiosemicarbazide, semicarbazide, phenylhydrazine, 2,4-dini-

trophenylhydrazine (10 mmole) were stirred in ethanol (20 mL),

and then sodium hydroxide (0.8 g, 20 mmole), in case of hydroxyl-

amine hydrochloride, was added. The reaction mixture was heated

to reflux for 7–12 h, and then the solvent was evaporated under

reduced pressure and poured into ice water. The obtained precipi-

4.1.2.2. 4-[5–(2-Chloro-6-methoxyquinolin-3-yl)-4,5-dihydro-1H-pyr-

azol-3-yl]aniline (9). Yellow solid (81%) mp ¼ 122–123 ^ꢁC; 3305,

2984 (NH₂), 3045 (CH aromatic), 2935 (CH aliphatic); ¹H NMR

(DMSO-d₆) d: 10.02 (brs, 1H, NH, D₂O-exchangeable), 8.39 (s, 1H,

quinoline-H4), 7.84 (d, J ¼ 9.2 Hz, 1H, quinoline-H8), 7.48 (s, 1H,

tate was filtered off, washed with copious amount of water and quinoline-H5), 7.45 (d, J ¼ 9.2, 1H, quinoline-H7), 7.32 (d, J ¼ 8.8 Hz,

652

M. HAGRAS ET AL.

Figure 14. Structure-activity relationship of the synthesised compounds as anti-proliferative agents.

2H, phenyl-H2, H6 protons), 6.55 (d, J ¼ 8.8 Hz, 2H, phenyl-H3, H5 375 (12.3%, M þ 2), 373 (18.9%, M^þ), 180 (100%); Anal. Calc. for:

protons), 5.36 (brs, 2H, NH₂, D₂O-exchangeable), 5.04 (t, J ¼ 10 Hz, (C₁₉H₁₄Cl₂N₂O₂): C, 61.14; H, 3.78; N, 7.51%; Found: C, 61.16; H,

3.81; N, 7.54%.

1H, pyrazole-H5), 3.85 (s, 3H, quinoline-OCH₃), 3.57 (dd,

J ¼ 10.8 Hz, J ¼ 16.4 Hz, 1H, pyrazole- H4 axial proton), 2.77 (dd,

J ¼ 10 Hz, J ¼ 16.4 Hz, 1H, pyrazole-H4 equatorial proton); ¹³C NMR

(DMSO-d₆) d: 159.4, 153.4, 150.3, 148.9, 142.3, 139.2, 135.7, 131.9,

129.4, 127.7, 127.1, 121.8, 116.6, 106.9, 55.2, 43.7, 36.7; MS (m/z)

354 (5.9%, M þ 2), 352 (18%, M^þ), 260 (46%), 192 (100%); Anal.

Calc. for: (C₁₉H₁₇ClN₄O): C, 64.68; H, 4.86; N, 15.88%; Found: C,

64.73; H, 4.91; N, 15.92%.

4.1.2.5. 4-[5–(2-Chloro-6-methoxyquinolin-3-yl)-4,5-dihydroisoxa-

zol-3-yl]aniline (12). Orange solid (61%) mp ¼ 127–128 ^ꢁC; 3300,

3104 (NH₂), 3041 (CH aromatic), 2959 (CH aliphatic); ¹H NMR

(DMSO-d₆) d: 8.26 (s, 1H, quinoline-H4), 7.84 (d, J ¼ 9.2 Hz, 1H,

quinoline-H8), 7.53 (s, 1H quinoline-H5), 7.42 (d, J ¼ 9.2, 1H, quin-

oline-H7), 7.36 (d, J ¼ 8.8 Hz, 2H, phenyl-H2, H6 protons), 6.55 (d,

J ¼ 8.8 Hz, 2H, phenyl-H3, H5 protons), 5.93 (t, J ¼ 6 Hz, 1H, isoxa-

4.1.2.3.

2-Chloro-6-methoxy-3-[3–(3,4,5-trimethoxyphenyl)-4,5- zole-H5), 5.62 (brs, 2H, NH₂, D₂O-exchangeable), 3.96 (dd,

J ¼ 10.8 Hz, J ¼ 17.2 Hz, 1H, isoxazole-H4 axial proton), 3.85 (s, 3H,

dihydro-1H-pyrazol-5-yl]quinoline (10). Yellow solid (77%)

mp ¼ 114–115 ^ꢁC; 3295 (NH), 3047 (CH aromatic), 2944 (CH ali-

phatic); ¹H NMR (DMSO-d₆) d: 8.41 (s, 1H, quinoline-H4), 7.86 (d,

J ¼ 9.2 Hz, 1H, quinoline-H8), 7.55 (s, 2H, phenyl-H2, H6 protons),

7.54 (brs, 1H, NH, D₂O-exchangeable), 7.48 (s, 1H, quinoline-H5),

7.43 (d, J ¼ 9.2 Hz, 1H, quinoline-H7), 5.14 (t, J ¼ 9.2 Hz, 1H, pyra-

zole-H5), 3.87 (s, 6H, 2OCH₃), 3.76 (s, 6H, 2OCH₃), 3.66 (dd,

J ¼ 11.2 Hz, J ¼ 16.4 Hz, 1H, pyrazole- H4 axial proton), 2.87 (dd,

J ¼ 9.2 Hz, J ¼ 16.4 Hz, 1H, pyrazole-H4 equatorial proton); ¹³C

NMR (DMSO-d₆) d: 157.9, 153.8, 153.4, 149.6, 144.7, 142.4, 136.8,

133.0, 128.1, 127.7, 126.4, 123.6, 115.3, 106.5, 56.9, 55.5, 55.1, 37.8,

29.9; MS (m/z) 429 (15%, M þ 2), 427 (46%, M^þ), 192 (100%); Anal.

Calc. for: (C₂₂H₂₂ClN₃O₄): C, 61.76; H, 5.18; N, 9.82%; Found: C,

61.82; H, 5.22; N, 9.84%.

quinoline OCH₃), 3.38 (dd, J ¼ 6.8 Hz, J ¼ 17.2 Hz, 1H, isoxazole-H4

equatorial proton); ¹³C NMR (DMSO-d₆) d: 162.4, 159.0, 150.6,

148.6, 142.7, 139.5, 136.1, 129.4, 128.4, 127.7, 127.1, 121.8, 116.6,

106.5, 59.0, 55.5, 38.5; MS (m/z) 355 (7.3%, M^þþ2), 353 (23.1%,

M^þ), 161 (37%), 192 (100%); Anal. Calc. for: (C₁₉H₁₆ClN₃O₂): C,

64.50; H, 4.56; N, 11.88%; Found: C, 64.54; H, 4.59; N, 11.91%.

4.1.2.5. 5–(2-Chloro-6-methoxyquinolin-3-yl)-3–(3,4,5-trimethoxy-

phenyl)-4,5-dihydroisoxazole

(13).

