Quinoline-based promising anticancer and antibacterial agents, and some metabolic enzyme inhibitors

Received: 21 March 2020

Revised: 15 May 2020

Accepted: 24 May 2020

|

DOI: 10.1002/ardp.202000086

F U L L P A P E R

Quinoline‐based promising anticancer and antibacterial

agents, and some metabolic enzyme inhibitors

Salih Ökten¹

Sultan Erkan⁵

| Ali Aydın²

| Cenk A. Andac⁶

| Ümit M. Koçyiğit³

| Osman Çakmak⁴

| İlhami Gülçin⁸

|

| Parham Taslimi⁷

¹Department of Maths and Science Education,

Kırıkkale University, Yahşihan, Kırıkkale,

Turkey

Abstract

A series of substituted quinolines was screened for their antiproliferative, cytotoxic,

antibacterial activities, DNA/protein binding affinity, and anticholinergic properties

by using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide cell pro-

liferation, lactate dehydrogenase cytotoxicity, and microdilution assays, the

Wolfe–Shimmer equality method, the Ellman method, and the esterase assay, re-

spectively. The results of the cytotoxic and anticancer activities of the compounds

displayed that 6‐bromotetrahydroquinoline (2), 6,8‐dibromotetrahydroquinoline (3),

8‐bromo‐6‐cyanoquinoline (10), 5‐bromo‐6,8‐dimethoxyquinoline (12), the novel N‐

nitrated 6,8‐dimethoxyquinoline (13), and 5,7‐dibromo‐8‐hydroxyquinoline (17)

showed a significant antiproliferative potency against the A549, HeLa, HT29,

Hep3B, and MCF7 cancer cell lines (IC₅₀= 2–50 μg/ml) and low cytotoxicity

(∼7–35%) as the controls, 5‐fluorouracil and cisplatin. The compound–DNA linkages

are hyperchromic or hypochromic, causing variations in their spectra. This situation

shows that they can be bound to DNA with the groove‐binding mode, with K_bvalue

in the range of 2.0 × 10³–2.2 × 10⁵M^–1. Studies on human Gram(+) and Gram(−)

pathogenic bacteria showed that the substituted quinolines exhibited selective an-

timicrobial activities with MIC values of 62.50–250 μg/ml. All tested quinoline de-

rivatives were found to be effective inhibitors of acetylcholinesterase (AChE) and

the human carbonic anhydrase I and II isoforms (hCA I and II), with K_ivalues of

46.04–956.82 nM for hCA I, 54.95–976.93 nM for hCA II, and 5.51–155.22 nM for

AChE. As a result, the preliminary data showed that substituted quinolines displayed

effective pharmacological features. Molecular docking studies were performed to

investigate the binding modes and interaction energies for compounds 2–17 with

AChE (PDB ID: 4EY6), hCA I (PDB ID: 1BMZ), and hCA II (PDB ID: 2ABE).

²Department of Basic Medical Science, Faculty

of Medicine, Yozgat Bozok University, Yozgat,

Turkey

³Department of Basic Pharmacy Sciences,

Faculty of Pharmacy, Cumhuriyet University,

Sivas, Turkey

⁴Department of Gastronomy, Faculty of Arts

and Design, İstanbul Rumeli University, Silivri,

İstanbul, Turkey

⁵Department of Chemistry and Chemical

Processing Technologies, Yıldızeli Vocational

School, Sivas Cumhuriyet University, Sivas,

Turkey

⁶Department of Pharmaceutical Chemistry,

Faculty of Pharmacy, Istanbul Istinye

University, Zeytinburnu, Istanbul, Turkey

⁷Department of Biotechnology, Faculty of

Science, Bartın University, Bartın, Turkey

⁸Department of Chemistry, Faculty of

Sciences, Atatürk University, Erzurum, Turkey

Correspondence

Salih Ökten, Department of Maths and Science

Education, Kırıkkale University, Yahşihan,

71451 Kırıkkale, Turkey.

Email: salihokten@kku.edu.tr;

sokten@gmail.com

K E Y W O R D S

acetylcholinesterase enzyme inhibition, antibacterial, anticancer activity, carbonic anhydrase

enzyme inhibition, DNA binding, molecular docking, quinolone

|

1

INTRODUCTION

intensive work on effective cancer treatment, it is an enormous life‐

threatening problem for human health in the world, as 11 million

Cancer leads to an uncontrolled cell growth to invade other tissues

and organs by spreading to the body through the blood. Despite the

people have been affected from cancer in the world and un-

fortunately, seven million people have died due to cancer.^[1]

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Arch Pharm. 2020;e2000086.

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https://doi.org/10.1002/ardp.202000086

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Nowadays, many scientists have made efforts to cure cancer or re-

duce its occurrence. Although many anticancer drugs have been

developed for the treatment of cancer, these drugs have some lim-

itations, such as side effects, tumor specificity, and tumor cell

resistance.^[2]Thus, the developments of new anticancer drug candi-

dates without side effects are necessary as alternatives to current

chemotherapeutic drugs.^[3]

hydrogen bond (HB), is formed between a covalently bonded ha-

logen atom (e.g., C–X, X = Cl, Br, I; XB donor) and a nucleophile (i.e.,

Lewis base; XB acceptor). Due to that the halogen bond has at-

tracted great attention in recent years, with hit‐to‐lead and lead‐

to‐candidate optimization aiming to increase drug‐target binding

affinity optimization. Generally, heavy organohalogens (i.e., orga-

nochlorines, organobromines, and organoiodines) are capable of

forming halogen bonds, whereas organofluorines are not capable

of doing so.^[32]

Quinoline heterocycles, developing rapidly, as well as their syn-

thetic versatility,^[4]are a highly attractive ring system in organic

chemistry due to their usage in developing new compounds posses-

sing a wide range of biological activities.^[5–10]Furthermore, the qui-

noline scaffold is widespread in natural products and drugs, and it is

considered an important pharmacophore and a privileged structure

in medicinal chemistry.^[11]Particularly, anticancer drugs bearing the

quinoline nucleus led to an increase in their anticancer activities

through different mechanisms involving apoptosis, cell cycle arrest,

and inhibition of angiogenesis.^[12]Moreover, the substantial antic-

ancer activities of several quinoline analogs through DNA intercala-

tion, causing interference in the replication, have been reported.^[9]

Despite the fact that a considerable number of biological, synthetic,

and semisynthetic quinolines, having important pharmacological

roles, have been reported in the literature, available methods for

preparation of synthetic quinoline derivatives starting from quinoline

pharmacophore are restricted.^[13–15]

Cyano‐substituted quinolines have important roles in biolo-

gical systems. Notably, quinoline compounds bearing cyano group

at the C‐3 position can act to deactivate the action of growth

factor receptor protein tyrosine kinases.^[33]Agents with cyano

groups also bind with biological systems as small molecule

inhibitors.^[34]The most common strategies for preparation of

quinolines are cyclization reactions of N‐functionalized benzene

or cyclohexane.^[35]The 2‐cyano‐substituted dihydro‐ and tetra-

hydroquinolines have been synthesized using the Reissert

reaction,^[36]whereas 8‐cyano‐substituted quinoline compounds

have been prepared by the treatment of cyano aniline with

ketone‐functionalized alkynes in polar solvents.^[37]Due to this,

the cyclizations using cyano‐substituted N‐functionalized aro-

matics allow only the synthesis of monocyano‐substituted quino-

lines,^[38]and the synthesis of polycyano‐substituted quinolines has

been restricted. However, we have accomplished di‐ or tricyano‐

substituted quinolines by substitution strategy to be used multi-

purpose, especially as a drug.^[39]

Many studies about pharmacological features of aryl or nitro/

amino‐substituted quinolines have been interested due to that they

have been starting materials for bioactive polycyclic systems.^[16,17]

Nitrated derivatives, which exhibit biological potency, that is,

antileishmanial^[18]or potent mutagenic activities,^[19]are important

key compounds for the preparation of amino derivatives for phar-

macological use.^[16,20]The aryl‐substituted quinolines displayed a

high antipesticide activity against the nematode Haemonchus con-

tortus,^[21]agricultural predatory activity, significant antibacterial ac-

tivity,^[22]anti‐inflammatory, analgesic, antipyretic activities, and

efficient inhibition of the COX‐2 enzyme.^[23]Moreover, a series of

substituted 2‐phenylquinolines exhibited a superior ERβ affinity in a

cell‐based transcriptional assay,^[24]potent antiplatelet activities,^[25]

antimitotic activity,^[26]and antiproliferative activity against HCT‐116

(colon cancer), MCF7 (breast cancer), and MDA‐MB‐435 (breast

cancer) with low GI₅₀values.^[27]

Previously, brominated quinolines, the starting compounds, were

synthesized according to reported procedures starting from 1,2,3,4‐

tetrahydroquinoline and 8‐hydroxyquinoline,^[8,30,40]followed by

transformation to their respective cyano, methoxy, phenyl, and

amino derivatives.^[7,14,17,39]The antiproliferative activity of 6,8‐

dibromotetrahydroquinoline and 5,7‐dibromo‐8‐hydroxyquinoline

against several cancer cell lines was determined by the BrdU cell

proliferation enzyme‐linked immunosorbent assay (BCPE).^[6–8]The

results showed that 6,8‐dibromotetrahydroquinoline and 5,7‐dibromo‐

8‐hydroxyquinoline significantly inhibited proliferation of HeLa, C6,

and HT29 cells, as compared with 5‐fluorouracil (5‐FU) at 5 μg/ml and

higher concentrations. Recently, the synthesized 6‐bromo‐5‐

nitroquinoline^[20]has been observed to exhibit a high biological ac-

tivity, with antiproliferative, cytotoxic, and apoptotic effects on several

cancer cell lines.^[15]Due to their inhibitory potency against cancer cell

lines, several bromo, nitro, methoxy, and nitrile derivatives were pre-

pared by starting from 6,8‐dibromotetrahydroquinoline and 5,7‐

dibromo‐8‐hydroxyquinoline according to procedures in the literature

reported by our group.^[15]In this study, we focused on the determi-

nation of anticancer activity against different cancer cell lines, anti-

bacterial activity against some Gram(+) and Gram(−) microorganisms

and acetylcholinesterase (AChE), and carbonic anhydrase enzyme in-

hibition potentials of these derivatives. In addition, the activity of the

substituted quinolines against AChE and human carbonic anhydrase I

and II isoform (hCA I and II) metabolic enzymes was supported by

molecular docking studies.

