Toward chelerythrine optimization: Analogues designed by molecular simplification exhibit selective growth inhibition in non-small-cell lung cancer cells

Accepted Manuscript

Toward chelerythrine optimization: analogues designed by molecular simplifi-

cation exhibit selective growth inhibition in non-small-cell lung cancer cells

Rosania Yang, Maurício T. Tavares, Sarah F. Teixeira, Ricardo A. Azevedo,

Diego C. Pietro, Thais B. Fernandes, Adilson K. Ferreira, Gustavo H.G.

Trossini, José A.M. Barbuto, Roberto Parise-Filho

PII:

S0968-0896(16)30588-0

DOI:

http://dx.doi.org/10.1016/j.bmc.2016.07.065

BMC 13178

Reference:

Bioorganic & Medicinal Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

29 June 2016

26 July 2016

28 July 2016

Please cite this article as: Yang, R., Tavares, M.T., Teixeira, S.F., Azevedo, R.A., Pietro, D.C., Fernandes, T.B.,

Ferreira, A.K., Trossini, G.H.G., Barbuto, J.A.M., Parise-Filho, R., Toward chelerythrine optimization: analogues

designed by molecular simplification exhibit selective growth inhibition in non-small-cell lung cancer cells,

Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.07.065

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Graphical Abstract

Toward chelerythrine optimization: analogues designed by

molecular simplification exhibit selective growth inhibition in non-

small-cell lung cancer cells

Rosania Yang,^a†Maurício T. Tavares,^a†Sarah F. Teixeira,^b†Ricardo A. Azevedo,^b

Diego C. Pietro,^aThais B. Fernandes,^aAdilson K. Ferreira,^bGustavo H. G. Trossini,^d

José A. M. Barbuto,^b,cRoberto Parise-Filho^a*

^aLaboratory of Design and Synthesis of Bioactive Substances (LAPESSB), Department of Pharmacy, Faculty of Pharmaceutical

Science, University of São Paulo, Av. Prof. Lineu Prestes, 580, São Paulo, SP 5508-000, Brazil

^bLaboratory of Tumor Immunology, Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, Av.

Prof. Lineu Prestes, 1730, São Paulo, SP 05508-900, Brazil

^cCell and Molecular Therapy Center NUCEL/NETCEM, Faculty of Medicine, University of São Paulo, Rua Pangaré, São Paulo,

SP 05360-120, Brazil

^dLaboratory of Experimental and Computational Integrated Techniques (LITEC), Department of Pharmacy, Faculty of

Pharmaceutical Science, University of São Paulo, Av. Prof. Lineu Prestes, 580, São Paulo, SP 5508-000, Brazil

†

Rosania Yang, Maurício T. Tavares and Sarah F. Teixeira contributed equally to this work.

*Corresponding author: Roberto Parise-Filho, roberto.parise@usp.br

Abstract

A series of novel chelerythrine analogues was designed and synthesized. Antitumor

activity was evaluated against A549, NCI-H1299, NCI-H292, and NCI-H460 non-

small-cell lung cancer (NSCLC) cell lines in vitro. The selectivity of the most active

analogues and chelerythrine was also evaluated, and we compared their cytotoxicity

in NSCLC cells and non-tumorigenic cell lines, including human umbilical vein

endothelial cells (HUVECs) and LL24 human lung fibroblasts. In silico studies were

performed to establish structure-activity relationships between chelerythrine and the

analogues. The results showed that analogue compound 3f induced significant dose-

dependent G₀/G₁cell cycle arrest in A549 and NCI-H1299 cells. Theoretical studies

indicated that the molecular arrangement and electron characteristics of compound

3f were closely related to the profile of chelerythrine, supporting its activity. The

present study presents a new and simplified chelerythrinoid scaffold with enhanced

selectivity against NSCLC tumor cells for further optimization.

Keywords

Chelerythrine, NSCLC, molecular simplification, chelerythrine analogues

Abbreviations

SCLC, small cell lung cancer; NSCLC, non-small-cell lung cancer; LNCaP, human

prostate adenocarcinoma; DU-145, human prostate carcinoma; MDA-MB-231,

human breast adenocarcinoma; BGC-823, human gastric cancer; SI, selectivity

index; SAR, structure-activity relationship; A549, human lung adenocarcinoma; NCI-

H1299, human lung carcinoma; NCI-H292, mucoepidermoid pulmonary carcinoma;

NCI-H460, non-small cell lung carcinoma; LL24, fibroblast lung cells; HUVEC,

human umbilical vein endothelial cell; TLC, thin-layer chromatography; DMSO-d₆,

deuterated dimethyl sulfoxide; CHN, elemental analysis; TFA, trifluoroacetic acid;

THF, tetrahydrofurane; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide; FBS, fetal bovine serum; RPMI, Roswell Park Memorial Institute; PBS,

phosphate-buffered saline; DMSO, dimethylsulfoxide; 3D, three-dimensional;

MMFF94, Merck molecular force field; PM6, parametrization method 6; HF/6-31G*,

Hartree-Fock 6-31G*; EPM, electrostatic potential map; DCM, dichloromethane;

HPLC, high-performance liquid chromatography; PANC-1, human pancreatic cancer;

HCC1937, human breast cancer.

1. Introduction

Cancer is a group of diseases that are characterized by uncontrolled cellular

growth [1]. Despite enormous efforts in the past decades to develop new strategies

for diagnosis and treatment, cancer is still a leading cause of death worldwide. This

group of diseases is responsible for approximately 8.2 million deaths annually. The

last census revealed that lung cancer was responsible for 1.59 million deaths,

making it one of the most frequent and fatal malignancies worldwide [2].

Lung cancer is divided into two major histological types: small-cell lung cancer

(SCLC) and non-small-cell lung cancer (NSCLC, i.e. adenocarcinoma, squamous

cell carcinoma, and large-cell carcinoma) [3]. Approximately 85-90% of lung cancer

cases are NSCLC [4]. Many affected patients do not respond to conventional

treatment [5]. The most common regimen for NSCLC treatment is based on a

combination of platinum compounds and taxanes [6,7]. However, these drugs are

associated with severe adverse effects, low selectivity, high toxicity, as well as

inappropriate selection of patient to the combinatorial therapeutic modality [8]. There

is a need to develop new compounds that can produce tumor remission and

circumvent anticancer drug resistance and that have fewer adverse effects [9].