Red

solid

(59%)

1

mp ¼ 133–134 ^ꢁC; 3062 (CH aromatic), 2978 (CH aliphatic); H NMR

(DMSO-d₆) d: 8.32 (s, 1H, quinoline-H4), 7.87 (d, J ¼ 9 Hz, 1H, quin-

oline-H8), 7.67 (s, 2H, phenyl-H2, H6 protons), 7.53 (s, 1H, quin-

oline-H5), 7.44 (d, J ¼ 9 Hz, 1H, quinoline-H8), 6.02 (t, J ¼ 6.9 Hz,

1H, isoxazole-H5), 4.05 (dd, J ¼ 5.2 Hz, J ¼ 11.1 Hz, 1H, isoxazole-H4

axial proton), 3.87 (s, 6H, 2OCH₃), 3.79 (s, 6H, 2OCH₃), 3.52 (dd,

J ¼ 4.8 Hz, J ¼ 17.4 Hz, 1H, isoxazole-H4 equatorial proton); ¹³C

NMR (DMSO-d₆) d: 162.1, 157.9, 153.4, 149.3, 144.4, 142.3, 136.5,

133.0, 128.1, 127.7, 126.4, 123.2, 115.6, 106.5, 61.8, 56.9, 55.2, 54.8,

31.2; MS (m/z) 430 (12%, M^þþ2), 428 (35%, M^þ), 192 (100%);

Anal. Calc. for: (C₂₂H₂₁ClN₂O₅): C, 61.61; H, 4.94; N, 6.53%; Found:

C, 61.66; H, 5.01; N, 6.54%.

4.1.2.4.

5–(2-Chloro-6-methoxyquinolin-3-yl)-3–(4-chlorophenyl)-

4,5-dihydroisoxazole (11). Yellow solid (64%) mp ¼ 145–146 ^ꢁC;

3031 (CH aromatic), 2957 (CH aliphatic), 1590 (C ¼ N); ¹H NMR

(DMSO-d₆) d: 8.31 (s, 1H, quinoline-H4), 7.88 (d, J ¼ 8.8 Hz, 2H, phe-

nyl-H2, H6 protons), 7.65 (d, J ¼ 9.6, 1H, quinoline-H8), 7.51 (s, 1H,

quinoline-H5), 7.43 (d, J ¼ 9.6 Hz, 1H, quinoline-H7), 6.99 (d, J ¼ 8.8,

2H, phenyl-H3, H5 protons), 6.02 (dd, J ¼ 6.8 Hz, J ¼ 11.2 Hz, 1H,

isoxazole-H5), 4.08 (dd, J ¼ 11.2, J ¼ 16.8, 1H, isoxazole-H4 axial

proton), 3.78 (s, 3H, quinoline OCH₃), 3.51 (dd, J ¼ 6.8, J ¼ 17.2, 1H,

isoxazole-H4 equatorial proton); ¹³C NMR (DMSO-d₆) d: 159.7,

4.1.2.6.

5–(2-Chloro-6-methoxyquinolin-3-yl)-3–(4-chlorophenyl)-

4,5-dihydro-1H-pyrazole-1-carbothioamide (14). Yellow solid (65%)

153.4, 149.3, 145.1, 142.3, 134.3, 133.3, 130.5, 129.1, 128.1, 127.4, mp ¼ 136–137 ^ꢁC; 3310, 3249 (NH₂), 3031 (CH aromatic), 2974 (CH

1

126.0, 123.6, 106.2, 59.0, 55.5, 45.4; MS (m/z) 377 (1.8%, M þ 4), aliphatic); H NMR (DMSO-d₆) d: 8.56 (s, 1H, quinoline-H4), 8.40 (d,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

653

J ¼ 8.4 Hz, 2H, phenyl-H2, H6 protons), 8.10 (d, J ¼ 9.4, 1H, quin- M^þ), 192 (100%); Anal. Calc. for: (C₂₀H₁₆Cl₂N₄O₂): C, 57.85; H, 3.88;

N, 13.49%; Found: C, 57.89; H, 3.94; N, 13.55%.

oline-H8), 8.05 (s, 1H, quinoline-H5), 8.0 (brs, 2H, NH₂, D₂O-

exchangeable), 7.95 (d, J ¼ 9.2 Hz, 1H, quinoline-H7), 6.96 (d,

J ¼ 8.4, 2H, phenyl-H2, H6 protons), 4.57 (t, J ¼ 7.6 Hz, 1H, pyra-

zole-H5), 3.75 (s, 3H, quinoline OCH₃), 3.58 (dd, J ¼ 12, J ¼ 16.8, 1H,

pyrazole- H4 axial proton), 2.93 (dd, J ¼ 12, J ¼ 18.2, 1H, pyrazole-

H4 equatorial proton); ¹³C NMR (DMSO-d₆) d: 179.8, 159.4, 153.8,

147.9, 144.7, 142.7, 134.3, 133.0, 130.5, 129.2, 128.1, 127.4, 126.4,

123.6, 106.5, 62.4, 55.9, 37.1; MS (m/z) 435 (1.2%, M þ 4), 433 (9%,

M^þþ2), 431 (13.2%, M^þ), 192 (100%); Anal. Calc. for:

(C₂₀H₁₆Cl₂N₄OS): C, 55.69; H, 3.74; N, 12.99%; Found: C, 55.76; H,

3.82; N, 13.03%.

4.1.2.10. 3–(4-Aminophenyl)-5–(2-chloro-6-methoxyquinolin-3-yl)-

4,5-dihydro-1H-pyrazole-1-carboxamide (18). Yellow solid (73%)

mp ¼ 130–131 ^ꢁC; IR (KBr) cm^ꢀ1: 3298, 3193 (NH₂), 3056 (CH aro-

1

matic), 2983 (CH aliphatic), 1680 (C¼O); H NMR (DMSO-d₆) d: 8.23

(s, 1H, quinoline-H4), 7.84 (d, J ¼ 9.2 Hz, 1H, quinoline-H8), 7.46 (d,

J ¼ 8.8, 2H, phenyl-H2, H6 protons), 7.41 (s, 1H, quinoline-H5), 7.38

(d, J ¼ 9.2 Hz, 1H, quinoline-H7), 6.54 (d, J ¼ 8.8 Hz, 2H, phenyl-H3,

H5 protons), 6.48 (brs, 2H, NH₂, D₂O-exchangeable), 5.55 (brs, 2H,

NH₂, D₂O-exchangeable), 5.35 (t, J ¼ 10.4 Hz, 1H, pyrazole-H5), 3.88

(dd, J ¼ 11.2 Hz, J ¼ 16.8 Hz, 1H, pyrazole- H4 axial proton), 3.84 (s,

3H, quinoline OCH₃), 3.14 (dd, J ¼ 10 Hz, J ¼ 12 Hz, 1H, pyrazole-H4

equatorial proton); ¹³C NMR (DMSO-d₆) d: 165.9, 159.4, 153.4,

151.0, 147.9, 142.7, 139.2, 137.1, 129.4, 128.4, 127.7, 126.7, 121.8,

116.3, 106.5, 64.2, 55.5, 37.1; MS (m/z) 397 (5.3%, M^þþ2), 395

(16.7%, M^þ), 203 (27%), 192 (100%); Anal. Calc. for: (C₂₀H₁₈ClN₅O₂):

C, 60.69; H, 4.58; N, 17.69%; Found: C, 60.76; H, 4.63; N, 17.74%.

4.1.2.7.