As halogen has a crucial role in the bioactivity of compounds

and provides an avenue for further structure elaboration, halogen‐

containing quinolines are of particular interest.^[28]Bromoquino-

lines are an important class of precursors for preparing hetero-

cyclic compounds with multifunctionality.^[7,29,30]These building

blocks have especially been used within medicinal chemistry as

starting materials for numerous compounds with a pharmacologi-

cal activity.^[7]Bromoquinolines have attracted more and more

attention in drug discovery due to their potential to make halogen

bonds. Halogen atoms are typically located at the circumference of

organic molecules and are positioned to be involved in inter-

molecular interactions.^[31]A halogen bond (XB), a highly versatile

and specific interaction that behaves similar to the classical

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ÖKTEN ET AL.

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SCHEME 1 A schematic presentation of substituted quinolines 2–14, starting with 1,2,3,4‐tetrahydroquinoline (1)

³J = 4.0 Hz). The signals for the aromatic protons H‐3 and H‐4

(δ_H7.84, ³J = 4.4 Hz and ³J = 8.8 Hz; 9.22, ⁴J = 1.6 Hz and ³J = 8.8 Hz,

respectively) were shifted more downfield when compared with

signals of the starting material. The proton of the benzene ring of 18

gave a singlet downfield at δ_H8.89, assigning bromine to C‐6. It was

seen that the signals of H‐5 and H‐7 disappeared after nitration,

when compared with signal systems of the starting material 16,

which is evidence for the existence of nitration at both C‐5 and C‐7

positions. The ¹H NMR signals of coupled compound 13 had the same

signal system as starting material 11, but signals of protons were

observed further downfield. In the ¹H NMR spectra of 14, signals of

H‐2 and H‐4 were observed as a doublet at δ_H9.12 and 8.24

(⁴J = 2.0 Hz), respectively, downfield as compared with chemical

shifting values of starting molecule 5.^[39]Moreover, the

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2

RESULTS AND DISCUSSION

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2.1

Chemistry

In our previous studies, brominated quinoline derivatives, 6,8‐

dibromotetrahydroquinoline (3) and 6,8‐dibromoquinoline (4), were

converted to corresponding nitrile derivatives (6–10) by treatment of

CuCN according to our reported procedures (Scheme 1).^[6,39]More-

over, in our reported studies, the treatment of 6,8‐dibromoquinoline

(4) with NaOCH₃furnished 6,8‐dimethoxyquinoline (11).^[6]Then,

5‐bromo‐6,8‐dimethoxyquinoline (12) was obtained by bromination

of 6,8‐dimethoxyquinoline (11) according to reported procedure

(Scheme 1).^[14]The direct nitration of 6,8‐dimethoxyquinoline (11)

with HNO₃/H₂SO₄mixture resulted in the formation of a novel 6,8‐

dimethoxy‐1‐nitroquinoline (N‐nitrated) (13) as sole product in a high

yield (97%; Scheme 2). 8‐Methoxyquinoline (16) was prepared by

treatment of 8‐hydroxyquinoline (15) with NaOH and (CH₃)₂SO₄at

0–80°C in 95% yield. Then, the novel dinitrate 8‐methoxyquinoline

was obtained by the direct nitration of 8‐methoxyquinoline (16) with

HNO₃/H₂SO₄mixture as the sole product in a high yield (87%;

Scheme 2). 3,6,8‐Tribromoquinoline (5) was nitrated with a mixture

of HNO₃/H₂SO₄at 0°C. The reaction yielded quantitatively 3,6,8‐

bromo‐5‐nitroquinoline (14) as the sole product (Scheme 1).

The structures of nitrate‐containing compounds 13 and 18 were

determined by ¹H NMR (nuclear magnetic resonance), ¹³C NMR,

Fourier transform infrared (FT‐IR), and elemental analysis. In the ¹H

NMR spectrum of 18, the characteristic doublet of doublet for H‐2 of

the quinoline scaffold was observed at 9.17 ppm (⁴J = 1.6 Hz and

SCHEME 2 A schematic presentation of substituted quinolines

16–19, starting with 8‐hydroxyquinoline (15)

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ÖKTEN ET AL.

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disappearance of the H‐5 doublet signal of the starting material 5^[39]

at δ_H8.14 and the appearance of a singlet signal at δ_H8.33 can be

possible evidence of NO₂group bounded to C‐5 position. In the FT‐IR

spectra of 3,6,8‐bromo‐5‐nitroquinoline (14), characteristic N═O

bromide (MTT) protocol. Growth inhibition (GI₅₀), total growth in-

hibition (TGI), and lethal concentration (LC₅₀) parameters of the

compounds were evaluated according to the NCI screening method,

and half‐maximal inhibitory concentrations (IC₅₀) of these molecules

were calculated using Four‐Parameter Logistic Function. When TGI

and IC₅₀values of the compounds were examined, it was found that

tested compounds exhibited selective antitumor properties against

all tested cell lines (Tables 1 and 2).

stretching signals were observed at 1,550 and 1,320 cm⁻¹

.

|

2.2

Biological activity

When TGI and IC₅₀values of the compounds were examined, it

was observed that 8‐bromo‐6‐cyanoquinoline (10) had the strongest

antitumor effect against all tested cell lines, with IC₅₀values

ranging between 1.5 and 22.5 μg/ml in a series of nitrile quinolines

|

2.2.1

Antiproliferative activities of the compounds

Many anticancer drug candidates have been withdrawn from market

due to their serious side effects, loss of sensitivity to drugs, and

limited use for many cancer types. The anticancer activities of qui-

noline derivatives were reported in many works. In our recent stu-

dies,^[6–8,15]the antiproliferative activities of 6,8‐dibromo‐1,2,3,4‐

tetrahydroquinoline (3), 5,7‐dibromo‐8‐hydroxyquinoline (17), 6‐

bromo‐8‐cyano‐1,2,3,4‐tetrahydroquinoline, and nitrated 3,6,8‐

tribromoquinoline (5) against HeLa, HT29, and C6 cell lines using

sulforhodamine‐B stain (SRB) and BCPE assays were determined. In

the present study, the substituted quinoline derivatives (Table 1)

were prepared according to reported procedures^{[6,30,39–42]}by our

research group and investigated for their anticancer effects and cy-

totoxicities against the A549 HeLa, Hep3B, HT29, MCF7, and FL cell

lines using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium

(Table

1 and 2). Whereas 6‐bromo‐8‐cyanoquinoline (9) and 6,8‐

dicyanotetrahydroquinoline (6) showed a good antiproliferation activity

against only one cell line, A549 (IC₅₀= 5.4 μg/ml) and HT29 (IC₅₀= 78.1

μg/ml), respectively, 6‐bromo‐8‐cyanotetrahydroquinoline (7) and 6,8‐

dicyanoquinoline (8) did not show inhibition against any tested cancer cell

lines (Table 1 and 2). Our previous results obtained by SRB assay^[15]

confirmed that 6‐bromo‐8‐cyanotetrahydroquinoline (7) did not display

an antiproliferative activity against HeLa and HT29 cells.

