Design, synthesis, anticancer activity, and solid lipid nanoparticle formulation of indole-and benzimidazole-based compounds as pro-apoptotic agents targeting bcl-2 protein

pharmaceuticals

Article

Design, Synthesis, Anticancer Activity, and Solid Lipid

Nanoparticle Formulation of Indole- and Benzimidazole-Based

Compounds as Pro-Apoptotic Agents Targeting Bcl-2 Protein

Manar I. Nagy ¹, Khaled M. Darwish ¹, Safaa M. Kishk ¹, Mohamed A. Tantawy ², Ali M. Nasr ^3,4

,

Mona Qushawy ^4,5, Shady A. Swidan ^6,7, Samia M. Mostafa ¹and Ismail Salama ^1,

*

1

Department of Medicinal Chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt;

manar.nagy@pharm.suez.edu.eg (M.I.N.); khaled_darwish@pharm.suez.edu.eg (K.M.D.);

safaa_keshk@pharm.suez.edu.eg (S.M.K.); samiamostafa2010@hotmail.com (S.M.M.)

National Research Center, Hormones Department, Medical Research Division, Dokki, Giza 12622, Egypt;

mohamed_tantawy@daad-alumni.de

Department of Pharmaceutics, Faculty of Pharmacy, Port Said University, Port Said 42526, Egypt;

a.nasr@pharm.psu.edu.eg

Department of Pharmaceutics, Faculty of Pharmacy, Sinai University, Alarish, North Sinai 45511, Egypt;

mqushawy@ut.edu.sa

Department of Pharmaceutics, Faculty of Pharmacy, University of Tabuk, Tabuk 71491, Saudi Arabia

Department of Pharmaceutics, Faculty of Pharmacy, The British University in Egypt, El-Sherouk City,

Cairo 11837, Egypt; shady.swidan@bue.edu.eg

The Center for Drug Research and Development (CDRD), Faculty of Pharmacy, The British University in

Egypt, El-Sherouk City, Cairo 11837, Egypt

2

3

4

5

6

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

7

ꢀꢁꢂꢃꢄꢅꢆ

Citation: Nagy, M.I.; Darwish, K.M.;

Kishk, S.M.; Tantawy, M.A.; Nasr,

A.M.; Qushawy, M.; Swidan, S.A.;

Mostafa, S.M.; Salama, I. Design,

Synthesis, Anticancer Activity, and

Solid Lipid Nanoparticle Formulation

of Indole- and Benzimidazole-Based

Compounds as Pro-Apoptotic Agents

Targeting Bcl-2 Protein. Pharmaceuticals

2021, 14, 113. http://doi.org/10.3390/

ph14020113

*

Correspondence: ismail_mohamed@pharm.suez.edu.eg; Tel.: +20-102-225-7643

Abstract: Cancer is a multifactorial disease necessitating identiﬁcation of novel targets for its treat-

ment. Inhibition of Bcl-2 for triggered pro-apoptotic signaling is considered a promising strategy

for cancer treatment. Within the current work, we aimed to design and synthesize a new series of

benzimidazole- and indole-based derivatives as inhibitors of Bcl-2 protein. The market pan-Bcl-2

inhibitor, obatoclax, was the lead framework compound for adopted structural modiﬁcations. The

obatoclax’s pyrrolylmethine linker was replaced with straight alkylamine or carboxyhydrazine

methylene linkers providing the new compounds. This strategy permitted improved structural

ﬂexibility of synthesized compounds adopting favored maneuvers for better ﬁtting at the Bcl-2 major

hydrophobic pocket. Anti-cancer activity of the synthesized compounds was further investigated

through MTT-cytotoxic assay, cell cycle analysis, RT-PCR, ELISA and DNA fragmentation. Cytotoxic

Academic Editors: Mary Meegan and

Maria Emília de Sousa

results showed compounds 8a

cancer cells (IC₅₀= 12.69 0.84 to 12.83

against A549/lung adenocarcinoma cells (IC₅₀= 23.05

,

8b and 8c with promising cytotoxicity against MDA-MB-231/breast

3.50 M), while 8a and 8c depicted noticeable activities

1.45 and 11.63 2.57 M, respectively). The

Received: 27 December 2020

Accepted: 26 January 2021

Published: 1 February 2021

±

µ

±

µ

signaling Bcl-2 inhibition pathway was conﬁrmed by molecular docking where signiﬁcant docking

energies and interactions with key Bcl-2 pocket residues were depicted. Moreover, the top active

compound, 8b, showed signiﬁcant upregulated expression levels of pro-apoptotic/anti-apoptotic of

genes; Bax, Bcl-2, caspase-3, -8, and -9 through RT-PCR assay. Improving the compound’s pharma-

ceutical proﬁle was undertaken by introducing 8b within drug-solid/lipid nanoparticle formulation

prepared by hot melting homogenization technique and evaluated for encapsulation efﬁciency, parti-

cle size, and zeta potential. Signiﬁcant improvement was seen at the compound’s cytotoxic activity.

In conclusion, 8b is introduced as a promising anti-cancer lead candidate that worth future ﬁne-

tuned lead optimization and development studies while exploring its potentiality through in-vivo

preclinical investigation.

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Attribution (CC BY) license (https://

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4.0/).

Keywords: Bcl-2 inhibitors; Indole-based analogues; benzimidazole; MTT cytotoxic assay; cell cycle

analysis; DNA fragmentation; ELISA; docking; solid/lipid nanoparticles

Pharmaceuticals 2021, 14, 113. https://doi.org/10.3390/ph14020113

https://ww w .mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 113

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1. Introduction

The failure of most cancer cells to undergo apoptosis confers them a survival advan-

tage over normal cells. Apoptosis is a genetically programmed cell death to get rid of an

undesirable cell, which is necessary for cell turnover [1]. Apoptosis is controlled by two

major modes: the extrinsic and the intrinsic pathways. Both pathways lead to activation of

caspases that result in morphological changes such as chromatin condensation, nuclear

fragmentation, formation of apoptotic bodies and ﬁnally cell death [2].

The extrinsic pathway comprises external signals, that bind to the cell’s surface death

receptors resulting in the formation of the death-induced signaling complex [

(B cell lymphoma gene 2) family proteins control the intrinsic pathway. These proteins

prevent the release of cytochrome C from mitochondria [ ]. The Bcl-2 family of proteins

3]. Bcl-2

4

can be classiﬁed as proapoptotic and antiapoptotic proteins. The conserved Bcl-2 homology

domain-3 (BH3) of pro-apoptotic Bcl-2 members is responsible for the mitochondrial

apoptosis [5]. Anti-apoptotic Bcl-2 proteins have a hydrophobic groove, containing both

of the conserved Bcl-2 homology domains BH1 and BH2 which sequesters the BH3 of

pro-apoptotic Bcl-2 members [6].

There are three approaches for targeting Bcl-2 in cancer therapy. The ﬁrst one depends

on the use of antisense oligonucleotide, which can bind to the complementary mRNA of

Bcl-2, resulting in blocking expression at the protein level. The second strategy develops

peptides against Bcl-2 protein [

can bind to the hydrophobic groove of anti-apoptotic Bcl-2 [

7

,

8

]. Lastly, the third approach utilizes small molecules that

]. These molecules target one

9

or more of P1–P4 sub-pockets in the BH3 groove of anti-apoptotic proteins. This binding

results in releasing pro-apoptotic BH3-only proteins that can activate Bax and Bak and lead

to apoptosis [10]. Several small molecules have been identiﬁed and synthesized, such as

venetoclax, disarib, and obatoclax (Figure 1).