3–(4-Aminophenyl)-5–(2-chloro-6-methoxyquinolin-3-yl)-

4,5-dihydro-1H-pyrazole-1-carbothioamide (15). Orange solid

(66%) mp ¼ 123–124 ^ꢁC; 3300, 3251 (NH₂), 3037 (CH aromatic),

2898 (CH aliphatic); ¹H NMR (DMSO-d₆) d: 8.22 (s, 1H, quinoline-

H4), 7.83 (d, J ¼ 9.2 Hz, 1H, quinoline-H8), 7.73 (brs, 2H, NH₂, D₂O-

exchangeable), 7.54 (d, J ¼ 8.8, 2H, phenyl-H2, H6 protons), 7.44 (s,

1H, quinoline-H5), 7.38 (d, J ¼ 9.2 Hz, 1H, quinoline-H7), 6.55 (d,

J ¼ 8.8 Hz, 2H, phenyl-H3, H5 protons), 5.70 (brs, 2H, NH₂, D₂O-

exchangeable), 5.14 (dd, J ¼ 3.2 Hz, J ¼ 11.2 Hz, 1H, pyrazole-H5),

3.94 (dd, J ¼ 11.2 Hz, J ¼ 18.4 Hz, 1H, pyrazole- H4 axial proton),

3.83 (s, 3H, quinoline OCH₃), 3.19 (dd, J ¼ 3.2 Hz, J ¼ 17.6 Hz, 1H,

pyrazole-H4 equatorial proton); ¹³C NMR (DMSO-d₆) d: 165.9,

159.3, 153.5, 150.6, 148.3, 142.3, 139.2, 137.1, 129.4, 128.1, 127.7,

126.7, 121.8, 116.4, 106.5, 64.9, 55.2, 36.8; MS (m/z) 413 (7.3%, M^þ

þ2), 411 (22%, M^þ), 219 (33%), 192 (100%); Anal. Calc. for:

(C₂₀H₁₈ClN₅OS): C, 58.32; H, 4.40; N, 17.0%; Found: C, 58.35; H,

4.44; N, 17.03%.

4.1.2.11. 5–(2-Chloro-6-methoxyquinolin-3-yl)-3–(3,4,5-trimethoxy-

phenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (19). Yellow solid

(63%) mp ¼ 117–118 ^ꢁC; IR (KBr) cm^ꢀ1: 3317, 3290 (NH₂), 3031 (CH

aromatic), 2934 (CH aliphatic), 1678 (C¼O); ¹H NMR (DMSO-d₆) d:

8.37 (s, 1H, quinoline-H4), 7.83 (d, J ¼ 9.3 Hz, 1H, quinoline-H8),

7.77 (s, 2H, phenyl-H2, H6 protons), 7.52 (d, J ¼ 9.3 Hz, 1H, quin-

oline-H7), 7.37 (s, 1H quinoline-H5), 6.52 (brs, 2H, NH₂, D₂O-

exchangeable), 5.14 (t, J ¼ 6.6 Hz, 1H, pyrazole-H5), 3.91 (s, 6H,

2OCH₃), 3.83 (s, 6H, 2OCH₃), 3.72 (dd, J ¼ 15.4 Hz, J ¼ 7.2 Hz, 1H,

pyrazole- H4 axial proton), 3.28 (dd, J ¼ 13.5 Hz, J ¼ 7.5 Hz, 1H, pyr-

azole-H4 equatorial proton); ¹³C NMR (DMSO-d₆) d: 166.6, 157.9,

153.8, 152.7, 149.3, 144.4, 142.3, 136.8, 133.3, 127.7, 127.1, 126.0,

123.2, 115.6, 106.5, 63.8, 56.9, 54.5, 54.2, 36.8; MS (m/z) 472 (25%,

M^þþ2), 470 (73%, M^þ), 192 (100%); Anal. Calc. for:

(C₂₃H₂₃ClN₄O₅): C, 58.66; H, 4.92; N, 11.90%; Found: C, 58.69; H,

4.98; N, 11.94%.

4.1.2.8. 5–(2-Chloro-6-methoxyquinolin-3-yl)-3–(3,4,5-trimethoxy-

phenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (16). Yellow

solid (69%) mp ¼ 121–122 ^ꢁC; 3301, 3145 (NH₂), 3064 (CH aro-

matic), 2987 (CH aliphatic); ¹H NMR (DMSO-d₆) d: 9.44 (brs, 2H,

NH₂, D₂O-exchangeable), 8.39 (s, 1H, quinoline-H4), 7.57 (d,

J ¼ 9.2 Hz, 1H, quinoline-H8), 7.57 (s, 2H, phenyl-H2, H6 protons),

7.44 (s, 1H, quinoline-H5), 7.44 (s, 1H, quinoline-H5), 6.94 (d,

J ¼ 9.2 Hz, 1H, quinoline-H7), 5.12 (t, J ¼ 10 Hz, 1H, 1H, pyrazole-

H5), 3.86 (s, 6H, 2OCH₃), 3.75 (s, 6H, 2OCH₃), 3.66 (dd, J ¼ 10.8 Hz,

J ¼ 16.4 Hz, 1H, pyrazole- H4 axial proton), 2.84 (dd, J ¼ 9.6 Hz,

J ¼ 16.8 Hz, 1H, pyrazole-H4 equatorial proton); ¹³C NMR (DMSO-

d₆) d: 169.0, 157.9, 155.5, 153.1, 149.3, 144.1, 142.7, 136.4, 133.0,

127.7, 127.1, 125.6, 123.9, 120.5, 106.2, 62.4, 57.9, 55.2, 54.8, 36.0;

MS (m/z) 488 (22%, M^þþ2), 486 (65%, M^þ), 167 (100%); Anal.

Calc. for: (C₂₃H₂₃ClN₄O₄S): C, 56.73; H, 4.76; N, 11.51%; Found: C,

56.80; H, 4.78; N, 11.53%.

4.1.2.12. 2-Chloro-3-[3–(4-chlorophenyl)-1-phenyl-4,5-dihydro-1H-

pyrazol-5-yl]-6-methoxyquinoline (20). Yellow solid (69%)

mp ¼ 145–146 ^ꢁC; IR (KBr) cm^ꢀ1: 3024 (CH aromatic), 2951 (CH ali-

phatic); ¹H NMR (DMSO-d₆) d: 8.57 (s, 1H, quinoline-H4), 8.41 (d,

J ¼ 8.4 Hz, 2H, chlorophenyl-H2, H6 protons), 8.10–7.94 (m, 4H,

quinoline-H5, H8 and phenyl-H2, H6), 7.80 (d, J ¼ 8 Hz, 2H, chloro-

phenyl-H3, H5 protons), 7.58 (t, J ¼ 5.6, 1H, phenyl-H4), 7.27 (d,

J ¼ 7.6 Hz, 1H, quinoline-H7), 6.99 (d, J ¼ 5.6 Hz, 2H, phenyl-H3,

H5), 5.78 (t, J ¼ 6.8, 1H, pyrazole-H5), 3.83 (dd, J ¼ 4.4, J ¼ 10, 1H,

pyrazole- H4 axial proton), 3.76 (s, 3H, quinoline OCH₃), 2.68 (dd,

J ¼ 7.2, J ¼ 9.2, 1H, pyrazole-H4 equatorial proton); ¹³C NMR

(DMSO-d₆) d: 159.4, 158.3, 153.6, 147.5, 134.2, 133.2, 132.3, 130.5,

129.5, 129.4, 129.3, 129.2, 128.2, 126.1, 123.8, 106.6, 57.6, 55.9,

36.9; MS (m/z) 452 (1.1%, M þ 4), 450 (6.7%, M þ 2), 448 (9.6%,

M^þ), 255 (100%); Anal. Calc. for: (C₂₅H₁₉Cl₂N₃O): C, 66.97; H, 4.27;

N, 9.37%; Found: C, 67.07; H, 4.34; N, 9.42%.