In a series of bromoquinolines, 6‐bromotetrahydroquinoline (2)

and 6,8‐dibromotetrahydroquinoline (3) significantly inhibited the

proliferation of all tested cancer cell lines, except A549, with IC₅₀

values ranging between 3.7 and 48.5 μg/ml. Whereas 6,8‐

dibromoquinoline (4) displayed an inhibition activity against only

TABLE 1 GI₅₀, TGI, LC₅₀, and IC₅₀of the test compounds against A549, FL, and HeLa cells

A549

GI₅₀

FL

HeLa

GI₅₀

2.0

1.7

5.7

9.7

7.1

8.1

12.8

3.9

1.7

4.2

5.1

3.1

4.4

7.5

5.3

Compounds (µg/ml)

TGI

LC₅₀

IC₅₀

GI₅₀

4.2

4.1

3.3

4.2

4.3

3.7

3.1

2.1

4.5

3.7

3.3

3.4

3.7

2.9

TGI

LC₅₀

IC₅₀

TGI

LC₅₀

IC₅₀

2

6.1

>1,000

511.9

>1,000

6.8

>1,000

255.4

435.2

206.3

>1,000

5.4

27.6

>1,000

312.7

27.1

4.3

19.8

4.3

3

5.6

16.8

16.6

3.6

19.4

3.7

4

>1,000

169.3

372.5

>1,000

18.5

314.4

>1,000

213.2

39.7

>1,000

207.8

288.1

>1,000

198.8

38.2

317.8

>1,000

4.3

>1,000

47.3

295.4

>1,000

912.5

>1,000

4.3

5

6

7

8

9

1.9

>1,000

7.3

>1,000

7.2

10

4.9

29.1

>1,000

240.9

22.5

12

31.2

>1,000

7.3

>1,000

5.8

51.7

>1,000

960.6

50.3

18.9

396.7

>1,000

178.9

361.1

>1,000

18.7

13

308.1

>1,000

2.0

28.9

28.3

52.7

51.2

14

25.3

24.6

11.2

11.1

17

17.9

17.6

18.9

18.7

18

10.1

>1,000

60.5

32.6

>1,000

31.8

>1,000

159.4

>1,000

151.4

50.3

19

>1,000

288.9

262.5

52.8

Cisplatin

5‐FU

69.8

59.1

61.6

Abbreviations: 5‐FU, 5‐fluorouracil; GI, growth inhibition; IC, inhibition concentration; LC, lethal concentration; TGI, total growth inhibition.

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TABLE 2 GI₅₀, TGI, LC₅₀, and IC₅₀of the test compounds against Hep3B, HT29, and MCF7 cells

Hep3B

GI₅₀

5.2

5.4

3.1

3.8

7.5

5.5

4.6

5.5

2.3

3.9

4.1

4.4

3.4

3.5

6.0

HT29

GI₅₀

4.1

2.6

1.2

2.5

1.4

2.5

3.1

1.6

1.1

2.9

4.5

2.9

1.9

3.8

6.2

MCF7

GI₅₀

2.8

3.7

1.9

1.7

2.5

2.6

1.3

2.4

1.7

2.3

2.9

2.5

2.4

2.5

Compounds (µg/ml)

TGI

LC₅₀

IC₅₀

TGI

LC₅₀

IC₅₀

TGI

LC₅₀

IC₅₀

2

49.6

>1,000

36.2

48.5

26.6

>1,000

199.2

21.8

20.7

>1,000

926.9

19.8

3

41.9

41.1

9.3

7.9

19.8

19.3

4

38.8

37.4

>1,000

557.2

>1,000

1.7

>1,000

11.2

>1,000

78.1

402.3

>1,000

927.2

4.8

>1,000

66.5

309.2

>1,000

683.6

4.6

5

958.4

>1,000

5.6

831.1

>1,000

5.6

6

7

>1,000

894.5

465.9

1.5

8

9

10

12

78.2

>1,000

849.7

74.7

26.1

>1,000

50.9

19.2

84.8

>1,000

154.0

75.6

13

22.7

22.3

39.0

30.1

20.4

19.5

14

200.9

66.5

186.8

62.7

7.6

7.1

8.7

8.5

17

6.5

227.6

5.4

17.5

>1,000

16.5

18

18.6

18.3

>1,000

40.4

23.7

22.3

19

>1,000

48.7

>1,000

63.8

Cisplatin

5‐FU

62.9

65.2

74.2

Abbreviations: 5‐FU, 5‐fluorouracil; GI, growth inhibition; IC, inhibition concentration; LC, lethal concentration; TGI, total growth inhibition.

Hep3B (IC₅₀= 37.4 μg/ml; TGI = 38.8 μg/ml), 3,6,8‐tribromoquinoline

(5) did not exhibit any antitumoral activity against any cancer cell

lines. However, nitrated derivative of tribromoquinoline (5) showed a

significant antiproliferative activity against FL, HeLa, HT29, and

MCF7 (IC₅₀values ranging from 7.1 to 24.6 μg/ml; TGI ranging from

7.6 to 25.3 μg/ml). The results of significant antiproliferative activ-

ities of 6,8‐dibromotetrahydroquinoline (3) and 5‐nitro‐3,6,8‐

tribromoquinoline (14) against HeLa and HT29 were confirmed by

our previous results obtained by SRB and BCPE assays.^[6,7,15]

23.7 μg/ml, respectively). 6,8‐Dimethoxyquinoline (4) exhibited a selec-

tive antiproliferative activity against only HT29 cell lines.^[6]In this study,

brominated 12 and N‐nitrated 13 at C‐5 forms of 6,8‐

dimethoxyquinoline (11) inhibited proliferation of all studied cancer

cells, except A549, with IC₅₀values ranging from 19.2 to 75.6 μg/ml

(Table 2). When the IC₅₀and TGI values of all the abovementioned

compounds are considered, effective ones have better or similar anti-

proliferative effects compared with the positive control group, cisplatin

and 5‐FU (Table 3). In addition, the active compounds can be used in

advanced pharmacological studies when low GI₅₀values (~1–5 μg/ml)

and high LC₅₀values (200 to >1,000 μg/ml) are considered (Table 1

and 2). Overall, the GI₅₀, TGI, and LC₅₀parameters of the respective

molecules are at the desired level and meet the NCI criteria.

We have obtained interesting results on antiproliferative effects of

substituted hydroxy‐ and methoxyquinoline derivatives. Our previous

study^[8]showed that 5,7‐dibromo‐8‐hydroxyquinoline (17) exhibited a

strong anticancer activity against HeLa, HT29, and C6 cancer cell lines

by SRB and BCPE assays. MTT assay also confirmed its significant effect

against HeLa and HT29 cells (IC₅₀= 18.7 and 5.4 μg/ml; TGI = 18.9 and

6.5 μg/ml, respectively). Moreover, 5,7‐dibromo‐8‐hydroxyquinoline

displayed a significant antiproliferative activity against A549, Hep3B,

and MCF7 cancer cell lines and FL healthy cells (with IC₅₀values ran-

ging between 5.8 and 62.7 μg/ml; TGI values ranging between 7.3 and

66.5 μg/ml). Interestingly, 5,7‐dibromo‐8‐methoxyquinoline (19) derived

from 5,7‐dibromo‐8‐hydroxyquinoline (17) did not inhibit proliferation

of any cancer cell lines. However, compound 18 bearing nitro group

instead of bromine in the same positions of 5,7‐dibromo‐8‐

methoxyquinoline (19) depicted a selective inhibitory activity against

Hep3B and MCF7 cells (IC₅₀= 18.3 and 22.3 μg/ml; TGI = 18.6 and

|

2.2.2

Cytotoxic activity of compounds and the

morphological changes in cancer cell lines

It is important for a substance to have a minimal toxicity against

normal cells. For this reason, antitumor and cytotoxic properties of

these compounds should be compared to find the forward pharma-

cological capacity of each. The cytotoxicities of the compounds in

cells were tested by the lactate dehydrogenase (LDH) assay, in-

directly demonstrating membrane damage. When the measurement

results of cytoplasmic LDH activity are evaluated for these

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TABLE 3 Minimum inhibitory concentrations (MIC, in μg/ml) of the compounds

Enterococcus

E. faecalis

(ATCC

Staphylococcus

aureus (ATCC

25923)

S. aureus

(ATCC

S. aureus

(ATCC

E. coli

Pseudomonas

faecalis (ATCC

Escherichia coli

(ATCC

35218)

eruginosa (ATCC

Compounds 19433)

29212)

29213)

46300)

(ATCC 25922)

27853)

2

500

250

1,000

500

1,000

125

500

125

250

1,000

500

1,000

>1,000

1,000

>1,000

500

1,000

250

500

125

250

500

125

500

62.5

250

125

500

1,000

500

125

500

62.5

250

125

250

500

1,000

>1,000

1,000

500

1,000

>1,000

1,000

>1,000

500

4

1,000

>1,000

1,000

500

5

6

1,000

500

7

8

>1,000

500

9

10

12

13

14

18

19

SCF

1,000

250

500

1,000

500

1,000

125

1,000

500

1,000

125

500

1,000

250

1,000

15.62

1,000

31.25

1,000

250

62.5

Abbreviations: ATCC, American Type Culture Collection; SCF, sulbactam (30 μg) + cefoperazone (75 μg), as a positive control.

compounds, it has been found that compounds 6, 8, 9, 12, 13, and 19

for A549 cancer cells; 7, 10, 13, 14, and 19 for Hep3B cancer cells; 4,

5, 6, 9, 12, 17, and 19 for MCF7 cells; 4, 5, 8, 9, and 18 for HeLa cells;

4, 5, 6, 7, 8, 9, 18, and 19 for HT29 cells; and 2, 3, 13, 14, 17, and 19

for FL cells cause approximately 7–19% membrane damage at their

IC₅₀concentration (Figure 1). If the compounds are compared with

controls (5‐FU and cisplatin) for this evaluation, the toxicity of mo-

lecules mentioned above is very close to the cytotoxicity values of

5‐FU and cisplatin. Therefore, they may be suitable for advanced

pharmacological assays (Figure 1).

antimicrobial drug (SCF = sulbactam [30 μg] + cefoperazone [75 μg])

used as positive controls. When the MIC values of newly synthesized

molecules displayed on Gram(+) bacteria were examined, it was

found that antibacterial effects of compounds 5, 12, 18, and 19

To investigate the morphology of cell‐treated compounds, we used

compound 3. When the effects of 3 used at 20 μg/ml concentration on

A549, Hep3B, HT29, MCF7, and FL cell morphology were visualized,

some changes were observed in the cells using phase‐contrast micro-

scopy. As shown in Figure 2, each cell line exposed to compound 3

exhibited a low cell confluence, floating cells, cellular and cytoplasmic

shrinkage, cytoplasmic extensions of bubble, and clumping of cells to-

gether. The morphological change most likely indicates the apoptotic

process. Moreover, the effect of substituted quinoline derivatives at

20‐μg/ml concentration on cells was so small that it did not change the

normal appearance of the cells, maintaining a normal morphology.

|

2.2.3

Antibacterial activities of the compounds

The effects of the compounds on some pathogenic bacteria causing

disease in the human body have been evaluated using the minimum

inhibition concentration (MIC) method. We considered our test mo-

lecules to be antibacterial at 250 μg/ml and below the MIC values.