Venetoclax is a highly potent BH3 mimetic molecule. It showed a subnanomolar

afﬁnity to Bcl-2 (Ki < 0.010 nM) with no measurable afﬁnities for other apoptotic proteins.

It showed cytotoxicity against non-Hodgkin’s lymphoma, chronic lymphocytic leukemia

(CLL) and acute leukemias [11]. Disarib is another selective Bcl-2 inhibitor [12]. Disarib

was proven to inhibit the growth of Bcl-2 high cancer cell lines and CLL patient primary cell

lines with minimum effect on Bcl-2 low cancer cell lines. In addition, disarib exhibited fewer

side effects in treated animals with respect to platelet count, body weight, and liver and

kidney functions [13]. Obatoclax is an oligopyrrole which antagonizes Bcl-2 and Bcl-xL [14].

Obatoclax binds to the BH3 domain of Bcl-2 and interrupts the interaction with proapoptotic

proteins. The combination of obatoclax and lapatinib, giﬁtinib, bortezomib, and entinostat

showed synergistic repression of cell growth in human breast cancer cells [15]. Furthermore,

obatoclax synergizes chemotherapeutic agents as cisplatin, in cancer cell lines [16].

For Bcl-2 inhibitors development, we have described several series of compounds

with chemical scaffolds that mimic obatoclax by either replacement of the pyrrole ring of

obatoclax with benzimidazole or extending the structure after the indole ring. Additionally,

comprehensive biological and molecular docking investigation, within the crystallised

Bcl-2 crystal structure, provided further insights regarding the molecular pro-apoptotic

mechanisms of the presented small synthesized molecules. Finally, formulating the most

promising lead within an optimized solid lipid nanoparticle formulation provided ben-

eﬁcial results concerning improved cytotoxic activity, drug release and pharmaceutical

properties, particularly drug’s aqueous solubility and bioavailability.

Pharmaceuticals 2021, 14, 113

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Figure 1. Mechanism of action of pro-apoptic Bcl-2 proteins small molecules inhibitors such as

venetoclax, disarib and obatoclax, where they can disrupt the interactions of Bcl-2 and BH3 proteins

such as BAK, leading to homodimerization of the BH3 proteins and activation of apoptotic cascade.

2. Results and Discussion

2.1. Compounds Design

The reported drug-like pan Bcl-2 inhibitor, obatoclax (GX15_070), was adopted as the

lead framework for structural modiﬁcations and manipulations. Obatoclax is BH3-mimetic

small molecule (317.4 Da) acting on several anti-apoptotic proteins such as Mcl-1, Bcl-2,

Bcl-W, and Bcl-XL [17]. Preclinical studies illustrated the drug capability in reversing BH3-

mediated binding of Bcl-2 family proteins to apoptotic proteins, Bak and Bax, allowing

unopposed Bak/Bax dimerization and subsequent initiation of intrinsic apoptotic signal-

ing [18

anticancer activity, including induction of autophagic cell death and necroptosis [19

Moreover, obatoclax can inhibit cell proliferation through seizing cell cycle development at

G1/S-phases [19 23]. Many reported studies conﬁrmed the clinical advent of obatoclax for

,

19]. Several additional mechanisms of action have been accounted for obatoclax

–

22].

,

inducing apoptosis within cells derived from solid tumors and hematological cancers such

as small cell lung cancer, acute myeloid leukemia, and Hodgkin’s lymphoma as well as

potentiating cytotoxicity of targeted therapy drugs and conventional chemotherapeutics.

Currently, there is no reported atomic resolution data concerning the obatoclax being

bounded to pre-survival Bcl-2 protein. Nevertheless, Zacarias-Lara et al. have reported

seven recognized Bcl-2 inhibitors, including obatoclax, to be investigated for their re-

Pharmaceuticals 2021, 14, 113

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spective afﬁnities and binding interactions through molecular docking calculations [24].

Interestingly, the authors suggested anchoring of obatoclax just adjacent the hydrophobic

cleft (H1-3) which may compromise the drug’s ability to dissociate BH3-only pro-apoptotic

proteins for subsequent programmed cell death. Driven by the above ﬁndings, the pre-

sented study aimed for developing new pro-apoptotic agents, based on obatoclax principal

pharmacophore, with an extra goal of improved Bcl-2 hydrophobic cleft accommodation.

Obatoclax belongs to the class of organic compounds, designated as dipyrrins, incorpo-

rating two pyrrole rings within their chemical architectures where the two 5-membered

rings are being fused via a trivalent methine (-CH=) bridge. Here within the presented

manuscript, three structural modiﬁcation strategies have been adopted to design the newly

synthesized compounds, Series-I-to-III (Figure 2). The ﬁrst modiﬁcation strategy was to

improve the obatoclax ﬂexibility through replacing the drug’s central pyrrolylmethine

linker with straight alkylamine or carboxyhydrazine methylene linkers furnishing the

members of the Series-I or -II, respectively. The aim of such tactic was to permit enhanced

maneuver for these newly synthesized compounds to better accommodate the Bcl-2 ma-

jor hydrophobic pockets (S1-to-5). Additionally, the terminal obatoclax pyrrole ring was

replaced with substituted phenyl groups to permit an enriched

π–π stacking and other

non-polar interactions with the hydrophobic residues at the Bcl-2 pocket.

Figure 2. Rationalized design of the proposed new pro-apoptotic agents (Series-I-to-III), relying on the pan Bcl-2 in-

hibitor, obatoclax.

Within a comparable bases, members of Series-I possess the benzimidazole scaffold as

the classical isostere of the indole moiety present within the obatoclax chemical architecture.

Such introduced second structural modiﬁcation relied on the ability of the benzimidazole

ring to permit extra H-bonding with the surface polar residues of the Bcl-2 pocket and

thus further ligand ﬁxation. Notably, both members of Series-I and -II were of comparable

overall size as that of the obatoclax, the thing that can limit the extension of the synthesized

compounds to the farthest hydrophobic pockets. Therefore, a third and last structural

modiﬁcation strategy has been adopted where the terminal substituted phenyl moieties

at Series-II were replaced with the highly extended substituted bisaryl pyrazole scaffold

Pharmaceuticals 2021, 14, 113

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resulting in the members of Series-III. The latter members possess the double advent of

great ﬂexibility and extended size. Finally, the adoption of the central pyrazole scaffold

was rationalized for its straightforward synthesis as well as easily derivatized during

the pyrazole ring closure. Based on the adopted three structural modiﬁcation strategies,

fourteen compounds were synthesized as well as evaluated for respective predicted anti-

cancer activities through in-vitro and in-vivo biological analyses as well as molecular

docking investigations.