Natural sources may assist in the search for more selective and potent

bioactive structures [10]. Natural products have been the mainstay of cancer

chemotherapy for the past 40 years. Such substances are likely to provide many

lead structures that can be used as prototypes for the construction of novel

compounds with enhanced biological properties [11]. Chelerythrine (Fig. 1) is a

natural quartenary benzophenanthridine alkaloid that has been shown to be an

interesting prototype for anticancer drug design. This alkaloid is commonly isolated

from Chelidonium majus L. and several other species of the Papaveraceae family

[12,13]. In addition to its antimicrobial [14-17], antiinflammatory [18], and

antialzheimer [19] properties, chelerythrine has potential as an anticancer compound

because of its cytotoxicity against several tumor cell lines, including human prostate

carcinoma, human breast adenocarcinoma, and human gastric cancer [20-23].

Figure 1. Chemical structure of chelerythrine.

Some authors have associated the antitumor activity of chelerythrine to its

ability to inhibit Bcl-X_L, an antiapoptotic protein of the Bcl-2 family [23-26]. Moreover,

some reports suggest that chelerythrine may act through protein kinase C inhibition

[27] and DNA intercalation [28]. Although it has been shown to have remarkable

anticancer properties, previous studies have shown that chelerythrine lacks

selectivity and has limitations on administration since it must be handled

intravenously [29]. However, chelerythrine still has an interesting scaffold that may

inspire the design of optimized structures with improved selectivity and

biopharmaceutical properties.

The molecular basis of the anticancer activity of chelerythrine was studied by

Bernardo and co-workers. These authors determined that a benzodioxole system

(Fig. 1, rings A and B) plays a role as an essential scaffold for the cytotoxic activity of

chelerythrine. Furthermore, the replacement of methoxyl groups (bonded to ring E in

Fig. 1) with hydroxyls increased the derivative’s activity [26]. Curiously, the

benzodioxole moiety can be found in many compounds with anticancer activity, such

as podophyllotoxin and its derivatives [30], dillapiole [31], and other synthetic

compounds that were previously explored by our group [32-35].

Therefore, we concentrated our efforts on optimizing the chelerythrine scaffold

in an attempt to identify new molecular entities with enhanced selectivity. As shown

in Fig. 2, we began by retaining the benzodioxole system that is found in

chelerythrine. Rings C and D and the formal charge of the nitrogen were removed to

investigate the influence of these modifications on selectivity and cytotoxicity.

Aromatic ring E (Fig. 2) was retained, but we introduced variations in its substituents.

The influence of methoxyl groups on the biologic profile conflicted with the hydroxyl,

in which natural products with a methoxyl/hydroxyl substitution pattern commonly

present interesting anticancer properties. Some examples that illustrate the influence

of these groups on cytotoxic activity include vanillin, curcumin, and silybin (all 3-

methoxy-4-hydroxylated), resveratrol and gossypol (3,4-dihydroxylated products),

and 3,4,5-trimethoxylated molecules (e.g., combrestatin) [36-43]. Thus, the

molecular diversity that can be incorporated over the chelerythrine scaffold may

allow the identification of more active and selective derivatives and provide insights

into the structure-activity relationship (SAR) of the proposed chemical library.

Figure 2. Proposed modifications of chelerythrine for the design of new selective cytotoxic

derivatives.

We also evaluated the ability of the newly derived compounds to promote

selective cytotoxic activity against various NSCLC cell lines (i.e., A549, NCI-H1299,

NCI-H292, and NCI-H460) compared with non-tumorigenic cell lines (i.e., LL24 and

human umbilical vein endothelial cells [HUVECs]). We also investigated the

mechanism of action of the most active compounds.

2. Results and Discussion

2.1. Synthesis

Secondary amines 3a-f and tertiary amines 5a-d were synthesized as shown in

Scheme 1. 3,4-Dihydroxybenzaldehyde was reacted with tert-butyl dimethylsilane

chloride in the presence of triethylamine to generate 1g. Aldehydes 1a-e and 1g

were condensed with piperonylamine, followed by reduction with NaBH(OAc)₃in a

one-pot fashion to afford the secondary amines 2a-e and 2g as free base. Because

the secondary amines were treated with ethereal HCl, they were given as amine

hydrochloride salts (3a-g). Compound 3g was reacted with TBAF for deprotection of

the hydroxyl groups affording 2f, which was then treated with ethereal HCl to afford

3f.

Scheme 1. Reagents and conditions: (a) tert-butyl dimethylsilane chloride (2.3 eq.), triethylamine (2

eq.), THF (4 mL), DCM (4 mL), r.t., 4 h; (b) piperonylamine (1.1 eq.), NaBH(OAc)₃(1.4 eq.), DCM (4

mL), r.t., N₂, 3-5 h; (c) ethereal HCl (6.8 M, 0.5 mL); (d) formaldehyde (8 mL), formic acid (8 mL),

110ºC, 2-4 h; (e) TBAF (2.2 eq.), THF (8 mL), 0ºC to 25ºC, 45 min.

An Eischeweiler-Clarke reaction from secondary amines 3a-d was performed

with formaldehyde under reflux and acidic conditions that were afforded by formic

acid, which was also the reductive agent. Tertiary amines 4a-d (free base) were

obtained from this reaction, and they were treated with ethereal HCl to afford the

amine hydrochloride salts (5a-d). The reactions gave target compounds 3a-f and 5a-

d, with yields in the range of 22-59%.

2.2. Cytotoxicity assay

Six secondary amines (3a-f), four tertiary amines (5a-d), and chelerythrine

were screened against the following NSCLC human cell lines: adenocarcinoma

(A549), non-small cell carcinoma (NCI-H1299 and NCI-H460), and mucoepidermoid

pulmonary carcinoma (NCI-H292). Non-tumorigenic HUVECs and LL24 human lung

fibroblast cells were used in the screening panel. The cytotoxicity of the compounds

was evaluated using the MTT assay [44]. Among these compounds, only

compounds 3b, 3c, and 3f had IC₅₀values < 200 µM in A549. The analogues 3a-3d

and 5a-5b had IC₅₀values < 200 µM in H1299, and only compound 3b had an IC₅₀

value < 200 µM in H292. Chelerythrine had IC₅₀values in the 3-8 µM range in all cell

lines in the panel (Table 1).

Table 1. In vitro inhibitory effects of chelerythrine and compounds 3a-f and 5a-d on the cellular

viability of A549, NCI-H1299, NCI-H292, NCI-H460, HUVEC, and LL24 cell lines.