The MIC values of the compounds were compared with the values of

FIGURE 1 % Cytotoxicity of these compounds and positive

controls against A549, Hep3B, MCF7, HeLa, HT29, and FL cells at

IC₅₀concentrations

7 of 16

ÖKTEN ET AL.

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hypochromic effect, and the increase in the absorption intensity of

molecules 4, 5, 6, 7, 8, 10, 13, 14, 18, and 19 caused a hyperchromic

appearance (Figure 3). The binding constants (K_b) of the compounds

show the affinity of the complex to DNA with the aid of the following

Wolfe–Shimmer equality equation: [DNA]/(εa − εf) = [DNA]/(εb − εf) + 1/

K_b(εb − εf), where the [DNA] symbol is the DNA concentration in the

base pairs and the εa, εf, and εb symbols are the molar absorption

coefficients of the A_observed/[Compound], free compound, and

compound–DNA solutions, respectively. K_bis the binding constant that

is related to the affinity between the compound and DNA, and it can be

calculated algebraically from the slope of the line drawn between

[DNA]/(εa − εf) and [DNA]. When the binding constants given in Table 4

are evaluated, it can be seen that the K_bvalues of the compounds are

between 2.0 × 10³and 2.2 × 10⁵M⁻¹. The binding constants of the

molecules in this group are ordered from large to small as follows:

18 > 7 = 6 > 8 > 4 > 14 > 12 > 5 > 9 > 13 > 10 > 19. When the data in

Table 4 are examined, it is understood that 18 binds DNA much more

strongly than others. However, the binding constants of the 17, 2, and 3

could not be calculated using UV–Vis spectrophotometric method.

|

2.2.5

Metabolic enzyme inhibition activity

FIGURE 2 The effect of compound 3 on the morphologies of

A549, Hep3B, HT29, MCF7, and FL cells. Exponentially growing cells

were incubated overnight with 20 μg/ml of compound 3 at 37°C.

Control cells were treated with only dimethyl sulfoxide. All

measurements were 100 μm

Anti‐AChE activity results

We have reported the inhibitory activity against AChE of substituted

quinoline derivatives and of control compound, tacrine (IC₅₀

=

224.93 nM for AChE) in Table 1, expressed as IC₅₀values. The sub-

stituted quinoline derivatives, 2–10, 12–14, and 17–18, were found

to be potent inhibitor compounds of cholinesterases with IC₅₀in the

nanomolar concentration scale. The excellent AChE inhibitors in the

assayed series of substituted quinoline analogs are all tested cya-

noquinoline derivatives 7, 6, 8, and 10, except 9 (IC₅₀= 111.34 nM),

and novel N‐nitrated 6,8‐dimethoxyquinoline 13 with IC₅₀values

ranging between 6.84 and 26.94 nM. 6‐Bromotetrahydroquinoline (2)

and 3,6,8‐tribromoquinoline (5) significantly inhibited AChE enzyme

at IC₅₀values of 47.94 and 88.95 nM, respectively, whereas 6,8‐

dibromotetrahydroquinoline (3; IC₅₀= 101.53 nM) and its aromatic

form 4 (IC₅₀= 136.19 nM) showed a moderate inhibition against

AChE enzyme, compared with tacrine (Table 5). The quinoline deri-

vatives 17–19 substituted at C‐5, C‐7, and C‐8, respectively, showed

a better inhibition than tacrine against AChE with an IC₅₀range of

72.23–104.23 nM.

against Enterococcus faecalis (VRE) ATCC 19433 (MIC values ranging

from 125 to 250 μg/ml), only compound 18 against E. faecalis ATCC

29212 (MIC = 125 μg/ml), compounds 13 and 18 against Staphylo-

coccus aureus ATCC 25923 (values ranging from 125 to 250 μg/ml),

compounds 8, 9, 12, 13, and 18 against S. aureus (MSSA) ATCC 29213

(MIC values ranging from 125 to 250 μg/ml), compounds 2, 4, 9, 12,

13, 14, 18, and 19 against S. aureus MRSA ATCC 46300

(62.5–250 μg/ml) were more or similar to the SCF antibiotic used as a

positive control (Table 3). According to the MIC values exhibited by

the synthesized molecules on Gram(−) bacteria, it was found that

none of our molecules had a sufficiently strong antibacterial effect on

the Escherichia coli ATCC 25922, E. coli ATCC 35218, and

P. aeruginosa ATCC 27853 strain (Table 3).

|

2.2.4

DNA binding properties of the compounds

Carbonic anhydrases inhibition activity results

An esterase assay method^[41,42]was used to investigate the inhibition

potentials of substituted quinoline derivatives against two physiolo-

gically relevant CA isoforms, the slower cytosolic isoform (hCA I) and

the more rapid cytosolic isoenzyme (hCA II). In this assay, acet-

azolamide (AZA) was used as a standard drug due to its utilization in

clinical application as a carbonic anhydrase inhibitor. CA I and II

isoforms' inhibition data of compounds are summarized and their

IC₅₀and K_ivalues expressed in nanomolar (nM) range are displayed

in Table 6.

DNA binding properties of the compounds were determined using the

ultraviolet (UV)–visible absorption (Vis) spectrophotometer. Binding

type and binding constants of the compounds were explained as follows.

A single maximum absorption peak was observed in the spectrum of 4,

6, 7, 9, 10, 12, 14, and 19, and no clear redshifts or blueshifts on this

peak were observed. When circulating tumor DNA (CT‐DNA) was ad-

ded in an increasing amount to the reaction mixture, the reduction in

the absorption intensity of molecules

9 and 12 resulted in a

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ÖKTEN ET AL.

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FIGURE 3 Ultraviolet–visible absorption spectra of 25‐μM selected compounds in the absence (a) and presence of 6.25 μM (b), 12.5 μM (c),

25 μM (d), 50 μM (e), 100 μM (f), 200 μM (g), 400 μM (h), and 800 μM (i) DNA. The direction of the arrow demonstrates increasing

concentrations of DNA. Inside graph is the plot of [DNA] versus [DNA]/(εa − εf) to find the binding constant of the complex–DNA adduct

Most of the substituted quinoline analogs remarkably inhibited

the slow cytosolic isoform hCA I, taking part in important physio-

logical and pathological processes in many tissues and

organs,^[43]with K_ivalues ranging between 39.52 and

574.52 nM. 6,8‐Dicyanotetrahydroquinoline (6) and 5,7‐dibromo‐8‐

methoxyquinoline (19) were determined as the best inhibitors for

this isoform, with K_ivalues of 39.52 and 48.05 nM, respectively. The

N‐nitrated (13) and brominated 6,8‐dimethoxyquinoline (12) at C‐5

and their starting material, 6,8‐dibromoquinoline (4), have displayed

a significant inhibition against cytosolic isoform hCA I with a K_i

value range of 103.64 20.63–125.50 14.78 nM (Table 6). More-

from 54.95 to 976.93 nM. The inhibitory potentials of substituted

quinoline derivatives against the hCA II had a similar behavior to

that against hCA I. In addition, AZA had a medium inhibition po-

tential against this isoform, with a K_ivalue of 1,104.43 nM.

TABLE 5 Acetylcholinesterase (AChE) inhibitory activity (IC₅₀and

K_i) of the substituted quinoline derivatives

IC₅₀(nM)

AChE

K_i(nM)

Compounds

r²

AChE

2

47.94

0.9376

0.9593

0.9818

0.9533

0.9952

0.9572

0.9491

0.9593

0.9583

0.9596

0.9882

0.9815

0.9911

0.9390

0.9106

0.9880

40.14 7.94

92.83 13.05

95.73 20.88

75.04 13.94

20.91 4.08

12.95 3.41

20.15 2.94

90.45 16.94

5.51 0.94

3

101.53

136.19

88.95

over, for 5‐acetamido‐1,3,4‐thiadiazole‐2‐sulfonamide (AZA),

a

broad‐specificity CA inhibitor and used for the treatment of altitude

sickness, cystinuria, idiopathic intracranial hypertension, glaucoma,

and epileptic seizure, a K_ivalue of 1,005.47 75.60 nM was re-

corded against hCA I. The high concentration of the hCA II led to

several diseases such as glaucoma, osteoporosis, and renal tubular

acidosis.^[41]Against rapid cytosolic isoenzyme hCA II, substituted

quinoline analogs (2–10, 12–14, and 17–13) had K_ivalues ranging

4

5

6

23.94

7

15.03

8

26.94

9

111.34

6.84

10

12

176.03

16.04

155.22 26.37

12.88 2.93

69.05 9.35

83.22 14.06

61.15 8.58

75.52 17.90

187.66 33.61

TABLE 4 The binding constants (K_b) of these compounds

13

Compound

K_b(M⁻¹

)

Compound

K_b(M⁻¹

)

14

95.26

4

5

6

7

8

9

7.2 × 10⁴

1.8 × 10⁴

1.6 × 10⁵

7.3 × 10⁴

1.6 × 10⁴

10

12

13

14

18

19

3.0 × 10³

1.9 × 10⁴

3.9 × 10³

2.2 × 10⁴

2.2 × 10⁵

2.0 × 10³

17

104.23

72.73

18

19

88.20

Tacrine^a

224.93

Note: The results were expressed in nanomolar (nM) range.