2.2. Chemistry

The synthesized compounds of the three designed series were obtained from straight-

forward schematic pathways. Structural diversity for synthesizing the target compounds

was performed at the ﬁnal steps of the synthetic pathways. Adopting such strategy

was beneﬁcial for minimizing the number of needed reactions that would provide each

individual analogue. Within Scheme 1 , Series-I benzimidazole derivatives were synthe-

sized from the commercially available, ortho-phenylene diamine (1) through Phillips-

Ladenburg reaction [25]. Cyclization was proceeded through reﬂuxing ortho-phenylene

diamine with chloroacetic acid, under acidic conditions, to yield the corresponding in-

termediate 2-(chloromethyl)-1H-benzimidazole (2). Introduction of 2-chloromethyl arm,

within the benzimidazole skeleton, served as a handy scaffold for introducing structural

diversity at the ﬁnal synthetic pathway. Subsequent S_N2 nucleophilic substitution at

2-methyl benzimidazole with various aromatic amines furnished the target substituted

2-aminomethyl-benzimidazole derivatives (3a–d) depicting extra aromatic signals at their

¹H-NMR spectra [26].

Scheme 1. Reagents and conditions for Series-I: (a) ClCH₂COOH, 4N HCL, reﬂux, overnight, 40%; (b) Ar-NH₂, 51–73%.

The indole-based derivatives (Series-II and -III) were synthesized starting from the

commercially available indole-2-carboxylic acid (

tion, reﬂuxing with concentrated H₂SO₄and ethanol yielded the corresponding ethyl,1H-

indole-2-carboxylate ( ) [27]. Subsequent, condensation of the obtained ester intermediate

with hydrazine hydrate furnished the hydrazide key intermediate ( ). Finally, Schiff’s base

condensation of with different commercially available and synthesized aromatic aldehy-

des, under acidic conditions, provided the target hydrazone analogues (7a and 8a ) [28].

Appearance of the characteristic methylene hydrogen at NMR spectra (δ_H= 8.16–8.98 ppm

4) (Scheme 2 ). Throughout Fischer reac-

4

5

6

–

d

–f

;

δ_C= 146.8–148.9 ppm) conﬁrmed successful Schiff’s base formation. Preparation of sev-

eral pyrazole-based aldehydes was proceeded through three-step chemical synthesis [29].

Synthesis of the pyrazole aldehydes, that served as the structural building blocks of the

Series-III compounds, was proceeded through initial acidic condensation of different ke-

tones with phenylhydrazine derivatives yielded the corresponding imine analogues (10a

(Scheme 3). Subsequently, Vilsmeier-Haack reaction, with the advent of phosphorous

oxychloride, provided the target pyrazole-based aldehydes (12a ) exhibiting characteristic

NMR signals of CHO groups (δ_H= 9.97–10.00 ppm; δ_C= 185.1–185.3 ppm).

–f)

–

f

Pharmaceuticals 2021, 14, 113

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Scheme 2. Reagents and conditions for Series-II and -III: (a) EtOH, Conc. H₂SO₄, reﬂux, 1.5 h, 92%; (b) Hydrazine hydrate,

absolute EtOH, reﬂux, 6 h, 44%; (c) Ar-CHO, EtOH, glacial acetic acid, reﬂux, 1.5–3 h, 30–45%.

◦

a) EtOH, Ar/R-COCH₃, 80 C, 1 h, 80–97%;

Scheme 3. Reagents and conditions for the synthesis of pyrazole aldehydes: (

(b) POCl₃, DMF, reﬂux, 4 h, 61–81%.

2.3. Biological Activity

2.3.1. In-Vitro Cytotoxic Activity

The potential anti-cancer activity of all fourteen ﬁnal synthesized compounds was

investigated through MTT cytotoxic assay against two different human solid cancer-

ous cell lines; A549/lung and MDA-MB-231/breast adenocarcinoma. The adopted cell-

proliferation assay assessed % cell viability in relation to the reducing activity of cellular

NADPH-dependent cellular oxidoreductases on tetrazolium dye, MTT [2,5-diphenyl-3-

(4,5-dimethylthiazol-2-yl)tetrazolium bromide salt], for furnishing an easily quantiﬁed

insoluble chromogenic formazan [30]. For each drug concentration, 0.1, 1, 10, and 100 µM,

the % cell-viability was estimated relative to negative control following a complete 48 h

of drug treatment. Both 5-ﬂuorouracil (5-FU) and doxorubicin (DOX) were set as posi-

Pharmaceuticals 2021, 14, 113

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tive index comparators. Moreover, the safety proﬁle of the synthesized compounds was

further investigated by examining their in-vitro cytotoxicity on non-cancerous cell line;

MDCK/kidney cells.

Findings within Figure 3A illustrate a dose-dependent impact of several synthesized

compounds on MDA-MB-231/breast cell proliferation. Rising drug concentration was

directly related with elevated cell growth inhibition up to the highest applied concentration

(100

cytotoxicity or a plateau effect, except for compound 3c on MDA-MB-231/breast cancer

where it enhanced cell proliferation at 100 M drug concentration. The steepest decline

within the cell proliferation was seen with 7c and 8a reaching down to very low % cell

viabilities (12% and 14%) at 100 M concentrations. The latter suggests great susceptibility

µM) (p < 0.05). Interestingly, most of the tested compounds had a dose dependent

µ

,

d

–d

µ

of MDA-MB-231/breast cancer cells towards indole derivatives, particularly those having

pyrazole-based scaffolds (Series-III). The rest of compounds exhibited comparable cytotoxi-

city patterns as those for positive reference controls, 5-FU and DOX, since minor %viability

ﬂuctuations were depicted across the applied drug concentrations. Regarding the drug’s

impact on A549/lung adenocarcinoma cell proliferation, both investigated and positive

control compounds showed quite lower overall anticancer activity as compared to their

actions up on the MDA-MB-231/breast cancer cells. Nevertheless, comparable differential

activity proﬁles were depicted among the investigated compounds, where compounds 8a

exhibited the highest activity proﬁles with steepest decrease in % cell viability (70–79%;

p < 0.001) at 100 M concentrations (Figure 3B). This again suggests promising anticancer

–c

µ

activity of the indole-based compounds on A549/lung cancer cells, particularly for the

ones incorporating the pyrazole scaffolds. The rest of the compounds exhibited comparable

pattern of cell growth inhibition across their concentration range which was also quite

similar to that of positive control drugs. However, it was only for compound 3b where its

anticancer activity on A549/lung cancer cells depicted irregular effects with tendency to

increase the cell proliferation. Evaluating drug’s cytotoxicity on non-cancerous kidney cells,

Figure 3C showed higher safety proﬁles for synthesized compounds compared to controls,

5-FU and DOX. Signiﬁcantly higher vitality patterns were illustrated for all compounds,

particularity over the concentration range 0.1–10 µM, as compared to those of reference

compounds (p < 0.001).