4.1.2.9.

5–(2-Chloro-6-methoxyquinolin-3-yl)-3–(4-chlorophenyl)-

4,5-dihydro-1H-pyrazole-1-carboxamide (17). White solid (58%)

mp ¼ 135–136 ^ꢁC; IR (KBr) cm^ꢀ1: 3294, 3195 (NH₂), 3024 (CH aro-

1

matic), 2972 (CH aliphatic), 1681 (C¼O); H NMR (DMSO-d₆) d: 8.57

(s, 1H, quinoline-H4), 8.51 (d, J ¼ 5.2 Hz, 2H, phenyl-H2, H6 pro-

tons), 8.93–8.04 (m, 3H quinoline-H5, H7and H8) , 7.93 (brs, 2H,

NH₂, D₂O-exchangeable), 7.19 (d, J ¼ 5.2 Hz, 2H, phenyl-H3, H5

protons), 4.61 (t, J ¼ 10 Hz, 1H, pyrazole-H5), 3.74 (s, 3H, quinoline

OCH₃), 3.57 (dd, J ¼ 11.6, J ¼ 19.6, 1H, pyrazole- H4 axial proton),

2.87 (dd, J ¼ 8.4, J ¼ 17.6, 1H, pyrazole-H4 equatorial proton); ¹³C

NMR (DMSO-d₆) d: 166.3, 160.1, 153.5, 146.5, 144.4, 141.2, 134.3,

133.0, 130.5, 129.1, 128.1, 127.4, 126.0, 123.9, 106.2, 59.0, 55.9,

4.1.2.13. 4-[5–(2-Chloro-6-methoxyquinolin-3-yl)-1-phenyl-4,5-dihy-

dro-1H-pyrazol-3-yl]aniline

(21).

Orange

solid

(55%)

mp ¼ 136–137 ^ꢁC; IR (KBr) cm^ꢀ1: 3315, 3297 (NH₂), 3066 (CH

aromatic), 2968 (CH aliphatic); ¹H NMR (DMSO-d₆) d: 8.41 (s, 1H,

quinoline-H4), 7.85 (d, J ¼ 9.2 Hz, 1H, quinoline-H8), 7.54 (s, 1H,

quinoline-H5), 7.44–7.37 (m, 3H, quinoline-H7 and aminophenyl-

H2, H6 protons) , 7.19–7.12 (m, 3H, phenyl-H3, H5 and H4), 6.90

36.0; MS (m/z) 419 (2.2%, M þ 4), 417 (14%, M^þþ2), 415 (22.2%, (d, J ¼ 8, 2H, aminophenyl-H3, H5 protons), 6.57 (d, J ¼ 8.4 Hz, 2H,

654

M. HAGRAS ET AL.

phenyl-H3, H5), 5.47 (brs, 2H, NH₂, D₂O-exchangeable), 5.16 (t, 4.1.2.17.

J ¼ 8 Hz, 1H, pyrazole-H5), 3.77 (s, 3H, quinoline OCH₃), 3.65 (dd,

2-Chloro-3-[1–(2,4-dinitrophenyl)-3–(3,4,5-trimethoxy-

phenyl)-4,5-dihydro-1H-pyrazol-5-yl]-6-methoxyquinoline

(25).

Orange solid (61%) mp ¼ 135–136 ^ꢁC; IR (KBr) cm^ꢀ1: 3039 (CH aro-

J ¼ 9.2 Hz, J ¼ 16.8 Hz, 1H, pyrazole- H4 axial proton), 3.17 (dd,

J ¼ 10.4 Hz, J ¼ 16.4 Hz, 1H, pyrazole-H4 equatorial proton); ¹³C

NMR (DMSO-d₆) d: 160.1, 153.4, 150.3, 148.3, 142.7, 139.5, 137.8,

134.3, 132.3, 129.1, 128.4, 127.4, 126.7, 122.9, 121.8, 116.6, 105.9,

59.0, 55.2, 37.5; MS (m/z) 430 (9.3%, M^þþ2), 428 (27.9%, M^þ), 236

(47%), 192 (100%); Anal. Calc. for: (C₂₅H₂₁ClN₄O): C, 70.01; H, 4.94;

N, 13.06%; Found: C, 70.08; H, 4.99; N, 13.12%.

1

matic), 2922 (CH aliphatic); H NMR (DMSO-d₆) d: 9.03 (s, 1H, nitro-

phenyl-H3), 8.94 (d, J ¼ 5.7 Hz, 1H, nitrophenyl-H5), 8.43 (s, 1H,

quinoline-H4), 7.97 (d, J ¼ 9.3 Hz, 1H, quinoline-H8), 7.52 (s, 2H,

methoxyphenyl-H2, H6), 7.50 (d, J ¼ 9.3 Hz, 1H, quinoline-H7), 7.42

(s, 1H, quinoline-H5), 7.34(d, J ¼ 5.7 Hz, 1H, 1H, nitrophenyl-H6),

5.30 (t, J ¼ 5.4 Hz, 1H, pyrazole-H5), 3.94 (s, 6H, 2OCH₃), 3.91 (s, 6H,

2OCH₃), 3.71 (dd, J ¼ 7.2 Hz, J ¼ 14.4 Hz, 1H, pyrazole- H4 axial pro-

ton), 2.52 (dd, J ¼ 6.3 Hz, J ¼ 12.3 Hz, 1H, pyrazole-H4 equatorial

proton); ¹³C NMR (DMSO-d₆) d: 157.9, 156.2, 155.5, 153.8, 153.1,

149.3, 144.7, 142.7, 136.8, 134.0, 133.3, 129.4, 129.1, 127.7, 127.4,

126.0, 123.6, 119.4, 115.6, 106.5, 56.9, 55.5, 55.2, 51.3, 37.1; MS (m/

z) 595 (9%, M^þþ2), 593 (28%, M^þ), 401 (100%); Anal. Calc. for:

(C₂₈H₂₄ClN₅O₈): C, 56.62; H, 4.07; N, 11.79%; Found: C, 56.65; H,

4.10; N, 11.84%.

4.1.2.14.