^aTacrine was used as a standard inhibitor for AChE enzyme.

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ÖKTEN ET AL.

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TABLE 6 The enzyme inhibition results

of substituted quinoline analogs against

human carbonic anhydrase isoenzymes I

and II (hCA I and II)

IC₅₀(nM)

hCA I

K_i(nM)

Compounds

r²

hCA II

r²

hCA I

hCA II

2

804.02

0.9583

1,004.85 0.9889 956.82 104.80

976.93 105.80

963.33 95.35

119.05 13.94

514.86 57.04

54.95 5.93

3

1,005.80 0.9374

834.93

105.94

486.77

52.84

0.9583 926.33 82.64

0.9588 103.64 20.63

0.9375 408.84 45.93

0.9460 46.04 8.63

0.9355 444.28 68.84

0.9905 635.33 71.26

0.9911 306.82 58.04

0.9799 294.62 21.74

0.9550 125.50 14.78

0.9485 104.94 11.66

0.9856 350.73 54.93

0.9646 347.91 18.66

0.9496 663.06 92.06

0.9930 51.68 5.95

4

83.04

0.9490

0.9374

0.9485

0.9284

0.9372

0.9733

0.9804

0.9882

0.9918

0.9084

0.9309

0.9406

0.9923

5

394.63

39.52

6

7

406.92

507.27

281.52

305.04

101.88

93.05

409.75

496.33

333.84

397.01

144.76

106.83

304.88

318.63

507.73

67.04

417.04 73.80

684.03 88.31

335.96 42.07

304.75 65.66

157.11 30.51

115.94 21.95

394.02 94.85

385.18 83.85

582.33 85.03

61.05 4.77

8

9

10

12

13

14

17

18

19

AZA^a

285.02

288.90

574.52

48.05

1,103.70 0.9586

1,188.01 0.9691 1,005.47 75.60 1,104.43 95.55

Note: The results were expressed in nanomolar (nM) range.

^aAcetazolamide (AZA) was used as a standard inhibitor for each hCA I and II enzymes.

|

2.3

Docking studies

|

2.3.1

Docking analyses for enzyme inhibition

TABLE 7 The estimated free energy of binding (kcal/mol) between

the test compounds and target proteins

For different substituted quinoline derivatives 1–19, the inhibitory

activity was determined by a molecular simulation method against

AChE, hCA I, and hCA II enzymes. The estimated free energies of

binding obtained from molecular docking results, which is a kind of

molecular simulation method, are listed for 1–19 in Table 7. In ad-

dition, to determine the activity against the AChE enzyme and hCA I

and hCA II enzymes of a chemical species, tacrine, and AZA, re-

spectively, which were taken as a reference, were docked with the

relevant target proteins.

Compounds

4EY6

−5.24

−4.53

−4.23

−4.83

−6.00

−6.80

−6.58

−4.47

−6.90

−3.71

−6.77

−2.78

−2.86

−4.94

−4.92

−3.53

–

1BMZ

−4.85

−4.60

−5.65

−5.26

−5.94

−5.05

−4.95

−5.40

−5.30

−5.51

−5.54

−5.39

−5.37

−4.94

−5.87

–

2ABE

−4.05

−4.08

−5.77

−4.62

−5.95

−4.55

−4.51

−4.86

−4.87

−4.84

−4.93

−4.64

−4.70

−4.30

−5.86

–

2

3

4

5

6

7

The inhibition efficiency of each compound and reference sub-

stances against selected target proteins is listed in Table 7. According

to Table 7, the obtained docking results and the experimental IC₅₀

values are quite compatible with each other. Also, it was found that

the estimated free energies of binding values of the compounds

without IC₅₀values in Tables 5 and 6 were lower than the energies of

the relevant reference substances. Compound 10 has the highest

activity with −6.90 kcal/mol binding energy against the 4EY6 target

protein determined for the AChE enzyme. The studied compounds

exhibited a similar activity against proteins selected for hCA I and

hCA II enzymes. When the molecular docking and experimental in-

hibition efficacy are compared, the only difference between mole-

cular simulation and experimental results can be seen in compounds

6 and 8 against 4EY6. Unlike experimental IC₅₀values, the estimated

8

9

10

12

13

14

17

18

19

Tacrine

Acetazolamide

−4.18

−3.92

10 of 16

ÖKTEN ET AL.

|

free energies of binding values of 8 are greater than that of 6, which

may be due to secondary chemical interactions between the com-

pounds and amino acid residues of the target proteins. Compound 8

formed more H‐bonds with the amino acid residues of the protein

than 6. Due to this status, the binding energy of compound 8 may

have been more. It can also be incorporated into the nonbonded

electron pair resonance structure on the nitrogen atom in compound

8, and this nitrogen atom has a less steric effect than compound 6.

The binding modes between amino acid residues of the first five

compounds (these compounds are written in red in Tables 5 and 6)

with a high activity against 4EY6, 2ABE, and 1BMZ proteins are given

in Figure 4.

2.3.2

Docking analyses for an anticancer effect of

compound 10

In an attempt to look into the mechanism of action of compound 10,

we extensively searched through the SEA receptor database

(sea.bkslab.org),^[44]which gave rise to a highly potential target, phos-

pholipase C gamma 1 (PLCγ1). PLCγ1, which is overexpressed in many

metastatic tumors,^[44–47]is involved in the generation of second mes-

sengers from phosphatidylinositol 4,5‐bisphosphate (PIP2) to signal cell

proliferation and differentiation.^[48]The downregulation (or inhibition) of

PLCγ1 in nude mice was reported to essentially suppress lung

metastasis.^[47]The inhibition of PLCγ1 by small molecules such as

FIGURE 4 Docking poses of the five most active inhibitors of each target protein

11 of 16

ÖKTEN ET AL.

|

triterpene esters^[49]was reported to diminish cancer cell proliferation. It

was also reported that some sulfonylpyridinediamine derivatives^[50]in-

hibit PLCγ1 at submicromolar concentrations. Furthermore, the inhibi-

tion of PLCγ1 was reported to induce autophagy in human colon cancer

and hepatocellular carcinoma cells, which was mediated by downstream

interference with the mTOR/ULK1 pathway as well as dissociation of the

beclin1–IP3R–Bcl‐2 complex.^[51]

group at C‐5 of 14 is critical for an antiproliferative activity. Compound

4 bearing bromine at C‐6 and C‐8 exhibits a selective antiproliferative

activity against Hep3B cells (IC₅₀= 37.4 μg/ml). Moreover, the cyano

group substituted at C‐6 in 4 resulted in having significant inhibition

potentials of 10 against all tested cancer cell lines. However, the pre-

sence of nitrile at C‐8 for quinoline bromides, that is, compound 9,

showed a selective inhibition against only A549 cells (IC₅₀= 5.4 μg/ml).

On the contrary, bromination of 1,2,3,4‐tetrahydroquinoline (1) at C‐6

and C‐8 in compounds 2 and 3 has increased antiproliferative activities

against MCF7, HT29, Hep3B, and HeLa cell lines with IC₅₀values of 3.7

and 48.5 μg/ml, except A549 cells. However, the strong antiproliferative

activity of 6,8‐dibromotetrahydroquinoline (3) significantly reduced

against tested cancer cell lines in case of cyano groups exchanged at C‐6

and/or C‐8 in compounds 6 and 7. Our previous study^[7]reported that

the methoxy group bound at both C‐6 and C‐8 in a quinoline cycle

showed a selective inhibition against HT29 cells, whereas bromination

at C‐5 or nitration at N‐1 positions of this compound led to display an

antiproliferation activity against MCF7, HT29, Hep3B, HeLa, and FL cell

lines. 8‐Hydroxyquinoline (15) brominated at C‐5 and C‐7 has a strong

inhibition of proliferation of all tested cell lines with IC₅₀values of 5.4

and 62.7 μg/ml. However, the reduction in significant inhibition of

compound 19 indicated that the methoxy group replaced the hydroxy

group at C‐8, which significantly decreased inhibition potential of 19.

Interestingly, 8‐methoxyquinoline nitrated at C‐5 and C‐7 displayed a

selective antiproliferation effect against MCF7, Hep3B, and FL cell lines

with IC₅₀values of 22.3, 18.3, and 31.8 μg/ml, respectively. According to

these results, it can be concluded that the cyano group bound at C‐6

and hydroxy group bound at C‐8 for quinoline bromides (10 and 17) and

bromination at C‐5 and nitration of N atom of 6,8‐dimethoxyquinoline

(12 and 13) led to significantly increased inhibition potentials against

cancer cell lines, but the presence of cyano and methoxy groups at C‐8

for quinoline bromides (6–8 and 19) reduced the inhibition activity.