For gaining more insights about the efﬁcacy/safety proﬁles of the synthesized com-

pounds, IC₅₀values for promising cytotoxic compounds were estimated over the three

cell lines. Only compounds exhibiting signiﬁcant cytotoxicity, with cut-off value < 85%

vitality at 100

and warrant further consideration. Interestingly, the selected compounds for MDA-MB-

231/breast cancer cells (3a 7b 7d, and 8a ) illustrated relevant IC₅₀values reaching

down to two-digit micromolar activity (Table 1). Members of Series-III showed the low-

est IC₅₀(down to 12.69 M) suggesting the preferential cytotoxicity of their substituted

pyrazole arms. Both 8d and 8f showed higher IC₅₀(21.64 and 31.46 M, respectively)

µM on both cancerous cell lines, were considered relevant for IC₅₀estimating

,

–d

µ

suggesting activity preferentiality for aromatic substitution, at C3 pyrazole ring, while

tolerating small-sized aliphatic chains. Compound 7d was comparable to the top-active

members of Series-III with IC₅₀17.38 µM, suggesting Series-II preferentiality for pyridine

ring substitution over methoxy phenyl synthon. Regarding IC₅₀on MDA-MB-231/lung

adenocarcinoma, only 8b and 8c were evaluated suggesting preferential activity for pyra-

zole substitution with branched aliphatic chains. Finally, the IC₅₀for selected Series-III

compounds on non-cancerous/kidney cells were much higher than the other two Series,

reaching up to IC₅₀92 µM activity for 8b. Based on all the above ﬁndings, signiﬁcant

cytotoxic activity has been assigned for members of Series-III which were also presented

with high safety proﬁle, particularly 8b, on non-cancerous cell line. Therefore, compound

8b was considered promising with double the beneﬁts of high efﬁcacy/safety proﬁles

making it worthy of further investigations.

Pharmaceuticals 2021, 14, 113

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B

Figure 3. In-vitro cytotoxicity of the fourteen ﬁnal synthesized compounds on cancerous and non-cancerous cell lines.

(A) MDA-MB-231/breast cancer cells; (B) A549/lung adenocarcinoma cells; (C) MDCK/kidney non-cancerous cells. At

1 × 10⁴cells/well cell density, cells were treated with several drug concentrations (0.1, 1, 10, and 100

µM) of investigated

compounds or positive comparator; 5-FU and DOX, for 48 h exposure time. Cytotoxicity was identiﬁed via MTT-based

colorimetric assay (490 nm) and % cell growth viability was estimated in triplicates as compared to vehicle negative controls

representing the untreated cells. Blue dotted line indicates this control.

Pharmaceuticals 2021, 14, 113

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Table 1. In-vitro ^adata of MTT-cell proliferation assay for promising synthesized compounds on

both cancerous and non-cancerous cell lines.

Compound ^b

No.

MDA-MB-231

A549

MDCK

IC₅₀(µM) ^c

3a

7b

7d

8a

8b

8c

29.68 ± 4.03

43.77 ± 1.09

17.38 ± 3.23

12.69 ± 0.84

12.71 ± 2.48

12.83 ± 3.50

31.46 ± 4.66

21.64 ± 0.28

ND

28.92 ± 1.49

ND

73.86 ± 2.30

92.75 ± 0.43

87.29 ± 3.00

ND

23.05 ± 1.45

11.63 ± 2.57

ND

8d

8f

ND

a

In-vitro data are reported as mean of three independent experiments

depicted % cell viability < 85%, on both cancerous cell lines, at their respective 100 µ

M concentrations; ^cIC₅₀values

±

SEM; ^bTested compounds were those

represent effective concentration of given small molecule being capable of inducing 50% inhibitory response of its

own intrinsic maximum response following 48 h of drug exposure; ND = Not-detected.

2.3.2. Morphological Assessment

The impact of 8b on cancerous cellular morphology was evaluated over different

concentrations following 48 h exposure time. In MDA-MB-231/breast cancer cells, normal

membrane integrity and control group nucleus morphology was depicted for cells within

vehicle negative control group (Figure 4A). Contrarily, cells treated with 8b exhibited

signiﬁcant cell morphology alteration, intercellular space dilatation, and cellular shrinkage

(Figure 4B). Alterations proceeded within a dose-dependent manner as escalated drug

concentrations showed cells being gradually shrunken, unable to adhere well, ﬂoated,

and clustered together (Figure 4C,D). At 100 µM concentration, the cell number became

highly declined and great loss of cellular polyhedral shape was observed as cells acquired

the tentacled spindle-shaped morphology (Figure 4E). These morphological changes were

more drastic with 8b as compared to 5-FU for similar concentrations (100 µM) (Figure 4F).

Comparable cell morphology changes were observed with A549/lung adenocarcinoma

(Figure 5 A–E). Nevertheless, an epithelial-mesenchymal transition was observed within

5-FU group, rather than 8b, at same drug concentration (100 µM) (Figure 5 F). The later

ﬁnding presents a signiﬁcant advantage for 8b as it did not trigger this evolutionarily-

conserved developmental program conferring metastatic properties upon cancerous cells

through enhanced mobility, invasion, and apoptotic stimuli resistance [31].

A

B

Figure 4. Cont.

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C

D

F

E

Figure 4. Morphological alterations of MDA-MB-231/breast cancer cells following 48 h incubation and photographed with

phase-contrast microscope. (

A

) vehicle control; (B) 0.1

µ

M 8b; (

C

) 1

µM 8b; (D) 10 µM 8b; (E) 100 µM 8b; (F) 100 µM 5-FU;

Magniﬁcation power ×20.

A

B

Figure 5. Cont.

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C

D

F

E

Figure 5. Morphological alterations of A549/lung carcinoma following 48 h incubation and photographed with phase-

contrast microscope. ( ) vehicle control; ( ) 0.1 M 8b; ( ) 1 M 8b; ( ) 10 M 8b; ( ) 100 M 8b; ( ) 100 M 5-FU;

Magniﬁcation power ×20.

A

B

µ

C

µ

D

µ

E

µ

F

µ

2.3.3. Apoptosis Rate and Cell Cycle Analysis

Further investigation of 8b mechanistic growth inhibitory action on MDA-MB-231/

breast cancer, both cell cycle, and apoptosis rate analysis were conducted at the compound’s

approximated IC₅₀value (13 µM). Using ﬂow cytometry, apoptosis rate analysis was pro-

ceeded through staining the cell surface-translocating phosphatidylserine with Annexin-V

ﬂuorescent conjugate, the calcium-dependent phospholipid binding protein [32]. However,

cell cycle analysis was achieved using propidium iodine ﬂuorescent dye to stoichiomet-

rically stain cell DNA contents allowing quantitation and identiﬁcation of all cell phase

rates; G0-G1, S, G2/M, and pre-G1 [33,34]. The impact of 8b on cell cycle distribution

depicted predominant cell population at G2/M stage (25.32%) was signiﬁcantly higher

than that of untreated cell line (6.15%; p < 0.001) (Figure 6A). The elevated cell population

at G2/M stage was complemented by signiﬁcant reduction at pre-G1 stage of treated cells

as compared to negative controls (1.72% vs. 18.93%; Figure 6B). For identifying the mode

of cell death promoted by 8b within MDA-MB-231/breast cancer cells, apoptosis rate

analysis was performed following 48 h exposure time. Compound 8b (13

µM) induced

both early and late stages of apoptosis in breast cancer cell line with signiﬁcantly elevated %

apoptotic cell levels as compared to controls (p < 0.01 and p < 0.001, respectively) (Figure 7)

Moreover, the average proportion of Annexin-V stained positive cells (total apoptotic

cells) elevated from 1.72% within untreated cells to 18.93% in treated ones (p < 0.001).

Interestingly, compound 8b showed no inﬂuence on the necrosis of MDA-MB-231/breast

.

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cancer cells. The above provided ﬁndings are supported by the previous cell cycle analysis

conﬁrming the potentiality of 8b as promising anticancer agent.