IC₅₀(µM) ± SD^a

Compound

A549

7.73 ± 0.58

> 200

NCI-H1299

4.28 ± 2.15

67.19 ± 18.41

73.71 ± 13.79

92.31 ± 32.57

106.55 ± 16.87

> 200

NCI-H292

4.96 ± 1.60

> 200

NCI-H460

5.14 ± 0.66

> 200

HUVEC

4.93 ± 1.73

> 200

LL24

3.73 ± 0.69

> 200

Chelerythrine

3a

3b

3c

3d

3e

3f

123.86 ± 32.20

118.31 ± 17.90

> 200

152.60 ± 17.92

> 200

112.19 ± 23.18

> 200

5a

5b

5c

5d

91.18 ± 21.85

76.42 ± 10.10

> 200

^aThe values are mean ± standard deviation. Tumor cell lines: adenocarcinoma (A549), non-small cell

carcinoma (NCI-H1299 and NCI-H460), and mucoepidermoid pulmonary carcinoma (NCI-H292). Non-

tumorigenic cells: HUVEC and LL24 human lung fibroblast cells.

All simplified analogues showed lower cytotoxicity than chelerythrine against

non-tumorigenic cell lines HUVEC and LL24. Moreover, selectivity index (SI), which

is defined as the relationship between the cytotoxic concentrations (IC₅₀) in

cancerous cell line and non-tumorigenic cell line, showed that simplified analogues

were more selective to tumor cells (A549, NCI-H1299 and NCI-H292) than

chelerythrine (table 2) [45–47]. As can be seen, the prototype presented SI lower

than 1 for most cell lines while its analogues showed SI average close to 2.

Noteworthy, SI could not be calculated to compounds that were not cytotoxic against

both, tumor and non-tumorigenic cells. Therefore, our results indicate that the

simplified compounds are safer than the prototype.

Table 2. Selectivity index for chelerythrine and compounds 3a-f and 5a-d for non-tumorigenic cell

lines such as HUVEC and LL24.

Selectivity Index (SI)^a

Compound

HUVEC

LL24

A549 NCI-H1299 NCI-H292 NCI-H460 A549 NCI-H1299 NCI-H292 NCI-H460

Chelerythrine 0,64

1,15

> 2,98

> 2,71

> 2,17

> 1,88

-

0,99

0,96

0,48

0,87

> 2,98

> 2,71

> 2,17

> 1,88

-

0,75

0,73

3a

3b

3c

3d

3e

3f

-

> 1,61

> 1,31

> 1,61

> 1,31

> 1,69

-

> 1,69

-

> 1,78

-

> 1,78

-

5a

5b

5c

5d

-

> 2,19

> 2,62

-

> 2,19

> 2,62

-

^aThe selectivity index is the ratio of the IC₅₀values of the treatments on HUVEC and LL24 cells to

those in the cancer cell lines. Tumor cell lines: adenocarcinoma (A549), non-small cell carcinoma

(NCI-H1299 and NCI-H460), and mucoepidermoid pulmonary carcinoma (NCI-H292). Non-

tumorigenic cells: HUVEC and LL24 human lung fibroblast cells.

From this preliminary screening, the molecular simplification of chelerythrine

resulted in compounds with less activity than the alkaloid. Interestingly, HUVEC and

LL24 cells, which are non-tumorigenic, were as sensitive to chelerythrine as most of

the NSCLC cell lines, with the exception of A549, which was less sensitive to

chelerythrine compared with the LL24 cell line (p ≤ 0.05, paired Student’s t-test). This

indicates that although chelerythrine has antitumor activity, it lacks selectivity for

tumor cell lines over non-tumorigenic cell lines [20].

Generally, secondary amines were more active against tumor cells than tertiary

amines. Secondary amines have a hydrogen bond acceptor and a hydrogen bond

donor, which is not observed in tertiary amines and might explain their lower activity.

Furthermore, for the synthesized compounds, a hydroxyl or methoxyl group at

positions R₂and R₃(e.g., methoxyl group at R₃with hydrogen at R₂; Scheme 1)

appeared to be preferred over hydrogen to produce cytotoxicity in A549 cells. The

most active compounds were 3b, 3c, and 3f (Scheme 1) in this cell line. A methoxyl

group at position R₄was found to not influence the compounds’ activity (compound

3d and 5d, Scheme 1).

NCI-H1299 was more sensitive to amine compounds than the other cell lines in

the panel. NCI-H1299 is known as p53-null, and the loss of p53 protein function has

been associated with enhanced cellular resistance to chemotherapeutic agents

[48,49]. The most active analogue was 3a (which has no substitution at positions R₁

to R₄), followed by analogue 3b (which has a methoxyl group at R₃) and compound

5b. This pattern demonstrated that the addition of methoxyl or hydroxyl groups at

positions R₁to R₄, such as a substitution on the nitrogen of the secondary amine to

form a tertiary amine, decreases the cytotoxicity of the analogues.

Although the compounds did not present higher cytotoxicity than chelerythrine,

their cytostatic activity should be considered in analyses of antitumor activity.

Despite most research groups emphasize the design of cytotoxic drugs, cytostatic

agents are important tools in cancer treatment [47-49]. Moreover, cytotoxic drugs are

more related to tumor resistance and adverse effects, which makes combinations

with cytostatic compounds an interesting strategy to reduce the dose of the cytotoxic

agent and thus lower its toxicity [53]. Furthermore, cytostatic drugs such as 5-

fluorouracil (5-FU) and gefitinib slow the emergence of cytotoxic drug resistance by

decreasing proliferation rates, preventing the rise of resistant cells during treatment,

and slowing tumor growth between chemotherapy cycles [54-59]. Altogether, those

facts demonstrate the relevance on combination of cytostatic and cytotoxic agents,

which may represent a more efficient and synergic therapeutic approach than the

use of single agents [52,56].

In these context, gefitinib is an epidermal growth factor receptor inhibitor that is

used for cancer treatment [54,60]. Gefitinib enhances the sensitivity of glioma cell

lines to radiotherapy and the cytotoxic agent temozolomide, showing the importance

and advantages of using cytostatic compounds [61–63].

In the present study, a clonogenic assay was performed to investigate the

cytostatic potential of chelerythrine analogues. A549 is a multidrug-resistant cell line

[64–66], and NCI-H1299 was shown to be more sensitive to the analogues.

Therefore, these cell lines were chosen for the clonogenic assay.

2.3. Clonogenic assay of the effects of the analogues on A549 and NCI-H1299 cell

lines

To provide further insights into the activity of the analogues against A549 and

NCI-H1299 cell lines, all of the compounds (3a-f and 5a-d) were evaluated in the

clonogenic assay with regard to their effects on A549 and NCI-H1299 cell growth.

Colony formation can mimic tumor cell development based on the ability of a single

cell to grow into a colony, thus providing physiological insights into tumor cell growth

[67]. In this context, 3f was the only chelerythrine analogue that presented a

cytostatic effect on clonogenic assay screening. Thus, the short-term inhibitory effect

of compound 3f on exponentially growing A549 and NCI-H1299 cancer cells was

measured after treatment with concentrations of 50, 75, and 100 µM for 24 h.