When the MIC values of substituted quinolines displayed on

Gram(+) bacteria were examined, it ws observed that some of the

compounds had a selective antibacterial effect. Whereas 5 showed

an antibacterial effect against only E. faecalis (VRE) ATCC 19433

Recently, it has been experimentally confirmed that the small mo-

lecule binding site of the PLCγ1 complex structure is in the nSH2 domain

that binds the phosphorylated tyrosine 766 (pY766) residue of the tyr-

osine kinase domain of growth factors.^[52]Therefore, DOCK studies were

performed on an X‐ray structure of nSH2 in complex with fibroblast

growth factor receptor 2 (FGFR2; PDB ID: 5EG3).^[53]As seen in

Figure 5a,b, compound 10 suitably and favorably docks into the FGFR2

binding site of PLCγ1. Apparently, binding of compound 10 prevents aa

sequence LEU(774)ASP(771)LEU(772)SER(773)GLN(774) of FGFR2 from

binding, significantly diminishing the binding interactions between PLCγ1

and FGFR2. Binding of 10 in the binding site of nSH2 of PLCγ1 is highly

likely to prevent the phosphorylated TYR(769) residue FGFR2 to conduct

its kinase activity on PLCγ1. To the best of authors' knowledge, there are

only a few small molecule inhibitors of PLCγ1 published in literature.

Therefore, we propose that compound 10 could represent a potential

drug candidate, which should be further investigated for metastatic

cancer treatment.

|

2.4

The structure–activity relationship study

The determination of antiproliferative effects of the substituents on

quinoline cycle indicated that the presence of bromine and cyano

groups at positions C‐6 and C‐8 in compounds 4 and 8 did not inhibit

the proliferation of cancer cell lines, and also three bromine groups

bound at C‐3, C‐6, and C‐8 in compound 5 did not increase the anti-

proliferative activity. The nitration of 5 at C‐5 led to show a selective

antiproliferation activity against HeLa, MCF7, and HT29 cell lines

(IC₅₀= 11.1, 8.5, and 7.1 μg/ml, respectively). In particular, the nitro

FIGURE 5 The docked structure of compound 10 in complex with the X‐ray structure coordinates of PLCγ1 (PDB ID: 5EG3).^[53](a) X‐ray

structure coordinates of a small FGFR2 peptide (PDB ID: 5EG3) and docked coordinates of 10 are both shown, and (b) only the docked

coordinates of 10 are shown in the binding site of PLCγ1. P‐TYR(769) in (a) is phosphorylated TYR residue of FGFR2, which phosphorylates

nSH2 to activate PLCγ1

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ÖKTEN ET AL.

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(125 μg/ml of MIC value), 2, 4, and 14 had an antibacterial effect

only against S. aureus ATCC 46300 (MIC = 250, 125, and 125 μg/ml,

respectively). Compound 18 displayed an inhibitory activity against

all tested Gram(+) bacteria at an MIC value of 125 μg/ml; however,

it did not have a sufficient antibacterial effect against all tested

Gram(−) bacteria. Whereas 13 showed an antibacterial effect

against S. aureus ATCC 25923, S. aureus ATCC 29213 (MSSA), and S.

aureus ATCC 46300 at an MIC value of 250 μg/ml, 10, 6, and 7 did

not have any antibacterial effect against tested Gram(+) and

Gram(−) bacteria. Against E. faecalis ATCC 19433, 12, 18, and 19

(MIC = 125 μg/ml) had a better antibacterial effect than the control

compound (SCF, MIC = 250 μg/ml). Against S. aureus ATCC 46300,

12 had the strongest inhibition at an MIC value of 62.5 μg/ml,

compared with all tested compounds and positive control (SCF;

MIC = 250 μg/ml; Table 4). Moreover, 9, 4, 14, and 18 had a stronger

effect (MIC = 125 μg/ml) against S. aureus ATCC 46300, compared

with positive control (SCF; MIC = 250 μg/ml; Table 4). Against E.

faecalis ATCC 29212, only 18 displayed an antibacterial effect. In

brief, against tested Gram(+) bacteria, substituted quinolines have a

moderate antibacterial effect. However, these compounds did not

show inhibition against any Gram(−) bacteria. It is evident that both

the nature and position of the substituents for different substituted

quinoline derivatives are able to cause variation in the AChE, cy-

tosolic hCA I, and hCA II enzyme inhibition activities of quinoline.

All quinoline derivatives have good inhibitory activities against

AChE, cytosolic hCA I, and hCA II enzymes, compared with standard

compounds, tacrine and AZA, respectively. Although the selectivity

and variation of the above‐mentioned enzyme inhibition potentials

of substituted quinolines were not observed, their anticancer and

antibacterial activities showed that substituted quinoline analogs

had selective antiproliferative and antibacterial effects. For ex-

ample, 7, 6 (except for its antiproliferative activity against HT29;

IC₅₀= 78.1 μg/ml), and 8 (except for its antiproliferative activity

against A549; IC₅₀= 5.4 μg/ml) did not display antiproliferation

against tested cancer cell lines and antibacterial activities on tested

Gram(+) and Gram(−) bacteria, whereas these compounds sig-

nificantly inhibited AChE (K_i= 12.95, 20.15, 20.91, and 90.45 nM,

Similarly, brominated quinolines, 3,6,8‐tribromoquinoline (5),

5,7‐dibromo‐8‐methoxyquinoline (19), and 6,8‐dibromoquinoline (4,

except its antiproliferative effect against Hep3B; IC₅₀= 37.4 μg/ml),

did not show any anticancer and antibacterial effect. However, these

compounds inhibited AChE (K_iranging from 75.04 to 95.73 μg/ml),

cytosolic hCA I (K_iranging from 51.68 to 408.84 μg/ml), and hCA II

(K_iranging from 61.05 to 514.86 μg/ml) enzymes. On the contrary,

brominated tetrahydroquinoline derivatives, 2 and 3, exhibited both

anticancer activity and enzyme (AChE, hCA I, and hCA II) inhibitory

potential.

The nitration of quinoline in any position led to increased en-

zyme inhibition, due to which 13, 18, and 14 had a significant enzyme

inhibitory effect against AChE (K_i= 12.88, 61.15, and 69.05 nM, re-

spectively), cytosolic hCA I (K_i= 104.94, 663.06, and 350.73 nM, re-

spectively), and hCA II (K_i= 115.94, 582.33, and 394.02 nM,

respectively).

|

3

CONCLUSION

Recently synthesized substituted quinoline bearing different func-

tional groups and novel nitrated methoxyquinoline analogs were

tested for their antibacterial, anticancer activities in vitro, and en-

zyme inhibition effects. We have shown that substituted quinolines

have a significant potential as enzyme inhibitors against AChE, hCA I,

and hCA II, and also a selective anticancer effect against A549, HeLa,

Hep3B, HT29, and MCF7 cancer cell lines. When TGI and IC₅₀values

of the compounds were examined, we found that 5,7‐dibromo‐8‐

hydroxyquinoline (17; IC₅₀values between 5.4 and 62.7 μg/ml; TGI

values between 6.5 and 66.5 μg/ml), 8‐bromo‐6‐cyanoquinoline (10;

IC₅₀values between 1.5 and 22.5 μg/ml; TGI values between 1.7 and

29.1 μg/ml), 6,8‐dibromotetrahydroquinoline (3; IC₅₀values between

3.7 and 41.1 μg/ml; TGI values between 3.1 and 41.9 μg/ml, except

A549 cell line), and novel synthesized N‐nitrated 6,8‐

dimethoxyquinoline (13; IC₅₀values between 19.5 and 51.2 μg/ml;

TGI values between 20.4 and 52.7 μg/ml, except A549 cell line) have

very strong antitumor effects against all tested cell lines (Table 1

and 2). All anticancer test results indicate that these compounds,

especially 10, can be promising anticancer agent candidates. More-

over, the docking study for anticancer activity suggested that 10

could represent a potential anticancer drug candidate for metastatic

cancer treatment due to its potential to suppress the PLCγ1. Novel

synthesized 5,7‐dinitro‐8‐methoxyquinoline (18) has the potential

of being an antibacterial agent against Gram(+) bacteria

(MIC = 125 μg/ml). Also, all of the substituted quinolines effectively

reduced enzyme activities of AChE, hCA I, and hCA II at the nano-

molar concentrations. The substituted quinoline analogs can be drug

candidates of the CAIs for therapy of some diseases such as epilepsy,

osteoporosis, glaucoma, gastric and duodenal ulcers, neurological

disturbances, and they can be drug candidates of the AChE inhibitor

for the therapy of Alzheimer's disease. The activity of quinoline de-

rivatives substituted with a number of different functional groups

against AChE and human carbonic anhydrase isoenzymes (hCA I and

respectively), cytosolic hCA

I (K_i= 444.28, 635.33, 46.04, and

306.82 nM, respectively), and hCA II (K_i= 417.04, 684.03, 54.95,

and 335.96 nM, respectively) enzymes. Moreover, 10 exhibited the

highest antiproliferative activities against all tested cancer cell lines

(IC₅₀values ranging from 1.5 to 22.5 μg/ml) and high enzyme in-

hibitory effect against AChE (K_i= 5.51 nM), hCA I (K_i= 296.62 nM),

and hCA II (K_i= 304.75 nM), compared with standard compounds.