(A)

8b

Control

(B)

60

50

40

30

20

10

0

Control

8b

***

%G0-G1

%S

%G2/M

%Pre-G1

Figure 6. Impact of compound 8b upon cell cycle distribution within MDA-MB-231/breast cancer cell line. Following

48 h-period incubation of cells with either 8b (13 M; approximated IC₅₀) or vehicle negative control [0.1% DMSO (v/v)],

cells were ﬁxed, stained with propidium iodide (PI), and assessed via ﬂow cytometry; ( ) DNA histograms for cell cycle

distribution of untreated cells (left panel) and 8b group (right panel) at in 2N (G0G1), >2N (S), 4N (G2/M), and <2N (pre-

G1), where cell number was correlated against gated counts/DNA area (FL2-A) using CellQuest^®software; (

) Bar-chart

µ

A

B

representation of cell accumulation percentage within each stage of cell development. All data are represented as mean of

three independent experiments ± SEM. Statistical analysis was performed using Student-t-test (***, p < 0.001).

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(A)

Control

8b

(B)

***

20

16

12

8

***

Control

8b

**

4

0

Early

Late

Apoptosis

Total

Necrosis

Figure 7. Impact of compound 8b on apoptosis rate within MDA-MB-231/breast cancer cell line (Annexin-V/FACS). (

totic impact of 8b following cell double staining via Annexin-V-FITC then PI, and then 48 h incubation time with either 8b

(13 M; approximated IC₅₀) or vehicle negative control [0.1% DMSO (v/v)]; ( ) Bar-chart representation of quantitative analysis

A) Apop-

µ

B

of apoptosis, across different stages, as well as necrosis. The four quadrants are recognised as: LL, LR, UR, and UL for viable,

early apoptotic, late apoptotic, and necrotic cell populations, respectively. All data are represented as mean of three independent

experiments ± SEM. Statistical analysis was conducted via Student-t-test (**, p < 0.01; ***, p < 0.001).

2.3.4. DNA Fragmentation Determination

In order to delineate the mechanistic aspects of MDA-MB-231/breast cancer cell

death mediated by 8b, the DNA fragmentation assay was conducted as being highly

characteristic for apoptosis. Signiﬁcant DNA fragmentation was depicted with 8b at IC₅₀

doses (13 µM) at various time-intervals; 48 h and 72 h following cell treatments. A typical

ladder pattern of internucleosomal fragmentation was observed from cell homogenates

within both incubation periods (Figure 8). Such ﬁndings further conﬁrm the signiﬁcant

activity of 8b as a potent inducer of apoptosis.

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Figure 8. DNA fragmentation of MDA-MB-231/breast cancer cell line treated with 8b. Fragmenta-

tions of genomic DNA in breast cancer cells were treated with 8b (13 µM) for 48 or 72 h exposure

time. Low-molecular-weight DNA from homogenized cells was resolved and visualized on ethidium

bromide-stained 1.5% agarose gel while being photographed by UV-illumination electrophoresis;

C = DMSO vehicle control.

2.3.5. Apoptotic Gene Expression and Protein Level Analysis

Compound 8b demonstrated strong cytotoxic impact upon breast cancer cell lines,

as it accomplished signiﬁcantly low IC₅₀value (12.71 µM) with conﬁrmed apoptosis

induction. Dissection of the compound’s proapoptotic activity was proceeded through gene

expression analysis for key genes, controlling apoptosis pathway, as well as protein level

determination. Following 8b treatment for 48 h, alterations within MDA-MB-231/breast

cancer cell expression of Bcl-2, Bax, caspase-3, -8, and -9 genes, as well as cytochrome c

and class-III β-tubulin proteins levels were determined using real-time polymerase chain

reaction (RT-PCR) or ELISA technique, respectively. Typically, Bcl-2, Bax, caspases, and

cytochrome c proteins contributes within the regulation of apoptotic signaling. Acting as

apoptotic activator (Bax) or inhibitor (Bcl-2), the Bcl-2 family proteins play their signiﬁcant

role in apoptosis [35]. The caspases family are cysteine proteases being classiﬁed as either

executioners; caspase-3, -6, or -7 or initiators; caspase-8 and -9 [36]. Regrading caspase-8,

its activation is proceeded through extrinsic death-receptor dependent apoptotic pathway,

while, caspase-9 activation is within the event of intrinsic mitochondrial cytochrome

c leakage [37]. Additionally, participation of activated caspase-3 is to be essential for

caspase-8 activation [38]. Class-III β-tubulin are pure prognostic biomarker within cancer

patients being associated with aggressive phenotypic/drug-resistant cancers and part of

complicated pro-survival molecular pathway, being triggered via poor nutrient supply and

hypoxia [39].

Findings within Figure 9A illustrated strong stimulated expression of pro-apoptotic

BAX gene (3.61 folds), and apoptotic genes; Caspase-3 (4.28 folds), Caspase-8 (1.53 folds), and

Caspase-9 (7.65 folds) as compared to negative controls (p < 0.05 and p < 0.001). Nevertheless,

signiﬁcant downregulation of anti-apoptotic gene Bcl-2 (0.165 folds) was depicted and have

been translated into elevated Bax/Bcl expression ratio (1

gene expression ratio can serve as early predictor for cancer in patients as well as sensitive

→

21.88; p < 0.001). The Bax/Bcl-2

monitor of cancer progression [40]. On the other hand, compound 8b illustrated increased

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levels of the apoptotic stimulator cytochrome c (0.675 ng/mL vs. 0.295 ng/mL; p < 0.01)

and down-regulated levels of class-III β-tubulin (0.23 vs. 0.73 ng/mL; 68.36% inhibition;

p < 0.001) as compared to controls (Figure 9 B). Gathering up all the provided evidence,

up-regulation of Caspase-3, -8, -9 and Bax, while down-regulation of Bcl-2 genes and class-III

β-tubulins are suggested believed to be related to compound 8b-induced apoptosis within

breast cancer cell line.

(A)

(B)

9

8

7

6

5

4

3

2

1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

***

Control

8b

Control

8b

***

*

***

0

Cyt. c

Class-III β-tubulin

Bcl-2

Bax

Casp-3 Casp-8 Casp-9

Figure 9. Apoptotic gene expressions and protein level analysis of MDA-MB-231/breast cancer cells exposed to 8b

) Expression of pro-apoptotic/anti-apoptotic of genes; Bax, Bcl-2, caspase-3, -8, and -9 using RT-PCR method; ( ) Evaluation

of cytochrome c and class-III -tubulin protein levels in MDA-MB-231/breast cancer cells using ELISA technique. Within

both assays, cultivated MDA-MB-231/breast cancer cells were treated with 8b at its IC₅₀concentration (13 M) for 48 h

SEM. Statistical analysis was

.

(A

B

β

µ

exposure period. All data are represented as average of three independent experiments

performed using Student-t-test (*, p < 0.05; ***, p < 0.001).