After 10 days, the A549 control group presented an average of 60 colonies.

A549 cells that were treated with compound 3f presented an average of 9, 19, and

34 colonies at concentrations of 100, 75, and 50 µM, respectively. The NCI-H1299

control group presented an average of 50 colonies. Treatment with compound 3f

resulted in an average of 11, 17, and 28 colonies at concentrations of 100, 75, and

50 µM, respectively. Surprisingly, compound 3f exhibited interesting cytostatic

activity. The colony-forming cells were concentration-dependently reduced by

compound 3f (Fig. 3), and the effect was more potent in A549 cells, in agreement

with the IC₅₀of 3f in these cell lines (Table 1).

Figure 3. (A, B) Images of the concentration-dependent effects of compound 3f on the formation of

A549 and NCI-H1299 tumor cell colonies, respectively. (C, D) Average of three different experiments.

*p ≤ 0.05, **p ≤ 0.01, compared with control group (paired Student’s t-test).

2.4. Effects of compound 3f on A549 and NCI-H1299 cell cycles

To determine the possible inhibitory mechanism of action of compound 3f on

tumor cell proliferation, a cell cycle distribution assay was performed, in which we

detected cellular DNA content by flow cytometry. The effects of 50, 75, and 100 µM

concentrations of compound 3f on cell cycle progression were examined after 24 h in

A549 and NCI-H1299 cells (Fig. 4).

Figure 4. (A, C) Effect of compound 3f on A549 and NCI-H1299 cell cycles, respectively. The red

histogram represents the control group. The blue histogram represents compound 3f at 100 µM. The

orange histogram represents compound 3f at 75 µM. The green histogram represents compound 3f at

50 µM. Phases G₀/G₁, S, and G₂/M are indicated. (B, D) Percent cell population distribution in each

phase of the cell cycle.

Compound 3f induced significant and concentration-dependent G₀/G₁cell cycle

arrest in A549 cells, with increases of 54.3%, 49.6%, and 45.9% at concentrations of

100, 75, and 50 µM, respectively. The control group presented 33.4% of the cell

population in the G₀/G₁phase. These effects occurred concomitantly with a decrease

in the percentage of cells in the other phases of the cell cycle (S and G₂/M).

The observed effect of compound 3f on the NCI-H1299 cell cycle was an

increase in the percentage of cells in the G₀/G₁phase, from 39.0% in the control

group to 55.8%, 65.0%, and 53.5% for concentrations of 100, 75, and 50 µM,

respectively. The percentage of the cell population in the S phase also increased,

from 7.8% in the control group to 17.7%, 20.4%, and 26.8% for concentrations of

100, 75, and 50 µM, respectively.

G₀/G₁cell cycle arrest may indicate that the cells are quiescent, a characteristic

phenotype of cells that do not proliferate [68], which was revealed in the clonogenic

assay of compound 3f in A549 and NCI-H1299 cells. The ability to promote G₀/G₁

cell cycle arrest was also observed in studies of the effects of gefitinib in the NSCLC

A549 cell line [69], PANC-1 human pancreatic cancer cells [70], and HCC1937

human breast cancer cells [71]. Gefitinib has been described as a cytostatic drug

[72], and G₀/G₁cell cycle arrest indicates the relationship between this phenomenon

and the potential cytostatic activity of a compound.

2.5. In silico SAR studies

The energy-minimized structures of chelerythrine and 3f were superimposed in

Fig. 5A, thus providing insights into SARs. Compound 3f presented a root mean

square deviation (RMSD) of 1.8 Å with the chelerythrine arrangement, suggesting

that the designed scaffold of compound 3f maintained a molecular arrangement that

was enclosed within the chelerythrinoid moiety. Thus, we compared the disposition

of the related portions of 3f and chelerythrine. Interestingly, the interatomic distance

between the methylenedioxy group and quaternary nitrogen of chelerythrine (6.61 Å,

Fig. 5B) was very similar to compound 3f (6.66 Å, Fig. 5C). The same correlation

was observed between the nitrogen and arylcarbon bound to the most distant

substituent of the structure, in which the interatomic distances for chelerythrine and

compound 3f were 4.77 and 4.97 Å, respectively (Fig. 5B, C). Steric and electronic

complementarity are known to be critical factors for drug-receptor interactions and

selectivity [73]. Preservation of the interatomic distance between the benzodioxole

moiety, nitrogen linker, and 3,4-dihydroxyaryl moiety of compound 3f appeared to

guarantee molecular recognition of the ligand by macromolecules.

As shown in Fig. 5A, compound 3f has several routable bonds, which is not

observed within the rigid pentacyclic system of chelerythrine. The dihedral angles of

both structures were calculated (Fig. 5D, E), and these angles (96.5° for

chelerythrine and 164.1° for compound 3f) may explain why the conformation of

compound 3f was intimately related to chelerythrine. The routable bonds of the

ligand allowed an arrangement of the structure (during optimization), including the

benzodioxole moiety, nitrogen linker, and 3,4-dihydroxyaryl moiety, that favored

orientations for interactions. Notably, the routable characteristic of the methylene

(arrow in Fig. 5E) allowed a better arrangement of the meta-hydroxyl group. Thus,

compound 3f could assume an even more chelerythrine-like conformation.

Figure 5. (A) Alignment of chelerythrine (carbon atoms in purple, oxygen in red, nitrogen in blue) and

compound 3f (carbon atoms in yellow, oxygen in red, nitrogen in blue). (B) Interatomic distance for

chelerythrine. Distances were measured between the methylene group (of benzodioxole) and

quaternary nitrogen and between the quaternary nitrogen and arylcarbon bound to the most distant

substituent of the structure. (C) Interatomic distance for compound 3f. Distances were measured

between the methylene group (of benzodioxole) and secondary nitrogen and between the secondary

nitrogen and arylcarbon bound to the most distant substituent of the structure. (D) Dihedral angles for

chelerythrine. (E) Dihedral angles for compound 3f. The black arrow indicates a routable methylene

that allows an opposite orientation of the meta-hydroxyl group that is more similar to the orientation

for chelerythrine.

Benzophenanthridine alkaloid has been reported about its ability of DNA

intercalation, since chelerythrine bonds between DNA base pairs and inside the

minor groove [74]. This mechanism has been associated to its antitumoral activity,

and in this context, conformational constraint and planarity may be important

characteristics for this property [75]. Therefore, less rigid compound, such as 3f,

could affect its ability to intercalate DNA and thus, might explain lower cytotoxic

activity than chelerythrine.