However, this molecule exhibited an antibacterial activity against

only S. aureus ATCC 46300 (MIC = 125 μg/ml). According to these

results, it can be concluded that nitrile group at the positions of C‐6

and C‐8 or only C‐8 of quinoline cycle decreased the anticancer

activity and antibacterial effects, whereas the presence of nitrile

group in quinoline cycle enhanced the enzyme inhibition against

AChE, cytosolic hCA I, and hCA II. However, the nitrile group bound

at only C‐6 promoted antiproliferation against cancer cell lines in

addition to enzyme inhibitory activity.

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ÖKTEN ET AL.

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II) was supported by molecular docking studies. Experimental IC₅₀

results of the compounds against these enzymes were found to be

highly compatible with each other.

4.1.3

The synthesis of 5,7‐dinitro‐8‐

methoxyquinoline

8‐Methoxyquinoline (16; 0.50 g, 3.14 mmol) was dissolved in 5 ml

sulfuric acid and cooled at −5°C with salt–ice bath. A mixture of

H₂SO₄(4 ml) and HNO₃(4 ml) acid was prepared and the acid mix-

ture was cooled at −5°C. The solution obtained was cooled at 0°C on

a salt–ice bath for a few minutes. While the 8‐methoxyquinoline (16)

solution was stirred with a magnetic stirrer, the H₂SO₄/HNO₃mix-

ture was added dropwise with the aid of a Pasteur pipette within 1 hr

so that the solution temperature does not exceed 0°C. After 2 hr, the

reaction was finished, the reaction mixture was poured into crushed

ice (30 g) in a beaker. After the ice melted, the mixture was extracted

with CH₂Cl₂(5 × 20 ml). The organic phase was neutralized with aq

NaHCO₃(10%) solution and dried over Na₂SO₄. The solvent was

removed in vacuo and filtrated over silica. Yellow‐colored needle

crystals were obtained as the sole product in a yield of 87% (0.25 g).

Mp. 140–143°C. ¹H NMR (400 MHz, CDCl₃, δ, ppm): δ_H9.22 (dd,

|

4

EXPERIMENTAL

|

4.1

Chemistry

|

4.1.1

General

Thin‐layer chromatography was carried out on Merck silica F254

0.255‐mm plates, and spots were visualized by UV at 254 nm. Flash

column chromatography was performed using Merck 60 (70–230

mesh) silica gel. The microwave reactions were run in CEM Discover

Labmate instrument. Melting points were determined on

a

Thomas–Hoover capillary melting points apparatus. Solvents were

concentrated at a reduced pressure. IR spectra were recorded on a

Bruker Vertex 70 v FT‐IR instrument. Mass spectra were recorded on

a spectrometer under electron‐impact (EI) and chemical ionization

conditions. The elemental analysis was recorded on an Ele-

mentarVario MICRO Cube instrument. NMR spectra were recorded

on Bruker 400 MHz for ¹H and at 100 MHz for ¹³C NMR.

J₄₂= 1.6 Hz, J₄₃= 8.8 Hz, 1H, H₄), 9.17 (dd, J₂₄= 1.6 Hz, J₂₃= 4.0 Hz,

1H, H₂), 8.89 (s, 1H, H₆), 7.84 (dd, J₃₂= 4.4 Hz, J₃₄= 8.8 Hz, 1H, H₃),

and 4.58 (s, 3H, OMe). ¹³C NMR (100 MHz, CDCl₃, δ, ppm): δ_C155.8,

151.3, 142.9, 139.4, 139.1, 132.9, 126.0, 124.7, 121.1, and 65.3

(–OMe). IR (solid KBr, v_max, cm⁻¹): 3,110, 3,085, 3,055, 2,960, 2,927,

1,607, 1,572, 1,518, 1,498, 1,467, 1,382, 1,318, 1,262, 1,239, 1,189,

1,160, 1,096, 987, 959, 909, 837, 798, 760, 716, 693, and 604. Anal.

calcd. for C₁₀H₇N₃O₅(249.0386): C, 48.20%; H, 2.83%; N, 16.86%.

Found: C, 48.27%; H, 2.79%; N, 16.80%.

The original spectra of the novel compounds are provided as

Supporting Information. The InChI codes of the investigated com-

pounds are also provided as Supporting Information.

|

4.1.2

The synthesis of substituted quinoline

|

compounds

4.1.4

Synthesis of 6,8‐dimethoxy‐1‐nitroquinoline

This study was carried out with substituted quinolines 2–10, 12–14,

and 17–19 according to our previous papers.^{[6,7,14,20,30]}Moreover,

two novel nitrated methoxyquinoline derivatives were synthesized

for this paper. In brief, the synthesis of 6‐bromo‐1,2,3,4‐

tetrahydroquinoline (2), 6,8‐dibromo‐1,2,3,4‐tetrahydroquinoline

(3), and 3,6,8‐tribromoquinoline (5) via direct bromination of

1,2,3,4‐tetrahydroquinoline (1) and 6,8‐dibromoquinoline (4) via

aromatization of 3 with DDQ has been reported in our previous

publications.^{[6,29,30,39,54]}The cyano and methoxyquinoline deriva-

tives (6–11) starting from 6,8‐dibromo‐1,2,3,4‐tetrahydroquinoline

(3) and 6,8‐dibromoquinoline (4) treated with NaOMe or CuCN

were synthesized according to our reported procedures.^[14,39]Fur-

thermore, the synthesis of 5,7‐dibromo‐8‐hydroxyquinoline (17)

and 5,7‐dibromo‐8‐methoxyquinoline (19) via bromination of 8‐

hydroxyquinoline (15) and 8‐methoxyquinoline (16), respectively,

was reported in our recent works.^[40,55]The isolated compounds,

2–10, 12–14, and 17–19, were fully characterized by melting point,

HRMS analysis, infrared, ¹H, ¹³C, HMBC (heteronuclear multiple

bond correlation), and HETCOR spectroscopy in these

papers.^{[6,29,30,39,40,43]}All tested compounds were purified by col-

umn chromatography. The purity of the tested compounds was

monitored with ¹H NMR spectroscopy.

6,8‐Dimethoxyquinoline (11; 0.50 g, 2.64 mmol) was dissolved in 5 ml

sulfuric acid and cooled at −5°C with salt–ice bath. A mixture of

H₂SO₄(3 ml) and HNO₃(3 ml) acid was prepared and the acid mix-

ture was cooled at −5°C. The solution obtained was cooled at 0°C on

a salt–ice bath for a few minutes. While the 6,8‐dimethoxyquinoline

(11) solution was stirred with a magnetic stirrer, the H₂SO₄/HNO₃

mixture was added dropwise with the aid of a Pasteur pipette within

1 hr so that the solution temperature does not exceed 0°C. After 1 hr,

the reaction was finished, the reaction mixture was poured into

crushed ice (30 g) in a beaker. After the ice melted, the mixture was

extracted with CH₂Cl₂(5 × 20 ml). The organic phase was neutralized

with aq NaHCO₃(10%) solution and dried over Na₂SO₄. The solvent

was removed in vacuo and filtrated over silica. Brown oil was ob-

tained as the sole product in yield of 97% (0.30 g). ¹H NMR

(400 MHz, dimethyl sulfoxide [DMSO]‐d₆, ppm): δ_H8.89 (d,

J₂₃= 4.4 Hz, 1, H₂), 8.85 (d, J₄₃= 8.8 Hz, 1H, H₄), 7.94 (dd, J₃₂= 5.2 Hz,

J₃₄= 8.4 Hz, 1H, H₃), 7.27 (d, J₅₇= 1.6 Hz, 1H, H₅), 7.22 (d,

J₅₇= 2.0 Hz, 1H, H₇), 4.10 (s, 3H, OMe), and 3.96 (s, 3H, OMe). ¹³C

NMR (100 MHz, DMSO‐d₆, ppm): δ_C160.2 (q), 152.4 (q), 143.2,

142.8, 134.5 (q), 131.1 (q), 123.5, 105.4, 98.6, 57.3 (OMe), and 56.6

(OMe). IR (solid KBr, v_max, cm⁻¹): 3,005, 2,937, 2,838, 1,667, 1,613,

1,579, 1,501, 1,452, 1,382, 1,234, 1,212, 1,158, 1,135, 1,120, 1,049,

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ÖKTEN ET AL.

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1,029, 998, 934, 868, 829, 784, 663, and 626. Anal. calcd. for

C₁₁H₁₁N₂O₄(235.0713): C, 56.17%; H, 4.71%; N, 11.91%. Found: C,

56.23%; H, 4.79%; N, 11.86%.

LC₅₀) were calculated according to the following formulas using Excel

software. The growth inhibition of 50% (GI₅₀) was calculated from

the following equation:

[(Ti – Tz)/(C – Tz)] × 100 = 50.

|

4.1.5

Synthesis of 3,6,8‐tribromo‐5‐nitroquinoline

This formula is the drug concentration resulting in a 50% re-

duction in the net growth increase in control cells during the drug

incubation. The total growth inhibition (TGI) was calculated from

Ti = Tz. LC₅₀, indicating a net loss of treated cells, was calculated from

the following equation:

A solution of 3,6,8‐tribromoquinoline (6; 0.70 g, 1.913 mmol) in 5 ml

of sulfuric acid was cooled at −5°C in a salt–ice bath and treated

cautiously with a solution of 50% nitric acid in 10 ml of sulfuric acid

at −5°C while the 3,6,8‐tribromoquinoline (6) solution was stirred.