±

2.4. Computational Study

To gain more insights about differential cytotoxic activity of synthesized compounds,

molecular docking simulations were conducted for all agents. The binary complex of X-ray

crystallized Bcl-2 (PDB ID: 6qgk; resolution 1.80 Å) [41], bounded to tetrahydroisoquinoline-

phenyl pyrazole derivative, was adopted for the docking studies. Adopting such complex

for the presented in-silico investigation was owing to structural similarities of crystallized

ligand with the synthesized compounds as well as its extended orientation within the

target protein. Typically, the Bcl-2 pocket is a narrow, long grooved site comprised of two

larger nearby sub-pockets (P2 and P4), in addition to, three small hydrophobic pockets

(P1, P3 and P5) [9]. The P1 through P4 sub-pockets are the key Bcl-2 binding sites for the

class of single BH3-only pro-apoptotic proteins. Owing to the hydrophobic nature of the

binding site residues, it has been challenging for inhibitors to competitively overcome the

natural substrate-Bcl-2 interactions. However, signiﬁcant polar residues (Tyr108, Asp111,

Glu136, Met115, and Arg146) represent relevant anchoring sites for stabilizing the bounded

ligands. The crystallized ligand showed extended accommodation within the target pocket,

reaching from P1-to-P4 subsites (Figure 10). Signiﬁcant non-polar contacts are depicted

with the hydrophobic residues of four sub-pockets, while the ligand’s polar functionalities

exhibit close proximity towards Tyr108, Asp111, and Arg146. Interactions with these polar

residues are suggested to be signiﬁcant for the ligand/target stability [41].

Proceeding throughout the docking studies, validation of the adopted docking pro-

tocol was achieved through redocking the crystallized ligand (PDB ID: J1Q) showing a

root-mean standard deviation (RMSD) below 2 Å. Depicting RMSD values below 2 Å

indicates that both the adopted algorithms and parameters were sufﬁcient for determin-

ing the best docking pose [42]. Regarding the docking results of the top cytotoxic drugs,

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limited binding of the Series-I compound (3b) towards Bcl-2 pockets was illustrated. The

benzimidazole-based ligand adopted V-shaped conformation with limited binding to P2

and P3 hydrophobic pockets. The benzimidazole scaffold predicted relevant anchoring

within P3 through H-bonding with Asp111, while the substituted phenyl group ﬁnely over-

laid at P2 pocket (Figure 11A). This orientation was further stabilized through π-π stacking

of the benzimidazole ring with Tyr108 (Supplementary Materials; Table S1). More extended

orientations were depicted for the Series-II indole derivatives (7a and 7b). Binding was ex-

tended at P2 through P4 sub-pockets showing their indole rings being docked at P2. Higher

docking score was assigned for 7d (S =

where double polar interactions between its pyridine ring and Arg146 side chain justiﬁed

signiﬁcant anchoring within pocket P4 (Figure 11B). Moreover, the pyridine ring was

−

4.8585 Kcal/mol vs.

−

4.76959 Kcal/mol for 7b),

further stabilized through double π-hydrogen interaction with Leu137 of P4 subsite sug-

gesting its great afﬁnity to Bcl-2 protein. Owing to its amide linker ﬂexibility, 7d indole ring

showed better overlay with the crystallized ligand than does the 3a benzimidazole scaffold.

For the pyrazole-based Series-III ligands, comparable binding modes, to the crys-

tallized ligand, were suggested (RMSD = 1.211 to 1.7374 Å) owing to a similar topology

of central pyrazole with substituted aromatic arms. Extended orientations and confor-

mations over P1-P4, with much closer proximity towards P5 subsite, have been pre-

dicted for Series-III ligands. Compound 8b showed one of the highest docking scores

(

S = −6.1846 Kcal/mol), which was explained by its exhibiting signiﬁcant hydrogen bond-

ing between its amide linker and Arg146 side chain at P4 subsite (Figure 10C). The same

polar interaction was predicted for the other Series-III members conﬁrming their superior

Bcl-2 afﬁnity (S = −5.7070 to

−

6.2460 Kcal/mol) and the importance of Arg146 in ligand

binding. Notably, compound 8f was suggested with the lowest member docking score

(

S = −4.9429 Kcal/mol) for lacking contacts with P1 due to its short methyl arm instead

of the substituted aromatic side chain in all Series-III members. Additionally, the 5-chloro

substituent on the pyrazole ring exhibited great solvent exposure suggesting high solvation

penalty (Figure 10D).

Figure 10. Structure of X-ray crystallized Bcl-2 (PDB ID: 6qgk) bounded to tetrahydroisoquinoline-phenyl pyrazole

derivative along the binding site. Five hydrophobic pockets (P1–5), constituting the binding site, are shown in 3D-surface

presentation and differently colored; P1 yellow, P2 magenta, P3 green, P4 red, P5 orange. Zoomed image is stereoview of

crystallized ligand (yellow sticks) at binding site. Sub-pockets are labeled with red letters and only signiﬁcant residues

(cyan lines) located within 4 Å radius of bound ligand are displayed and labeled with sequence number.

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Figure 11. Docking results of synthesized compounds within Bcl-2 protein target (PDB ID: 6qgk). Left panels illustrate

the proposed docking poses for the investigated compounds (magenta sticks) at the hydrophobic surface binding site of

Bcl-2 target (cyan cartoon); (A) 3a; (B) 7d; (C) 8b; (D) 8f. On the right is the overlay of docked synthesized compounds

(magenta sticks) and crystallized ligand (yellow sticks), depicting their comparative orientations within the binding site.

Only signiﬁcant residues (cyan lines) located within 4 Å radius of docked synthesized compounds are displayed and labeled

with sequence number and colored. Hydrogen bonds are displayed as black-dashed lines.

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2.5. Solid Lipid Nanoparticle Formulation Studies

In an attempt to the clinical suitability of 8b, the in-silico pharmacokinetic proper-

ties and drug-likeness of compound was investigated using free web-based tool Swis-

sADME (http://ww w .swissadme.ch/). Notably, the predicted poor water-solubility of 8b

(LogS_SILICOS-IT =

−

7.62; 1.06

×

10⁻⁵mg/mL; 2.38

×

10⁻⁸mol/L) was identiﬁed as a

signiﬁcant parameter that might hinder the compound’s full potential cytotoxic activity.

Thus, Solid lipid nanoparticles (SLNs) formulation was adopted as a simple cost-effective

approach for optimizing the compound’s kinetic parameters. The main target of SLNs is to

enhance the drug absorption, enhance the pharmacological response, and decrease the side

effects. SLNs are colloidal dispersions made of solid lipid (possessing high melting point)

and a hydrophilic surfactant [43]. SLNs are considered a new generation of submicron-

sized lipid emulsions where solid lipids are utilized instead of liquid lipids (oil) [44]. Due

to their unique characteristics, such as small size, large surface area, and increased drug

loading capacity, SLNs are attracting great attention of formulators world-wide to improve

performance and bioavailability of pharmaceuticals [45]. It was reported that SLNs have

the ability to release entrapped drug in controlled manner and enhance stability of the

entrapped drug [46]. Therefore, formulating 8b as SLNs was expected to enhance its

bioavailability at the tumor site and hence improve its cytotoxic activity.

2.5.1. Design, Preparation and Optimization of Drug-SLN

Eight drug-SLNs formulations were prepared via hot-melting homogenization tech-

nique. The formulations were designed by 2³-factorial design using Stat-Ease^®V.11 soft-

ware (Design-Expert^TM; Minneapolis, MN, USA) (Table 2). The selected factors (indepen-

dent variables) were type of lipid (A; X₁) and Surfactant (B; X₂), as well as the concentration

of surfactant (C; X₃). According to the adopted 2³-factorial design, eight formulations were

prepared and evaluated for encapsulation efﬁciency (Y₁: EE%), particle size (Y₂: PS) and

zeta potential (Y₃: ZP). Compositions of eight drug-SLNs formulations are presented in

Table 3. Drug analysis at different concentration was done using HPLC at

λ

max

254 nm,

showing linear relationship between the drug concentration and peak area, obeying Beer-

Lambert’s law (R²= 0.999) (Supplementary Materials; Figure S1).