The electron studies of the chelerythrine and 3f scaffolds revealed similar

electron behavior. As shown in Fig. 6A, the dipole moment vector (µ) of both

chelerythrine and compound 3f had a benzodioxole orientation but distinct intensity.

The obtained value for chelerythrine was µ = 4.82 Debye, and the obtained value for

compound 3f was µ = 6.16 Debye. Subtle differences in the dipole moment vector

can affect interactions between micro- and macromolecules. Additionally, alterations

on dipole vector of ligands can enhance their selectivity index for a protein isoform

compared to other ones [76]. Additionally, the electrostatic potential maps (EPMs)

that were calculated for chelerythrine and compound 3f (Fig. 6B, C) showed similar

dispositions of the electrostatic charges of both molecules. The poor electron

characteristic of the nitrogen in compound 3f could be related to its molecular

recognition process. Previous studies reported that the formal charge of the nitrogen

of chelerythrine favors its anticancer activity [20,77]. Notably, the electron studies of

the 3f scaffold considered its protonated form. This procedure was performed

because of the pka of the moiety (pka = 8.28). This pka value indicates that the

ionized species of 3f could be the predominant species under physiological

conditions.

Figure 6. Electron findings for chelerythrine (left) and compound 3f (right). Carbon atoms are

displayed in dark gray. Oxygen is displayed in red. Nitrogen is displayed in blue. Hydrogen is

displayed in white. (A) Minimized conformations of chelerythrine and compound 3f. The total dipole

moment vectors (µ) of chelerythrine and compound 3f are shown as yellow arrows and presented µ =

4.82 Debye and µ = 6.16 Debye, respectively. The molecules are presented as ball-and-wire models.

(B) Electrostatic potential maps of chelerythrine and compound 3f are shown according to color

(atomic charge color ramp: +60 kJ/mol [red] to +580 kJ/mol [blue]) as solid surfaces. (C) Electrostatic

potential maps of chelerythrine and compound 3f shown in mesh surfaces. The molecules are

presented as ball-wire models.

3. Conclusions

Novel chelerythrine secondary and tertiary amine derivatives were designed

and synthesized. These compounds were evaluated for their inhibitory activity and

selectivity against NSCLC A549, NCI-H1299, NCI-H292, and NCI-H460 cell lines.

Compound 3f presented activity against A549 and NCI-H1299 cells. Further studies

demonstrated that compound 3f had concentration-dependent activity against tumor

cell colony formation by increasing G₀/G₁cell cycle arrest in both cell lines, indicating

its potential to inhibit cell growth. These results, together with the low toxicity of

compound 3f in LL24 human lung fibroblasts and HUVECs, indicate a preferential

activity of compound 3f toward A549 and H1299 tumor cell lines. In silico SAR

studies suggested that the similar enclosed structural arrangements and electron

characteristics of compound 3f and chelerythrine could be related to the molecular

recognition process of the compounds and their cytostatic activity. These findings

provide new insights into the SARs of chelerythrine and compound 3f, suggesting

that compound 3f has good potential for the further development of a new class of

amine derivatives of chelerythrine with promising antitumor potential.

4. Experiments

4.1. Chemistry

Reagents and solvents were obtained from commercial sources, and some of

them were used without further purification. The progression of the reactions was

monitored by thin-layer chromatography (TLC) on precoated aluminum sheets (SiGel

60 F254, Merck) by ultraviolet and/or staining solution revelation. Chromatographic

purification was performed on silica gel columns (SiGel 60, 220-440 mesh, Sigma-

Aldrich). Proton and carbon nuclear magnetic resonance (¹H NMR and ¹⁵C NMR)

spectra were recorded on a Bruker Advanced DPX-300 instrument (300 MHz and 75

MHz, respectively) in CDCl₃or DMSO-d₆solution. Chemical shifts are given in parts

per million (ppm), with tetramethylsilane or DMSO-d₆as an internal standard. The

following abbreviations are used: s = singlet, d = doublet, t = triplet, m = multiplet, dd

= doublet of doublets. Coupling constants (J values) are given in Hertz (Hz). The

detailed spectra of each compound could be found in Supplementary Material.

Elemental analyses of 3a-f and 5a-d were performed on a Perkin Elmer 2400 CHN

instrument and were within ±0.5% of the theoretical values unless otherwise noted.

HPLC was performed on a Shimadzu Proeminence HPLC system using a C18

column (5µm, 1504.6 mm, Gemini) and gradient elution (0.1% CH₃OH/TFA), with

ultraviolet detection set at 230 nm to verify the purity of compounds 3a-f and 5a-d. A

purity of >95% was established for all of the compounds.

4.1.1. Procedure A for the synthesis of compound 1g

To a mixture of 3,4-dihydroxybenzaldehyde (414 mg, 3 mmol) and triethylamine

(834.7 µL, 6 mmol) in tetrahydrofurane (4 mL) was added a mixture of tert-butyl

dimethylsilane chloride (1.057 g, 7 mmol) and dichloromethane (4 mL). The mixture

was stirred at room temperature under a N₂atmosphere until the consumption of

aldehyde was observed. Saturated aqueous NaHCO₃was added, and the mixture

was extracted with CHCl₃. The organic layer was washed with brine and dried over

with Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by

column chromatography (CHCl₃/hexane). The eluent proportion varied according to

the synthesized compound.

4.1.2. Procedure B for the synthesis of secondary amines (2a-g)

To a solution of piperonylamine (332.2 mg, 2.2 mmol) in CH₂Cl₂(4 mL) was

added a mixture of aldehyde (2 mmol), acetic acid (146 µL, 1.15 mmol), and CH₂Cl₂

(4 mL) at room temperature. The mixture was stirred at the same temperature under

a N₂atmosphere; after 10 min, NaBH(OAc)₃(593.6 mg, 2.8 mmol) was added to the

mixture. The reaction mixture was stirred under the same conditions until the

consumption of aldehyde was observed. Saturated aqueous NaHCO₃was added,

and the mixture was extracted with CHCl₃. The organic layer was washed with brine

and dried over with Na₂SO₄, filtered, and concentrated in vacuo. The residue was

purified by column chromatography (EtOAc/hexane) to give the secondary amines.

4.1.3. Procedure C for the synthesis of compounds 3a-g and 5a-d

To a solution of the resulting secondary amines (from procedure B or E) or

tertiary amines (from procedure D) in ether (4 mL) was added 0.5 mL of saturated

ethereal HCl (6.8 M) to give the amine hydrochloride salts as solids.