2-Chloro-6-methoxy-3-[1-phenyl-3–(3,4,5-trimethoxy-

phenyl)-4,5-dihydro-1H-pyrazol-5-yl]quinoline (22). Brown solid

(58%) mp ¼ 136–137 ^ꢁC; IR (KBr) cm^ꢀ1: 3024 (CH aromatic), 2941

(CH aliphatic); ¹H NMR (DMSO-d₆) d: 8.46 (s, 1H, quinoline-H4),

8.00 (d, J ¼ 9.2 Hz, 1H, quinoline-H8), 7.88 (d, J ¼ 9.6 Hz, 1H, quin-

oline-H7), 7.73(d, J ¼ 8 Hz, 2H, methoxyphenyl-H2, H6 protons),

7.58–7.46 (m, 6H, aromatic protons), 5.14 (t, J ¼ 9.2 Hz, 1H, pyra-

zole-H5), 3.87 (s, 6H, 2OCH₃), 3.76 (s, 6H, 2OCH₃), 3.66 (dd,

J ¼ 5.2 Hz, J ¼ 11 Hz, 1H, pyrazole- H4 axial proton), 2.86 (dd,

J ¼ 11.2 Hz, J ¼ 15.2 Hz, 1H, 1H, pyrazole-H4 equatorial proton); ¹³C

NMR (DMSO-d₆) d: 159.3, 157.9, 154.2, 153.4, 150.3, 149.3, 144.4,

142.4, 136.8, 133.3, 130.1, 129.1, 128.1, 127.4, 126.4, 123.6, 115.9,

106.2, 61.8, 56.9, 55.5, 37.8; MS (m/z) 505 (22%, M þ 2), 503 (68%,

M^þ), 311 (100%); Anal. Calc. for: (C₂₈H₂₆ClN₃O₄): C, 66.73; H, 5.20;

N, 8.34%; Found: C, 66.79; H, 5.25; N, 8.38%.

4.1.3. General procedure for synthesis of compounds (26–31)

A mixture of chalcone 5–7 (10 mmole) and thiourea or guanidine

hydrochloride (10 mmole) was stirred in absolute ethanol (20 mL),

and then sodium hydroxide (0.8 g, 20 mmole) was added. The

reaction mixture was heated under reflux for 8 h. After completion

of reaction, as detected by TLC, the solvent was concentrated

under reduced pressure, and then poured into ice water (50 mL).

The obtained solid was filtered off, washed and recrystallized from

ethanol to afford the desired compounds 26–31.

4.1.2.15. 2-Chloro-3-[3–(4-chlorophenyl)-1–(2,4-dinitrophenyl)-4,5-

dihydro-1H-pyrazol-5-yl]-6-methoxyquinoline (23). Yellow solid

(63%) mp ¼ 143–144 ^ꢁC; IR (KBr) cm^ꢀ1: 3047 (CH aromatic), 2973

(CH aliphatic); ¹H NMR (DMSO-d₆) d: 9.54 (s, 1H, nitrophenyl-H3),

8.74 (d, J ¼ 4.4 Hz, 1H, nitrophenyl-H5), 8.69 (d, J ¼ 4.4 Hz, 1H, 1H,

nitrophenyl-H6), 8.61 (s, 1H, quinoline-H4), 8.10 (d, J ¼ 6.8 Hz, 2H,

chlorophenyl-H2, H6 protons), 8.20 (s,1H, quinoline-H5), 7.95 (d,

J ¼ 5.2 Hz, 1H, quinoline-H8), 7.76 (d, J ¼ 5.6 Hz, 1H, quinoline-H7),

7.56 (d, J ¼ 5.2 Hz, 2H, chlorophenyl-H3, H5 protons), 4.61 (t,

J ¼ 10, 1H, pyrazole-H5), 3.74 (s, 3H, quinoline OCH₃), 3.59 (dd,

J ¼ 9.2, J ¼ 16.4, 1H, pyrazole- H4 axial proton), 2.78 (dd, J ¼ 12,

J ¼ 17.2, 1H, pyrazole-H4 equatorial proton); ¹³C NMR (DMSO-d₆)

d: 153.4, 150.3, 146.8, 144.7, 141.2, 137.8, 136.4, 134.3, 133.3,

130.8, 130.1, 129.4, 128.4, 128.2, 127.4, 126.4, 125.6, 123.9, 106.5,

56.9, 55.9, 37.1; MS (m/z) 542 (2.7%, M^þþ4), 540 (16%, M þ 2),

538 (24.6%, M^þ), 345 (100%); Anal. Calc. for: (C₂₅H₁₇Cl₂N₅O₅): C,

55.78; H, 3.18; N, 13.01%; Found: C, 55.85; H, 3.24; N, 13.08%.

4.1.3.1.

6–(2-Chloro-6-methoxyquinolin-3-yl)-4–(4-chlorophenyl)-

pyrimidine-2(1H)-thione (26). Yellow solid (54%) mp ¼ 155–156 ^ꢁC;

IR (KBr) cm^ꢀ1: 3308 (NH), 3056 (CH aromatic), 2969 (CH aliphatic);

¹H NMR (DMSO-d₆) d: 14.16 (brs, 1H, NH, D₂O-exchangeable), 8.61

(s, 1H, quinoline-H4), 8.34 (d, J ¼ 8.8 Hz, 1H, quinoline-H8), 8.14 (d,

J ¼ 8.4 Hz, 2H, phenyl-H2, H6 protons), 7.86 (d, J ¼ 8.8 Hz, 1H, quin-

oline-H7), 7.55 (d, J ¼ 8.4 Hz, 2H, phenyl-H3, H5 protons), 7.42 (s,

1H, pyrimidine-H5), 7.33 (s, 1H, quinoline-H5), 3.81 (s, 3H, quin-

oline OCH₃); ¹³C NMR (DMSO-d₆) d: 166.6, 160.1, 159.0, 157.9,

153.8, 147.5, 144.7, 142.3, 134.3, 133.3, 130.5, 129.1, 128.4, 128.1,

126.4, 123.9, 106.2, 55.9; MS (m/z) 418 (2.4%, M þ 4), 416 (15.4%,

M þ 2), 414 (22.5%, M^þ), 220 (33%), 192 (100%); Anal. Calc. for:

(C₂₀H₁₃Cl₂N₃OS): C, 57.98; H, 3.16; N, 10.14%; Found: C, 58.04; H,

3.22; N, 10.19%.

4.1.3.2.

yl)pyrimidine-2(1H)-thione

4–(4-Aminophenyl)-6–(2-chloro-6-methoxyquinolin-3-

(27). Yellow solid (63%)

4.1.2.16.