After 1 hr in an ice bath, the red mixture was allowed to stand at

room temperature. The red‐colored solution was poured into crushed

ice (30 g) in a beaker. After the ice melted, the mixture was extracted

with CH₂Cl₂(5 × 15 ml). The organic phase was neutralized with aq

NaHCO₃(10%) solution and dried over Na₂SO₄. The solvent was

removed in vacuo. Yellow‐colored needle crystals were obtained as

the sole product in yield of 100% (0.78 g). M.p. 215–216°C. ¹H NMR

(400 MHz, CDCl₃, δ, ppm): δ_H9.12 (d, ⁴J = 2.0 Hz, 1 H, H‐2), 8.33 (s, 1

H, H‐7), and 8.24 (d, ⁴J = 2.0 Hz, 1 H, H‐4). ¹³C NMR (100 MHz,

CDCl₃, δ, ppm): δ_C153.9, 146.2, 142.1, 136.1, 131.8, 129.0, 122.5,

122.0, and 113.5. IR (solid KBr, v_max, cm⁻¹): 3,074, 2,956, 2,921,

1,663, 1,650, 1,550, 1,455, 1,349, 1,320, 1,080, 1,014, 934, 893, 871,

806, 783, 737, 675, and 617. Anal. calcd. for C₉H₃Br₃N₂O₂

(407.7745): C, 26.31%; H, 0.74%; N, 6.82%. Found: C, 26.22%; H,

0.73%; N, 6.80%.

[(Ti – Tz)/Tz] × 100 = −50.

|

4.2.2

Cytotoxicity assay

The cytotoxic potentials of the compounds were determined by a

cytosolic LDH measurement kit according to the manufacturer's

procedures.^[56]Briefly, 5 × 10³cells were conveyed into each well as

triplicates and exposed with IC₅₀concentrations of the compounds at

37°C with 5% CO₂overnight. The percentage of cytotoxicities was

obtained by using the following equation:

[(Experimental

control)] × 100,

value – Low

control)/(High

control − Low

where the experimental value pertains to the cells treated with

the test compound, the high control (maximum LDH release) means

the 2% Triton X‐100‐treated cells, and the low control (spontaneous

LDH release) are the untreated cells.

|

4.2

Biological assays

|

DNA binding studies

4.2.1

MTT cell proliferation assay

4.2.3

HT29 (human colorectal adenocarcinoma), HeLa (human cervix

adenocarcinoma), MCF7 (human breast adenocarcinoma), A549

(human lung carcinoma), and Hep3B (human hepatocellular carci-

noma) cancer cell lines, and FL (human amnion cells) normal cell line

were maintained in a suitable medium containing fetal bovine serum

and antibiotic solution. A cell suspension was adjusted at 1 × 10⁶

cells in 10 ml and 100 μl was transferred into each well of culture

plates. The compounds were dissolved in sterile DMSO at final

concentrations of 10–200 μg/ml and the cells were incubatedat

37°C with 5% CO₂overnight. The antitumor activities of the com-

pounds were determined using MTT cell proliferation assay. In MTT

assay, the percent inhibitions of test and control molecules were

determined. The percent inhibition was equal % inhibitions with the

following formula:

The binding constants (K_b) against calf thymus DNA and physiolo-

gical interactions of disubstituted tacrine derivatives were ex-

amined by using UV–Vis spectroscopy technique. To prepare stock

calf thymus DNA solution, 2.5 mg DNA was dissolved in 10.0 ml

Tris‐HCl buffer (20 mM Tris‐HCl, 20 mM NaCl, pH 7.0) and stored at

+4°C for up to 7 days. The DNA concentration in solution was

calculated by using ε value (6,600 M⁻¹·cm⁻¹at 260 nm) that belongs

to DNA. In addition, the purity of the calf thymus DNA solution was

controlled with the help of a change of absorbance obtained from

the ratio of A260/A280. As the value was equal to 1.87, the DNA

was considered to be sufficiently pure. To obtain 25 μM of a

working solution, disubstituted tacrine derivatives were diluted

with Tris‐HCl buffer and then all of the compounds were incubated

at 24°C for 30 min before the measurement. To ensure sufficient

solubility in solution throughout measurement, a special solvent

system (1:9 DMSO/Tris‐HCl buffer) was prepared. Eight measure-

ment points at room temperature for disubstituted tacrine deriva-

tives were recorded by using 1‐cm‐path quartz cuvettes. The

number of disubstituted tacrine derivatives was kept constant while

increasing the CT‐DNA concentrations (6.5–800 μM) in the UV

absorption titrations.

Inhibition(%) = (A_sample– A_control)/(A_control) × 100,

where A_sampleis the absorbance of treated cells and A_controlis the

absorbance of the untreated cells. The IC₅₀values of the compounds

were obtained by using Excel software and noted in μg/ml at 95%

confidence intervals. The dose–response parameters (GI₅₀, TGI, and

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ÖKTEN ET AL.

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Dock computations for the anticancer effect

4.2.4

Microdilution assay

4.3.2

of compound 10

The minimal inhibitory concentration (MIC) values of disubstituted ta-

crine derivatives toward some human bacterial strains, (Gram[+], E.

faecalis ATCC 19433, E. faecalis ATCC 29212, S. aureus ATCC 25923, S.

aureus ATCC 29213, and S. aureus ATCC 46300; Gram[−], E. coli ATCC

25922, E. coli ATCC 35213, and P. eruginosa ATCC 27853], were ex-

amined with the help of a microwell dilution method. According to this

method, the inocula of bacteria were obtained using 12‐hr LB broth

cultures. The optical density at 600 nm (OD₆₀₀) was adjusted to

0.08–0.1 and 0.5 McFarland bacterial suspensions were obtained. Each

disubstituted tacrine derivative was dissolved in DMSO (20 mg/ml). A

concentration gradient range of 7.81–1,000 μg/ml in uncovered micro-

plate wells containing nutrient broth was made by using serial two‐fold

dilutions of these compounds. This plate was inoculated with bacteria

and incubated at 35°C for 24 hr. At the end of this period, the growth of

microorganisms was determined visually, and the point where no visible

growth was accepted as the MIC.

X‐ray structure coordinates for phospholipase C gamma 1 (PLCγ1) in

complex with fibroblast growth factor receptor 2 (FGFR2) were ob-

tained from Protein Data Bank (PDB ID: 5EG3).^[53]Molecular docking

studies were performed using AutoDock_Vina v1.1.2.^[66]Ligands and

the receptor were prepared for docking by MGLTools v1.5.4, and the

dock results were visualized by the same program.^[67]Gasteiger

partial atomic charges were assigned to the receptor as well as the

ligand. Rotatable bonds in all ligands were kept flexible, whereas

those of the receptor were kept rigid. Ligand and receptor co-

ordinates prepared by MGLTools were saved in PDBQT form. A

gridbox, with size 30 Å (X) × 25 Å (Y) × 28 Å (Z) and center co-

ordinates 15.209 (X) × −24.299 (Y) × 8.2133 (Z), was used to imple-

ment dock computations. Parameters exhaustiveness = 8 and

num_modes = 10 were used for AutoDock Vina computations.

CONFLICT OF INTERESTS

The authors declare that there are no conflicts of interests.

|

4.2.5

Enzyme inhibition studies

ORCID

Both hCA isoenzymes' inhibition effects of novel compounds were

measured according to the method of Verpoorte et al.,^[57]conforming

to previous studies,^[58,59]and recorded at 348 nm spectro-

photometrically using p‐nitrophenyl acetate substrate. On the con-

trary, the AChE inhibitory effect of novel compounds was

determined according to the procedure of Ellman et al.,^[60]con-

forming to previous studies,^[61,62]and recorded at 412 nm spectro-

photometrically using acetylthiocholine iodide as a substrate for the

enzymatic reaction. The 5,5′‐dithio‐bis(2‐nitrobenzoic) acid com-

pound was used for the measurement of the AChE activity.

Salih Ökten

http://orcid.org/0000-0001-9656-1803

http://orcid.org/0000-0002-9550-9111

Ali Aydın

Ümit M. Koçyiğit

Osman Çakmak

Sultan Erkan

http://orcid.org/0000-0001-8710-2912

http://orcid.org/0000-0001-9293-5572

http://orcid.org/0000-0001-6744-929X

http://orcid.org/0000-0002-4545-3500

http://orcid.org/0000-0002-3171-0633

http://orcid.org/0000-0001-5993-1668

Cenk A. Andac

Parham Taslimi

İlhami Gülçin

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SUPPORTING INFORMATION

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How to cite this article: Ökten S, Aydın A, Koçyiğit ÜM, et al.

Quinoline‐based promising anticancer and antibacterial

agents, and some metabolic enzyme inhibitors. Arch Pharm.

2020;e2000086. https://doi.org/10.1002/ardp.202000086

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Cancer 2019, 19, 747.

Article Doi

Quinoline-based promising anticancer and antibacterial agents, and some metabolic enzyme inhibitors

DOI: 10.1002/ardp.202000086

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