4.1.4. Procedure D for the synthesis of tertiary amines (4a-d)

To a solution of 3a-d (1.5 mmol) in formic acid (8 mL) was dropwise added

formaldehyde (8 mL). The mixture was refluxed until the consumption of 3a-d was

observed. A solution of 40% NaOH was added to neutralize the formic acid, and the

mixture was extracted with EtOAc. The organic layer was washed with brine, dried

over with Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by

column chromatography (EtOAc/hexane) to give the tertiary amines. Saturated

ethereal HCl was used to give the amine hydrochloride salts as white solids.

4.1.5. Procedure E for the synthesis of compound (2H-1,3-benzodioxol-5-

ylmethyl)[(3,4-dihydroxyphenyl)methyl]azanium chloride (3f)

To a suspension of 3g (215.31 mg, 0.4 mmol) in tetrahydrofurane (4 mL) was

added a mixture of tetrabutylammonium fluoride (230.01 mg, 0.88 mmol) with 4 mL

of THF. The reaction was stirred at 0ºC for 5 min and at room temperature for 40

min. The solvent was removed under reduced pressure, and the mixture was

extracted with EtOAc after the addition of saturated aqueous NaHCO₃. The organic

layer was dried over with Na₂SO₄, filtered, and concentrated in vacuo. The residue

was purified as described for procedure C to give the secondary amine hydrochloride

salt 3f.

3,4-bis[(tert-butyldimethylsilyl)oxy]benzaldehyde (1g)

1

White solid; yield: 40%; H NMR (300 MHz, DMSO-d₆) δ: 9.81 (1H, s, H-1),

7.37 (1H, d, J = 2.0 Hz, H-3), 7.35 (1H, d, J = 1.6 Hz, H-7), 6.94 (1H, d, J = 8.7 Hz,

H-4), 1.00 (18H, s, H-11-13,17-19), 0.24 (12H, d, J = 5.8 Hz, H-8,9,14,15).

(2H-1,3-benzodioxol-5-ylmethyl)(benzyl)azanium chloride (3a)

1

White solid; yield: 58%; H NMR (300 MHz, DMSO-d₆) δ: 9.60 (2H, s, H-

16,16’), 7.56-7.53 (2H, m, H-12,14), 7.46-7.40 (3H, m, H-11,13,15), 7.18 (1H, s, H-

6), 7.00 (1H, dd, J¹= 8.0 Hz, J²= 1.2 Hz, H-4), 6.95 (1H, d, J = 7.9 Hz, H-3), 6.05

13

(2H, s, H-7), 4.09 (2H, s, H-9), 4.05 (2H, s, H-8); C NMR (75 MHz, DMSO-d₆) δ:

152.9 (C1), 152.5 (C2), 137.2 (C10), 135.3 (C11, C15), 134.0 (C5), 133.7 (C12,

C14), 130.5 (C13), 129.5 (C4), 115.7 (C6), 113.4 (C3), 106.5 (C7), 54.7 (C8), 54.6

(C9); Anal. Calcd for C₁₅H₁₆ClNO₂: C, 64.87; H, 5.81; N, 5.04. Found: C, 64.43; H,

5.61; N, 5.09. HPLC: t = 11.9 min, purity >95%.

(2H-1,3-benzodioxol-5-ylmethyl)[(4-methoxyphenyl)methyl]azanium chloride (3b)

1

White solid; yield: 55%; H NMR (300 MHz, DMSO-d₆) δ: 9.68 (2H, s, H-

16,16’), 7.48 (2H, d, J = 8.6 Hz, H-11,15), 7.21 (1H, s, H-6), 7.00 (1H, dd, J¹= 8.0

Hz, J²= 1.3 Hz, H-3), 6.97 (1H, s, H-4), 6.93 (2H, d, J = 7.8 Hz, H-12,14), 6.04 (2H,

s, H-7), 4.00 (4H, t, J = 5.7 Hz, H-8,9), 3.77 (3H, s, H-17); ¹³C NMR (75 MHz,

DMSO-d₆) δ: 159.6 (C13), 147.6 (C1), 147.2 (C2), 131.7 (C11, C15), 125.3 (C5),

124.2 (C10), 123.7 (C4), 113.9 (C12, C14), 110.4 (C6), 108.2 (C3), 101.2 (C7), 55.2

(C17), 49.2 (C8), 48.9 (C9); Anal. Calcd for C₁₆H₁₈ClNO₃: C, 62.44; H, 5.89; N, 4.55.

Found: C, 62.44; H, 5.80; N, 4.69. HPLC: t = 6.1 min, purity >98%.

(2H-1,3-benzodioxol-5-ylmethyl)[(3,4-dimethoxyphenyl)methyl]azanium chloride (3c)

1

White solid; yield: 59%; H NMR (300 MHz, DMSO-d₆) δ: 9.60 (2H, s, H-

16,16’), 7.28 (1H, s, H-15), 7.20 (1H, s, H-6), 7.04-6.93 (4H, m, H-3,4,11,12), 6.04

(2H, s, H-7), 4.00 (4H, s, H-8,9), 3.77 (3H, s, H-18), 3.76 (3H, s, H-17); ¹³C NMR (75

MHz, DMSO-d₆) δ: 149.1 (C14), 148.6 (C1), 147.6 (C13), 147.2 (C2), 125.3 (C10),

124.2 (C5), 124.0 (C11), 122.7 (C4), 113.9 (C15), 111.5 (C6), 110.4 (C12), 108.1

(C3), 101.2 (C7), 55.6 (C18), 55.5 (C17), 49.2 (C8), 49.2 (C9); Anal. Calcd for

C₁₇H₂₀ClNO₄: C, 60.44; H, 5.97; N, 4.15. Found: C, 60.36; H, 6.05; N, 4.36. HPLC: t

= 7.7 min, purity >99%.

(2H-1,3-benzodioxol-5-ylmethyl)[(3,4,5-trimethoxyphenyl)methyl]azanium

chloride

(3d)

1

White solid; yield: 54%; H NMR (300 MHz, DMSO-d₆) δ: 9.66 (2H, s, H-

16,16’), 7.20 (1H, s, H-6), 7.01 (1H, d, J = 8.0 Hz, H-4), 6.96 (1H, s, H-3), 6.94 (2H,

s, H-11,15), 6.04 (2H, s, H-7), 4.03-4.01 (4H, m, H-8,9), 3.79 (6H, s, H-18,19), 3.66

(3H, s, H-17); ¹³C NMR (75 MHz, DMSO-d₆) δ: 152.7 (C12, C14), 147.6 (C1), 147.3

(C2), 137.6 (C13), 127.3 (C10), 125.2 (C5), 124.3 (C4), 110.4 (C6), 108.2 (C3),

107.6 (C11, C15), 101.2 (C7), 60.0 (C17), 56.0 (C18, C19), 49.6 (C9), 49.4 (C8);