4-[5–(2-Chloro-6-methoxyquinolin-3-yl)-1–(2,4-dinitro-

phenyl)-4,5-dihydro-1H-pyrazol-3-yl]aniline (24). Yellow solid (57%)

mp ¼ 139–140 ^ꢁC; IR (KBr) cm^ꢀ1: 3300, 3292 (NH₂), 3021 (CH aro-

mp ¼ 146–147 ^ꢁC; IR (KBr) cm^ꢀ1: 3311, 3299 (NH₂), 3039 (CH aro-

matic), 2974 (CH aliphatic); ¹H NMR (DMSO-d₆) d: 12.74 (brs, 1H,

NH, D₂O-exchangeable), 8.09 (s, 1H, quinoline-H4), 7.86 (d,

J ¼ 8.8 Hz, 1H, quinoline-H8), 7.45 (d, J ¼ 8.8 Hz, 1H, quinoline-H7),

7.41 (s, 1H, pyrimidine-H5), 7.15 (d, J ¼ 8.4 Hz, 2H, phenyl-H2, H6

protons), 6.49 (d, J ¼ 8.4 Hz, 2H, phenyl-H2, H6 protons), 6.41 (s,

1H, quinoline-H5), 5.32 (brs, 2H, NH₂, D₂O-exchangeable), 3.88 (s,

3H, quinoline OCH₃); ¹³C NMR (DMSO-d₆) d: 165.9, 159.7, 159.0,

157.6, 153.4, 148.3, 142.7, 139.5, 131.6, 129.5, 128.1, 127.4, 126.7,

123.2, 121.2, 116.3, 106.2, 55.5; MS (m/z) 396 (5.4%, M^þþ2), 394

(16.6%, M^þ), 202 (39%), 302 (100%); Anal. Calc. for:

(C₂₀H₁₅ClN₄OS): C, 60.83; H, 3.83; N, 14.19%; Found: C, 60.87; H,

3.88; N, 14.22%.

1

matic), 2923 (CH aliphatic); H NMR (DMSO-d₆) d: 8.85 (s, 1H, nitro-

phenyl-H3), 8.83 (d, J ¼ 2.8 Hz, 1H, nitrophenyl-H5), 8.41 (s, 1H,

quinoline-H4), 8.29 (d, J ¼ 2.8 Hz, 1H, nitrophenyl-H6), 8.0 (d,

J ¼ 9.2 Hz, 1H, quinoline-H8), 7.77 (d, J ¼ 9.2, 1H, quinoline-H7),

7.47 (s, 1H, quinoline-H5), 7.40 (d, J ¼ 8.8 Hz, 2H, aminophenyl-H2,

H6), 6.84 (d, J ¼ 8.8 Hz, 2H, aminophenyl-H3, H5), 5.36 (brs, 2H,

NH₂, D₂O-exchangeable), 4.98 (t, J ¼ 6 Hz, 1H, pyrazole-H5), 3.88 (s,

3H, quinoline OCH₃), 3.70 (dd, J ¼ 10 Hz, J ¼ 14.4 Hz, 1H, pyrazole-

H4 axial proton), 2.82 (dd, J ¼ 9.6 Hz, J ¼ 17.2 Hz, 1H, pyrazole-H4

equatorial proton); ¹³C NMR (DMSO-d₆) d: 162.4, 157.6, 156.9,

153.5, 150.6, 149.3, 145.8, 142.4, 139.2, 137.5, 133.3, 131.9, 129.1,

128.4, 127.7, 127.1, 123.9, 121.5, 116.3, 106.9, 57.2, 55.5, 37.5; MS

(m/z) 520 (9.3%, M þ 2), 518 (27.9%, M^þ), 326 (38%), 192 (100%);

Anal. Calc. for: (C₂₅H₁₉ClN₆O₅): C, 57.87; H, 3.69; N, 16.20%; Found:

C, 57.93; H, 3.74; N, 16.26%.

4.1.3.3. 6–(2-Chloro-6-methoxyquinolin-3-yl)-4–(3,4,5-trimethoxy-

phenyl)pyrimidine-2(1H)-thione

(28).

Yellow

solid

(54%)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

655

mp ¼ 141–142 ^ꢁC; IR (KBr) cm^ꢀ1: 3275 (NH), 3028 (CH aromatic), pressure and poured into ice water (50 mL). The obtained precipi-

1

2932 (CH aliphatic); H NMR (DMSO-d₆) d: 10.83 (brs, 1H, NH, D₂O- tate was filtered off, washed and recrystallized from ethanol to

yield the titled compounds 32–34.

exchangeable), 8.75 (s, 1H, quinoline-H4), 7.83 (d, J ¼ 9.3 Hz, 1H,

quinoline-H8), 7.52 (d, J ¼ 9.3 Hz, 1H, quinoline-H7), 7.37 (s, 1H,

pyrimidine-H5), 7.18 (s, 2H, phenyl-H2, H6 protons), 7.07 (s, 1H,

quinoline-H5), 3.94 (s, 6H, 2OCH₃), 3.89 (s, 6H, 2OCH₃); ¹³C NMR

(DMSO-d₆) d: 166.6, 163.8, 159.1, 153.8, 152.8, 148.9, 144.7, 142.7,

136.8, 134.3, 133.6, 128.4, 127.7, 126.4, 123.6, 115.6, 106.5, 56.5,

54.8, 54.4; MS (m/z) 471 (3%, M^þþ2), 469 (10%, M^þ), 70 (100%);

Anal. Calc. for: (C₂₃H₂₀ClN₃O₄S): C, 58.78; H, 4.29; N, 8.94%; Found:

C, 58.79; H, 4.33; N, 8.99%.

4.1.4.1.

6–(2-Chloro-6-methoxyquinolin-3-yl)-4–(4-chlorophenyl)-

pyrimidin-2(1H)-one (32). Yellow solid (76%) mp ¼ 180–181 ^ꢁC; IR

(KBr) cm^ꢀ1: 3332 (NH), 3078 (CH aromatic), 2932 (CH aliphatic); H

1

NMR (DMSO-d₆) d: 11.96 (brs, 1H, NH, D₂O-exchangeable), 8.51 (s,

1H, quinoline-H4), 8.33 (d, J ¼ 8.2 Hz, 1H, quinoline-H8), 8.06 (d,

J ¼ 8.8 Hz, 2H, phenyl-H2, H6 protons), 7.77 (d, J ¼ 8.2 Hz, 1H, quin-

oline-H7), 7.28 (s,1H, pyrimidine-H5), 7.20 (s, 1H, quinoline-H5),

7.08 (d, J ¼ 8.8 Hz, 2H, phenyl-H3, H5 protons), 3.74 (s, 3H, quin-

oline OCH₃); ¹³C NMR (DMSO-d₆) d: 166.3, 160.1, 159.0, 157.9,

153.8, 147.5, 144.7, 142.7, 134.3, 133.6, 130.5, 129.8, 128.1, 127.1,

126.0, 123.6, 105.5, 55.2; MS (m/z) 402 (1.4%, M^þþ4), 400 (9%,

M þ 2), 398 (13%, M^þ), 204 (33%), 192 (100%); Anal. Calc. for:

(C₂₀H₁₃Cl₂N₃O₂): C, 60.32; H, 3.29; N, 10.55%; Found: C, 60.40; H,

3.35; N, 10.61%.

4.1.3.4.