Table 2. The formulation factors and responses of 23 factorial design for drug-solid lipid nanoparticles (SLNs).

Factors and Responses Level Used

Factor

Name

Low (−1)

High (+1)

A: X₁

B: X₂

C: X₃

Type of lipid

Type of surfactant

Surfactant conc. (% w/v)

Compritol 888 ATO (COMP)

Cremophor RH40

1%

Glyceryl Monostearate (GMS)

Poloxamer 188

1.5%

Response

Name

Goal

Y₁

Y₂

Y₃

EE (%)

PS (nm)

ZP (mV)

Maximize

Minimize

Maximize

Table 3. The eight designed drug-SLNs according to 2³-factorial design.

X₁

X₂

X₃

SNP Formulation No.

A: Lipid Type

B: Surfactant Lipid

C: Surfactant Conc. (% w/v)

1

2

3

4

5

6

7

8

COMP

GMS

COMP

GMS

Cremophor RH40

Poloxamer 188

Cremophor RH40

Poloxamer 188

Cremophor RH40

Poloxamer 188

1.5

1

1.5

1

GMS

1.5

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2.5.2. The Effect of Formulation Factors in the Responses

The prepared formulations of drug-SLNs were evaluated for the preselected responses;

Encapsulation Efﬁciency (Y1: EE%), the particle size (Y2: PS) and zeta potential (Y3: ZP).

As represented in Table 4, it was found that there was a difference in the results of the

responses which gave an indication that the formulation factors have a great effect of

the responses.

Table 4. The measured responses of prepared drug-SLNs according to 2³factorial design.

SNP Formulation

No.

(Y1)

EE%

(Y2)

PS (nm)

(Y3)

ZP (mV)

Polydispersity

Index (PDI)

F1

F2

F3

F4

F5

F6

F7

F8

37.1 ± 2.45

53.4 ± 1.65

95.3 ± 1.34

82.8 ± 2.55

86.8 ± 3.32

67.7 ± 1.52

94.6 ± 2.67

92.8 ± 2.38

140.9 ± 3.2

135.1 ± 1.0

329.4 ± 6.1

189.8 ± 3.3

181.5 ± 2.1

100.4 ± 0.4

537.3 ± 10.4

226.1 ±10.5

−12.3 ± 0.77

−13.6 ± 0.32

−21.7 ± 0.15

−36.1 ± 1.90

−16.4 ± 1.10

−39.4 ± 0.095

−37.5 ± 0.58

−29.3 ± 1.17

0.352 ± 0.03

0.282 ± 0.01

0.563 ± 0.03

0.432 ± 0.04

0.442 ± 0.02

0.433 ± 0.01

0.582 ± 0.04

0.639 ± 0.12

With the prepared formulations of drug-SLNs, signiﬁcant impact on the preselected

responses was depicted in response to the formulation factors. The EE% of all prepared

drug-SLNs ranged from 37.1

model Equation (1), the EE% was increased by increasing the level of X1 and X2 from

to +1. These results infer that the formulations prepared by GMS and Poloxamer 188 had

a higher EE% than those prepared using COMP or Cremophor RH40. Moreover, EE%

showed an inverse relationship with X3: surfactant concentration where higher EE% was

seen with 1.5% w/v surfactant.

±

2.45% for F1 to 95.3

±

1.34% for F3. As represented by

−

1

Y₁(EE%) = 76.31 + 8.16 X₁+ 16.06 X₂− 5.21 X₃

(1)

For better illustration, a 3D-response surface plot showing the impact of formulation

factors on EE% was constructed (Figure 12). Interestingly, GMS-prepared SLNs exhib-

ited higher EE% than those constituted by COMP. These ﬁndings might be for the large

encapsulation space in case of GMS resulting from less-ordered SLNs structure by the

virtue of their long carbon chains (C21) [10,30]. Additionally, higher EE% was assigned

for Poloxamer 188-prepared SLNs as compared to Cremophor RH40 which may be cor-

related to the higher hydrophilic-lipophilic balance (HLB) value proposed by Poloxamer

188 [47]. Latter ﬁndings are in good agreement with Qushawy et al., where the prepared

carbamazepine-SLNs depicted increased EE% through using GMS and Poloxamer 188

as solid lipid and surfactant, respectively [48]. The increase of surfactant concentration

from 1% to 1.5% resulted in decreased EE% which may be for increased drug solubility

within the aqueous phase [49]. The same results were depicted by Joseph et al. where

olanzapine-SLNs EE% showed an inverse relationship with surfactant concentration [50].

Regarding the PS of all prepared drug-SLNs, values ranged from 135.1

±

1.0 nm for

F2 to 537.3 10.4 nm for F7. The model Equation (2) of PS, revealed that PS had a direct

±

relationship with X1 and X2 while, an inverse relationship with X3. As shown by Figure 13,

the 3D response surface plot studied the impact of formulation factors (X1, X2 and X3) on

PS of the prepared SLNs. It was found that PS was increased in case of CMS than in case of

COMP which might be correlated to the fact of using solid lipid with high melting point,

resulted in slow crystallization and large particle size [51]. The results in good agreement

with Priyanka and Hasan found that the particle size of prepared montelukast SLNs was

inﬂuenced by lipid type and the decreasing order of particle size for the three lipids was

Compritol < GMS < stearic acid [52].

Y2 (PS) = 250.06 + 33.34 X₁+ 88.51 X₂− 67.84 X₃

(2)

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(A)

(B)

(C)

Figure 12. 3D-response surface plots showing combined effect of formulation factors on encapsulation efﬁciency (EE%;

Y₁). The adopted 2³model analysis evaluated two variable parameters while keeping (

A) type of lipid (X₁); (B) type of

surfactant (X₂); (C) concentration of surfactant (X₃) constant.

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(A)

(B)

(C)

Figure 13. 3D-response surface plots showing combined effect of formulation factors on particle size (PP; Y₂). The adopted

2³model analysis evaluated two variable parameters while keeping (

A) type of lipid (X₁); (B) type of surfactant (X₂);

(C) concentration of surfactant (X₃) constant.

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The PS of the prepared SLNs was also affected by surfactant type, where using

Poloxamer 188 resulted in SLNs larger sizes than those prepared by Cremophor RH40.

This may be reasoned to the conception of using surfactant with higher HLB can result

in preparation of SLNs with larger size [53]. Additionally, as the surfactant concentration

elevated from 1% to 1.5%, the particle size of SLNs decreased. This might be assigned

to reduction within interfacial tension between the emulsion phases by increasing the

concentration of surfactant which led to smaller PS of SLNs after congealing [50,52].

Moving towards the ﬁnal parameter, The ZP of all prepared drug-SLNs was ranged

from

−

12.3

±

0.77 mV for F1 to

−

39.4

±

0.095 mV for F6. zeta potential (ZP) is deﬁned as

potential difference existing within the stationary layer between the dispersion medium

and the dispersed phase (solid particles) [54]. The value of ZP provides indication for the

preparation stability, where higher values correlate to higher formulation stability [55].