Anal. Calcd for C₁₈H₂₃ClNO₅: C, 58.78; H, 6.03; N, 3.81. Found: C, 58.28; H, 5.91; N,

3.74. HPLC: t = 8.0 min, purity >99%.

(2H-1,3-benzodioxol-5-ylmethyl)[(4-hydroxy-3-methoxyphenyl)methyl]azanium

chloride (3e)

1

White solid; yield: 45%; H NMR (300 MHz, DMSO-d₆) δ: 9.50 (2H, s, H-

16,16’), 9.22 (1H, s, H-17), 7.21 (1H, d, J = 1.4 Hz, H-6), 7.18 (1H, d, J = 1.4 Hz, H-

15), 7.00 (1H, dd, J¹= 8.0 Hz, J²= 1.3 Hz, H-3), 6.94 (1H, d, J = 7.9 Hz, H-4), 6.88

(1H, dd, J¹= 8.0 Hz, J²= 1.3 Hz, H-15), 6.79 (1H, d, J = 8.0 Hz, H-11), 6.04 (2H, s,

13

H-7), 3.98 (4H, t, J = 5.5 Hz, H-8,9), 3.78 (3H, s, H-18); C NMR (75 MHz, DMSO-

d₆) δ: 147.6 (C1), 147.4 (C14), 147.2 (C2), 147.1 (C13), 125.4 (C10), 124.2 (C5),

122.9 (C11), 122.3 (C4), 115.2 (C12), 114.4 (C15), 110.4 (C6), 108.2 (C3), 101.2

(C7), 55.7 (C18), 49.4 (C9), 49.1 (C8); Anal. Calcd for C₁₆H₁₈ClNO₄: C, 59.35; H,

5.60; N, 4.33. Found: C, 59.05; H, 5.58; N, 4.43. HPLC: t = 8.1 min, purity >98%.

(2H-1,3-benzodioxol-5-ylmethyl)[(3,4-dihydroxyphenyl)methyl]azanium chloride (3f)

1

Yellow solid; yield: 22%; H NMR (300 MHz, DMSO-d₆) δ: 9.28 (2H, s, H-

16,16’), 9.12 (2H, s, H-17,18), 7.13 (1H, s, H-6), 6.98 (1H, d, J = 8.0 Hz, H-4), 6.93

(1H, d, J = 8.0 Hz, H-3), 6.89 (1H, s, H-15), 6.76 (2H, s, H-11,12), 6.03 (2H, s, H-7),

3.99 (2H, t, J = 6 Hz, H-8), 3.89 (2H, t, J = 6 Hz, H-9); ¹³C NMR (75 MHz, DMSO-d₆)

δ: 147.6 (C1), 147.3 (C2), 146.0 (C14), 145.2 (C13), 125.2 (C10), 124.1 (C5), 122.2

(C11), 121.3 (C4), 117.5 (C15), 115.5 (C12), 110.2 (C6), 108.2 (C3), 101.3 (C7),

49.4 (C8), 49.3 (C9); Anal. Calcd for C₁₅H₁₆ClNO₄: C, 58.17; H, 5.21; N, 4.52. Found:

C, 58.06; H, 5.86; N, 4.26. HPLC: t = 8.1 min, purity >96%.

(2H-1,3-benzodioxol-5-ylmethyl)({3,4-bis[(tert-butyldimethylsilyl)oxy]phenyl}methyl)

azanium chloride (3g)

1

White solid; yield: 24%; H NMR (300 MHz, DMSO-d₆) δ: 9.39 (2H, s, H-

16,16’), 7.13 (1H, s, H-6), 7.06 (1H, s, H-4), 7.00-6.95 (3H, m, H-3,11,12), 6.88 (1H,

d, J = 8.2 Hz, H-15), 6.04 (2H, s, H-7), 4.00 (4H, s, H-8,9), 0.95 (18H, d, J = 4.5 Hz,

H-20-22,26-28), 0.19 (12H, d, J = 3.7 Hz, H-17,18,23,24); ¹³C NMR (75 MHz,

DMSO-d₆) δ: 147.5 (C1), 147.2 (C14), 146.7 (C2), 146.0 (C13), 125.2 (C10), 124.9

(C5), 124.0 (C11), 123.7 (C4), 122.5 (C12), 120.5 (C15), 110.2 (C6), 108.1 (C3),

101.2 (C7), 49.1 (C8), 48.8 (C9), 25.6 (C20-22, C26-28), 18.0 (C25), 17.9 (C19), -4.3

(C17. C18, C23, C24).

(2H-1,3-benzodioxol-5-ylmethyl)(benzyl)methylazanium chloride (5a)

1

White solid; yield: 29%; H NMR (300 MHz, CDCl₃) δ: 12.55 (1H, s, H-16),

7.69-7.64 (2H, m, H-12,14), 7.45-7.43 (3H, m, H-11,13,15), 7.23 (1H, s, H-6), 7.09

(1H, d, J = 7.8 Hz, H-4), 6.83 (1H, d, J = 7.9 Hz, H-3), 5.99 (2H, s, H-7), 4.30-4.08

13

(4H, m, H-8,9), 2.59 (3H, s, H-17); C NMR (75 MHz, CDCl₃) δ: 149.1 (C1), 148.4

(C2), 131.3 (C11, C15), 130.0 (C10), 129.4 (C12, C14), 128.8 (C5), 125.6 (C13),

122.0 (C4), 111.4 (C6), 108.8 (C3), 101.7 (C7), 59.0 (C8), 58.9 (C9), 38.5 (C17);

Anal. Calcd for C₁₆H₁₈ClNO₂: C, 65.86; H, 6.22; N, 4.80. Found: C, 65.40; H, 5.78; N,

4.74. HPLC: t = 12.2 min, purity >95%.

(2H-1,3-benzodioxol-5-ylmethyl)[(4-methoxyphenyl)methyl]methyl azanium chloride

(5b)

White solid; yield: 33%; ¹H NMR (300 MHz, DMSO-d₆) δ: 10.95 (1H, s, H-16),

7.55 (1H, s, H-15), 7.55 (1H, s, H-11), 7.28 (1H, d, J = 1.1 Hz, H-6), 7.06 (1H, dd, J¹

= 8.0 Hz, J²= 1.3 Hz, H-4), 7.01 (1H, s, H-3), 6.96 (2H, d, J = 7.8 Hz, H-12,14), 6.06

(2H, s, H-7), 4.32-4.25 (2H, m, H-8), 4.12-4.04 (2H, m, H-9), 3.78 (3H, s, H-18), 2.44

(3H, d, J = 4.2 Hz, H-17); ¹³C NMR (75 MHz, DMSO-d₆) δ: 159.9 (C13), 148.0 (C1),

147.4 (C2), 132.9 (C11, C15), 125.5 (C5), 123.5 (C10), 121.8 (C4), 114.0 (C12,

C14), 111.3 (C6), 108.3 (C3), 101.4 (C7), 57.7 (C8), 57.4 (C9), 55.2 (C18), 37.3

(C17); Anal. Calcd for C₁₇H₂₀ClNO₃: C, 63.45; H, 6.26; N, 4.35. Found: C, 63.03; H,

6.22; N, 4.31. HPLC: t = 6.0 min, purity >98%.