4–(2-Chloro-6-methoxyquinolin-3-yl)-6–(4-chlorophenyl)-

pyrimidin-2-amine (29). Yellow solid (72%) mp ¼ 195–196 ^ꢁC; IR

(KBr) cm^ꢀ1: 3321, 3284 (NH₂), 3049 (CH aromatic), 2972 (CH ali-

phatic); ¹H NMR (DMSO-d₆) d: 8.49 (s, 1H, quinoline-H4), 8.37 (d,

J ¼ 8.8 Hz, 1H, quinoline-H8), 7.79 (d, J ¼ 8.4 Hz, 2H, phenyl-H2, H6

protons), 7.50 (s,1H, pyrimidine-H5), 7.11 (s, 1H, quinoline-H5), 7.05

(d, J ¼ 8.4 Hz, 2H, phenyl-H3, H5 protons), 6.78 (d, J ¼ 8.8 Hz, 1H,

quinoline-H7), 5.41 (brs, 2H, NH₂, D₂O-exchangeable), 3.72 (s, 3H,

quinoline OCH₃); ¹³C NMR (DMSO-d₆) d: 162.8, 160.1, 159.4, 157.9,

153.8, 146.5, 144.4, 142.7, 134.0, 132.6, 130.5, 129.1, 128.1, 127.4,

126.0, 123.6, 106.5, 55.5; MS (m/z) 376 (1.4%, M þ 4), 374 (7.4%,

M^þþ2), 372 (11%, M^þ), 204 (33%), 192 (100%); Anal. Calc. for:

(C₂₀H₁₄Cl₂N₄O): C, 60.47; H, 3.55; N, 14.10%; Found: C, 60.54; H,

3.61; N, 14.17%.

4.1.4.2.

4–(4-Aminophenyl)-6–(2-chloro-6-methoxyquinolin-3-

yl)pyrimidin-2(1H)-one (33). Yellow solid (76%) mp ¼ 171–172 ^ꢁC;

IR (KBr) cm^ꢀ1: 3322, 3285 (NH₂), 3023 (CH aromatic), 2917 (CH ali-

phatic); ¹H NMR (DMSO-d₆) d: 10.41 (brs, 1H, NH, D₂O-exchange-

able), 8.12 (s, 1H, quinoline-H4), 7.84 (d, J ¼ 8.8 Hz, 2H, phenyl-H2,

H6 protons), 7.59 (d, J ¼ 8.4 Hz, 1H, quinoline-H8), 7.30 (d,

J ¼ 8.4 Hz, 1H, quinoline-H7), 7.20 (s, 1H, pyrimidine-H5), 6.62 (d,

J ¼ 8.8 Hz, 2H, phenyl-H3, H5 protons), 6.15 (s, 1H, quinoline-H5),

5.32 (brs, 2H, NH₂, D₂O-exchangeable), 3.79 (s, 3H, quinoline

OCH₃); ¹³C NMR (DMSO-d₆) d: 169.8, 160.1, 159.0, 157.6, 153.8,

148.9, 142.4, 139.5, 131.9, 129.1, 128.4, 127.7, 127.1, 124.2, 121.8,

116.3, 105.9, 55.2; MS (m/z) 380 (9.1%, M þ 2), 378 (28.6%, M^þ), 92

(25%), 286 (100%); Anal. Calc. for: (C₂₀H₁₅ClN₄O₂): C, 63.41; H, 3.99;

N, 14.79%; Found: C, 63.44; H, 4.02; N, 14.83%.

4.1.3.5.

4–(4-Aminophenyl)-6–(2-chloro-6-methoxyquinolin-3-

yl)pyrimidin-2-amine (30). Yellow solid (58%) mp ¼ 175–176 ^ꢁC; IR

(KBr) cm^ꢀ1: 3327, 3289 (NH₂), 3028 (CH aromatic), 2918 (CH ali-

phatic); ¹H NMR (DMSO-d₆) d: 8.51 (s, 1H, quinoline-H4), 8.13 (s,

1H, pyrimidine-H5), 7.84 (d, J ¼ 8.4 Hz, 1H, quinoline-H7), 7.62 (d,

J ¼ 8.8 Hz, 2H, phenyl-H2, H6 protons), 7.43 (d, J ¼ 8.4 Hz, 1H, quin-

oline-H8), 7.25 (s, 1H, quinoline-H5), 6.82 (d, J ¼ 8.8 Hz, 2H, phenyl-

H3, H5 protons), 5.50 (brs, 2H, NH₂, D₂O-exchangeable), 5.30 (brs,

2H, NH₂, D₂O-exchangeable), 3.71 (s, 3H, quinoline OCH₃); ¹³C

NMR (DMSO-d₆) d: 164.2, 162.4, 159.4, 157.6, 153.8, 148.9, 142.3,

139.9, 132.3, 129.4, 128.4, 127.7, 126.7, 121.5, 119.0, 116.6, 105.9,

55.2; MS (m/z) 379 (11.4%, M^þþ2), 377 (35.6%, M^þ), 185 (66%),

302 (100%); Anal. Calc. for: (C₂₀H₁₆ClN₅O): C, 63.58; H, 4.27; N,

18.54%; Found: C, 63.66; H, 4.33; N, 18.59%.

4.1.4.3. 6–(2-Chloro-6-methoxyquinolin-3-yl)-4–(3,4,5-trimethoxy-

phenyl)pyrimidin-2(1H)-one

(34).

Yellow

solid

(74%)

mp ¼ 176–177 ^ꢁC; IR (KBr) cm^ꢀ1: 3319 (NH), 3055 (CH aromatic),

1

2912 (CH aliphatic); H NMR (DMSO-d₆) d: 11.96 (brs, 1H, NH, D₂O-

exchangeable), 8.52 (s, 1H, quinoline-H4), 8.35 (d, J ¼ 9 Hz, 1H,

quinoline-H8), 7.79 (d, J ¼ 9 Hz, 1H, quinoline-H7), 7.32 (s, 1H, pyr-

imidine-H5), 7.24 (s, 2H, phenyl-H2, H6 protons), 7.13 (s, 1H, quin-

oline-H5), 3.88 (s, 6H, 2OCH₃), 3.82 (s, 6H, 2OCH₃); ¹³C NMR

(DMSO-d₆) d: 162.4, 161.1, 159.4, 153.5, 153.1, 148.9, 144.7, 142.3,

136.8, 134.3, 137.3, 128.4, 128.1, 126.7, 123.9, 115.9, 106.5, 56.9,

55.2, 54.8; MS (m/z) 455 (1.6%, M^þþ2), 453 (6%, M^þ), 167 (100%);

Anal. Calc. for: (C₂₃H₂₀ClN₃O₅): C, 60.86; H, 4.44; N, 9.26%; Found:

C, 60.92; H, 4.50; N, 9.29%.

4.1.3.6. 4–(2-Chloro-6-methoxyquinolin-3-yl)-6–(3,4,5-trimethoxy-

phenyl)pyrimidin-2-amine

(31).

brown

solid

(69%)

mp ¼ 162–163 ^ꢁC; IR (KBr) cm^ꢀ1: 3303, 3291 (NH₂), 3024 (CH aro-

1

matic), 2949 (CH aliphatic); H NMR (DMSO-d₆) d: 8.53 (s, 1H, quin-

oline-H4), 7.95 (d, J ¼ 9 Hz, 1H, quinoline-H8), 7.54 (s, 1H,

pyrimidine-H5), 7.51 (d, J ¼ 9 Hz, 1H, quinoline-H7), 7.42 (s, 1H,

quinoline-H5), 7.25 (s, 2H, phenyl-H2, H6 protons), 6.8 (brs, 2H,

NH₂, D₂O-exchangeable), 3.91 (s, 6H, 2OCH₃), 3.83 (s, 6H, 2OCH₃);

¹³C NMR (DMSO-d₆) d: 168.3, 162.1, 159.4, 153.8, 153.5, 149.6,

144.7, 142.4, 136.7, 134.0, 133.0, 128.1, 127.4, 126.4, 123.6, 115.3,

106.2, 56.9, 55.2, 54.8; MS (m/z) 454 (0.6%, M þ 2), 452 (2%, M^þ),

192 (100%); Anal. Calc. for: (C₂₃H₂₁ClN₄O₄): C, 61.00; H, 4.67; N,

12.37%; Found: C, 61.07; H, 4.70; N, 12.44%.