(2H-1,3-benzodioxol-5-ylmethyl)[(3,4-dimethoxyphenyl)methyl]methyl

azanium

chloride (5c)

1

White solid; yield: 23%; H NMR (300 MHz, CDCl₃) δ: 12.63 (1H, s, H-16),

7.61 (1H, s, H-15), 7.15 (1H, s, H-6), 7.04 (1H, d, J = 7.9 Hz, H-11), 6.96 (1H, d, J =

8.1 Hz, H-4), 6.85 (2H, d, J = 7.8 Hz, H-3,12), 6.01 (2H, s, H-7), 4.25-4.17 (2H, m, H-

8), 4.02-3.89 (8H, m, H-9,18,19), 2.54 (3H, d, J = 4.8 Hz, H-17); ¹³C NMR (75 MHz,

CDCl₃) δ: 150.3 (C14), 149.7 (C1), 149.1 (C13), 148.4 (C2), 125.6 (C10), 123.5 (C5),

121.9 (C11), 121.2 (C4), 114.1 (C15), 111.4 (C6), 111.0 (C12), 108.8 (C3), 101.6

(C7), 59.1 (C8), 58.9 (C9), 56.5 (C19), 55.9 (C18), 38.4 (C17); Anal. Calcd for

C₁₈H_23clNO₄: C, 61.45; H, 6.30; N, 3.98. Found: C, 61.44; H, 6.34; N, 4.12. HPLC: t =

9.3 min, purity >99%.

(2H-1,3-benzodioxol-5-ylmethyl)(methyl)[(3,4,5-trimethoxyphenyl)methyl]

azanium

chloride (5d)

1

White solid; yield: 24%; H NMR (300 MHz, CDCl₃) δ: 12.63 (1H, s, H-16),

7.16 (1H, s, H-6), 7.05 (1H, d, J = 8.2 Hz, H-4), 7.01 (2H, s, H-11,15), 6.85 (1H, d, J

= 7.9 Hz, H-3), 6.01 (2H, s, H-7), 4.24-4.16 (2H, m, H-8), 4.10-4.01 (2H, m, H-9),

13

3.92 (6H, s, H-19,20), 3.86 (3H, s, H-18), 2.59 (3H, s, H-17); C NMR (75 MHz,

CDCl₃) δ: 153.7 (C12, C14), 149.2 (C1), 148.4 (C2), 139.1 (C13), 125.7 (C10), 124.4

(C5), 124.3 (C4), 121.7 (C6), 111.4 (C3), 108.8 (C15), 108.2 (C11), 101.7 (C7), 60.8

(C18), 59.4 (C8), 59.2 (C9), 56.7 (C19, C20), 38.6 (C17); Anal. Calcd for

C₁₉H₂₄ClNO₅: C, 59.76; H, 6.34; N, 3.67. Found: C, 59.50; H, 6.27; N, 3.79. HPLC: t

= 7.7 min, purity >97%.

4.2. Cytotoxicity assay

The cytotoxicity of the compounds was tested against A549, NCI-H1299, NCI-

H292, NCI-H460, HUVEC, and LL24 cell lines by the MTT assay. The A549, NCI-

H1299, NCI-H292, NCI-H460, and HUVEC cells were suspended in RPMI-1640

(Cultlab; pH 7.0) supplemented with 10% of inactivated fetal bovine serum (FBS), 2

mM L-glutamine, and 1% glutamine. LL24 cells were suspended in RPMI-1640

(Cultlab; pH 7.0) supplemented with 20% FBS. Afterward, 10⁴cells were added in

triplicate to each well of a 96-well plate in 100 µL. The cells were then incubated at

37ºC for 2 h with 5% CO₂. The compounds, previously diluted in sterile DMSO, were

diluted in RPMI-1640, with a maximum of 1% DMSO for each concentration. The

medium was aspirated after 2 h of incubation, and then 100 µL of the diluted

compounds was added, and the cells were treated for 24 h. Afterward, 5 μL of MTT

(5 mg/mL) in phosphate-buffered saline (PBS) was added to each well, and the

plates were incubated at 37ºC with 5% CO₂for 2 h. The medium was aspirated, and

then 100 μL of DMSO was added to dissolve the formazan crystals. Each

experiment was run in triplicate. The optical density was read at 540 nm with an

enzyme-linked immunosorbent assay plate reader. The percent cell survival was

calculated as the following: % cell survival = (Abs_treatment/Abs_control)  100. The IC₅₀

value for each compound was calculated by nonlinear regression (curve fit) of log

(concentration) vs. percent survival using Prism 5.0 software (GraphPad, La Jolla,

CA, USA).

4.3. Clonogenic assay of 3f in A549 and NCI-H1299 cells

A549 and NCI-H1299 cells were seeded into 12-well plates in triplicate at a

density of 200 cells/well in 500 µL of supplemented medium and incubated at 37ºC

for 2 h with 5% CO₂. After 2 h of adhesion, the medium was aspirated, and the cells

were treated with 500 µL of compound 3f at concentrations of 50, 75, and 100 µM for

24 h. The supernatants were then replaced with fresh supplemented medium, and

the plate was incubated at 37ºC with 5% CO₂for 10 days. The wells were washed

twice with PBS. The cell clones were fixed with 6% glutaraldehyde and stained for 30

min with a solution that contained 0.5% Cresyl violet in PBS, followed by rinsing with

water to remove dye excess. Colonies were counted manually using a cutoff of 50

viable cells to consider it a colony. The results represent the standard of three

different experiments under the same conditions.

4.4. Analysis of effects of compound 3f on A549 and NCI-H1299 cell cycles

A549 and NCI-H1299 cells were plated into 12-well plates at a density of 2 

10⁵cells/well in 500 µL of supplemented medium. The medium was aspirated after 2

Article Doi

Toward chelerythrine optimization: Analogues designed by molecular simplification exhibit selective growth inhibition in non-small-cell lung cancer cells

DOI: 10.1016/j.bmc.2016.07.065

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Article abstract of DOI:10.1016/j.bmc.2016.07.065

Full text of DOI:10.1016/j.bmc.2016.07.065

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