4.2. Biological evaluation

4.2.1. In vitro cytotoxic activity

Evaluation of cytotoxic activity of the synthesised compounds was

carried out using MTT assay protocol^46–48against a group of can-

cer cell lines namely; colorectal carcinoma (HCT-116),

Hepatocellular carcinoma (HepG-2) and breast cancer (MCF-7) and

colchicine was used as a standard drug. The complete procedure

was depected in Supplementary data.

4.1.4. General procedure for synthesis of compounds (32–34)

A mixture of chalcone 5–7 (10 mmole) and urea (0.6 g, 10 mmole)

was stirred in ethanol (20 mL), and then hydrochloric acid (2 mL) 4.2.2. In vitro tubulin polymerisation assay

was added. The mixture was heated at reflux for 12 h. After com- The effect of the synthesised compounds on tubulin polymerisa-

pletion the reaction, the solvent was concentrated under reduced tion was assessed turbidimetrically using a fluorescent plate

656

M. HAGRAS ET AL.

reader method⁴⁹. The complete procedure was depected in

Supplementary data.

interphase impairment of microtubule function. Bioorg Med

Chem 2014;22:5050–9.

7. Kavallaris M. Microtubules and resistance to tubulin-binding

agents. Nat Rev Cancer 2010;10:194–204.

8. Giannakakou P, Sackett D, Fojo T. Tubulin/microtubules: still

a promising target for new chemotherapeutic agents. J Natl

Cancer Inst 2000;92:182–3.

4.2.3. Cell cycle analysis

The effect of the most promising compound 25 on cell cycle was

evaluated

using

flowcytometer^50,53,54

as

illustrated

in

Supplementary data.

9. Wilson L, Jordan MA. Microtubule dynamics: taking aim at a

moving target. Chem Biol 1995;2:569–73.

10. Jordan MA, Wilson L. Microtubules as a target for anticancer

drugs. Nat Rev Cancer 2004;4:253–65.

11. Botta M, Forli S, Magnani M, Manetti F. Molecular modeling

approaches to study the binding mode on tubulin of micro-

tubule destabilizing and stabilizing agents. Tubulin-Binding

Agents. Berlin: Springer; 2008:279–328.

4.2.4. Annexin V-FITC apoptosis assay

The effect of compound 25 on apoptosis induction was analysed

using Annexin V-FITC/PI apoptosis detection kit using flowcytome-

ter^51,55,56as illustrated in Supplementary data.

12. Altaha R, Fojo T, Reed E, Abraham J. Epothilones: a novel

class of non-taxane microtubule-stabilizing agents. Curr

Pharm Des 2002;8:1707–12.

13. Prota AE, Bargsten K, Diaz JF, et al. A new tubulin-binding

site and pharmacophore for microtubule-destabilizing anti-

cancer drugs. Proc Natl Acad Sci USA 2014;111:13817–21.

14. Lu Y, Chen J, Xiao M, et al. An overview of tubulin inhibitors

that interact with the colchicine binding site. Pharm Res

2012;29:2943–71.

15. Li L, Jiang S, Li X, et al. Recent advances in trimethoxy-

phenyl (TMP) based tubulin inhibitors targeting the colchi-

cine binding site. Eur J Med Chem 2018;151:482–94.

16. Zhou J, Giannakakou P. Targeting microtubules for cancer

chemotherapy. Curr Med Chem Anticancer Agents 2005;5:

65–71.

17. Pettit GR, Lippert JW, Herald DL, et al. Antineoplastic agents

440. Asymmetric synthesis and evaluation of the combretas-

tatin A-1 SAR Probes (1S,2S)- and (1R,2R)-1, 2-dihydroxy-1-

(2⁰,3⁰-dihydroxy-4⁰-methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxy-

phenyl)-ethane. J. Nat. Prod 2000;63:969–74.

18. Pettit GR, Lippert JW. Antineoplastic agents 429. Syntheses

of the combretastatin A-1 and combretastatin B-1 prodrugs.

Anticancer Drug Design 2000;15:203–16.

19. Pettit GR, Temple JC, Narayanan V, et al. Antineoplastic

agents 322. synthesis of combretastatin A-4 prodrugs. Anti-

Cancer Drug Des 1995;10:299–309.

20. Kirwan IG, Loadman PM, Swaine DJ, et al. Comparative pre-

clinical pharmacokinetic and metabolic studies of the com-

bretastatin prodrugs combretastatin A4 phosphate and A1

phosphate. Clin Cancer Res 2004;10:1446–53.

21. Ding Z, Cheng H, Wang S, et al. Development of MBRI-001,

a deuterium-substituted plinabulin derivative as a potent

anti-cancer agent. Bioorg Med Chem Lett 2017;27:1416–9.

22. Lloyd GK, Mueller P, Zippelius A, Huang L. Abstract A07: pli-

nabulin: evidence for an immune-mediated mechanism of

action. AACR 2016;76:A07–A07.

4.3. Docking studies

The Crystal structure of the target receptor (tubulin) [PDB ID:

1SA0, resolution 3.00 Å] was obtained from Protein Data Bank

(http://www.pdb.org). The docking process was carried out using

MOE2014 software. At first, the crystal structure of the target was

prepared by removing water molecules and retaining the two

essential chains and the co-crystallised ligand, N-deacetyl-N-(2-

mercaptoacetyl)-colchicine (DAMA-colchicine). Then, the protein

structure was protonated, and the hydrogen atoms were hided.

Next, the energy was minimised, and the binding pocket of the

protein was defined.

The 2D structures of the synthesised compounds and reference

ligand (DAMA-colchicine) were sketched using ChemBioDraw Ultra

14.0 and saved as MDL-SD format⁵⁷. Then, the saved files were

opened using MOE and 3D structures were protonated. Next,

energy minimisation was applied. Before docking process, valid-

ation of the docking protocol was carried out by running the

simulation only using the co-crystallised ligand (DAMA-colchicine)

which showed low RMSD value. The molecular docking of the syn-

thesised was performed using a default protocol against the tar-

get receptor. In each case, 30 docked structures were generated

using genetic algorithm searches, London dG was used for scoring

and forcefield (MMFF94) for refinement. The London dG scoring

function estimates the free energy of binding of the ligand from a

given pose. The output from of MOE was further analysed and

visualised using Discovery Studio 4.0 software.^58–63

Disclosure statement

No potential conflict of interest was reported by the author(s).

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Article Doi

Discovery of new quinolines as potent colchicine binding site inhibitors: design, synthesis, docking studies, and anti-proliferative evaluation

DOI: 10.1080/14756366.2021.1883598

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Full text of DOI:10.1080/14756366.2021.1883598

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