Within the presented model Equation (3), the ZP of prepared drug-SLNs increased by

increasing the level of lipid and surfactant types yet decreased by increasing the level of

surfactant concentration.

Y₃(ZP) = 25.79 + 9.79 X₁+ 0.4375 X₂− 1.44 X₃

(3)

Corresponding 3D-response surface plot showed the effect factors on ZP responses

(Figure 14). The type of lipid had a signiﬁcant impact on negative value of ZP. The value

was increased by using GMS than in case of COMP which might be related to longer

carbon-chain of GMS resulted in the larger size of SLNs and larger surface area. It was

found that negative zeta potential of the prepared SLNs was slightly decreased by using

Cremophor RH40 while increased in case of Poloxamer 188 which may be attributed to

differences within respective HLB values [56]. Moreover, ZP was decreased with increased

surfactant concentration which may be due to the masking of surface charge by increasing

the surfactant concentration [50

drug-SLNs ranged from 0.282

from PDI values indicated a narrow size distribution [54].

,

±

57]. It worth mentioning that the PDI values of all prepared

0.01 to 0.639 0.12 for F2 and F8, respectively. Findings

±

2.5.3. Optimization of Formulation Variables to Select the Best Formula

Purpose of optimization was to maximize EE%, minimize PS, as well as maximize

values of the ZP. The Design-Expert V.11 software was used for obtaining an optimum

level of each single formulation factor to achieve the desired goals for every response

and obtain the optimized formulation of drug-SLNs. According to 2³-factorial design,

formulation F8 was the optimized formulation in which prepared using GMS as a solid

lipid and poloxamer 188 as a surfactant in concentration 1.5%. The predicted values of

responses for the optimized formulation were 95.325% for EE% (Y1), 284.075 nm for PS

(Y2), and

−

34.575 mV for ZP (Y3). Interestingly, these predicted values were close to

the actual values of responses with desirability value 0.781 which indicate the validity of

2³-factorial design. The transmission electron microscopic (TEM) image of F8 illustrated

the spherical shape of the prepared SLNs within the nano size (Figure 15A). Size analysis

for TEM image, using Nano-Measurer^®V.1.2 software (Shanghai, China), exhibited narrow

size distribution (Figure 15 B). These ﬁndings were in good agreement with Qushawy et al.

preparing carbamazepine-SLNs using GMS and stearic acid where the prepared SLNs were

spherical in shape with nano size [44].

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(A)

(B)

(C)

Figure 14. 3D-response surface plots showing combined effect of formulation factors on zeta potential (ZP; Y₃). The

adopted model analysis evaluated two variable parameters while keeping (A) type of lipid (X₁); (B) type of surfactant (X₂);

(C) concentration of surfactant (X₃) constant.

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(A)

(B)

Figure 15. The surface morphology of the optimized formulation (F8). (A) TEM image; (B) size distribution analysis of

TEM image.

2.5.4. In-Vitro Release Study of Optimized Formulation (F8) in Comparison with 8b

The in-vitro drug release proﬁle of drug-SLNs was conducted for evaluating the

stability and release behavior of drug-SLNs. The drug release from F8 was prolonged over

48 h, where the total released amount of drug from F8 was 91.47

±

2.78%, while being

21.48 ± 1.17% for 8b (p < 0.001) (Figure 16). Thus, drug-SLNs may be expected to assist as

stable nanoparticles for prolonged time and help in increasing the accumulation of drug in

tumor site [58]. These results may be attributed to presence of the drug in more solubilized

form and the prolonged effect might be the result of the drug diffusion from the lipid

matrix [59].

100

80

60

40

20

0

8b

F8

0

10

20

30

40

50

Time (hr)

Figure 16. In-vitro drug release analysis of drug-optimized SLNs formula (F8) in comparison with 8b as free drug form. All

data are represented as mean of three independent experiments ± SEM.

2.5.5. Cytotoxicity Study of Optimized Formulation

The cytotoxic efﬁcacy of compound 8b was assayed in its pure form and in optimized

SLN formulation (F8). From the Sulforhodamine B (SRB) assay, the compound’s IC₅₀within

MDA-MB-231/breast cancer cell line was signiﬁcantly decreased from 12.43 ± 0.50 µM

to 9.27

±

0.34 µM for 8b and formulated form, respectively (p < 0.05) (Figure 17). Based

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on the fact that 8b is lipophilic (Consensus LogP_o/w= 5.16) with poor aqueous solubility,

it was predicted to possess low bioavailability [60]. Incorporation of the drug into lipid-

based nanoparticles might offer signiﬁcant improvement in its anticancer efﬁcacy as the

lipid nature of the SLN increases its solubility and allows the presence of the drug in its

amorphous form. The latter might cause an increase within the drug 8b penetration into the

tumor cells [61]. Wang and colleagues found similar results when evaluated the cytotoxic

effect of Resveratrol on MDA-MB-231/breast cancer cell line [62]. The authors mentioned

that the enhanced cytotoxic effect of Resveratrol SLNs as compared to pure resveratrol may

be due to the carrier hydrophobic nature facilitating the intra-cellular uptake.

Figure 17. The Sulforhodamine B (SRB) cytotoxicity assay of the tested 8b as compared to drug-

optimized SLNs formula (F8) on MDA-MB-231/breast cancer cells. At 5

cells were treated with various 8b concentrations (0.01, 0.1, 1, 10, and 100

×

10³cells/well cell density,

M) for 72 h exposure time

µ

frame. Cytotoxic effect was detected via protein-bound SRB colorimetric assay (540 nm) and % cell

growth viability was estimated in triplicates as compared to vehicle negative controls representing

the untreated cells.

3. Materials and Methods

3.1. General Experimental

All chemicals, reagents and solvents were purchased from Sigma-Aldrich, Fisher

Scientiﬁc, Alfa-Aesar, Fluka and Acros Chemicals. Whenever required, solvents were

dried prior to use as described by the handbook Puriﬁcation of Laboratory Chemicals

and stored over 4Å molecular sieves under nitrogen. Flash column chromatography

was performed with silica gel (230–400 mesh) (Merck) and TLC was performed on pre-

coated silica gel plates (Merck Kiesel gel 60F254, BDH). Melting points were determined

on an electrothermal instrument (Gallenkamp) and are uncorrected. Compounds were

visualized by irradiation with UV light at 254 nm and 365 nm. The NMR spectra of all

new compounds were recorded on a Bruker AVANCE DPX500 spectrometer operating at

500 and 125 MHz for ¹H and ¹³C NMR, respectively, and auto calibrated to the deuterated

solvent reference peak (Supplementary Materials; Figure S2). Chemical shifts are given in

δ

relative to tetramethylsilane (TMS); the coupling constants (J) are given in Hertz. TMS

was used as an internal standard (

= 0 ppm) for ¹H NMR and CDCl₃served as an internal

standard (

= 77.0 ppm) for ¹³C NMR. Multiplicity is denoted as s (singlet), d (doublet),

δ

t (triplet), q (quartet), m (multiplet) or combinations thereof. The compounds imines

11a–f and aldehydes 12a–f were prepared according to Kishk et al. [29]. The DI Analysis

Shimadzu QP2010-Plus^®GC/MS (Shimadzu^TM, Tokyo, Japan) was adopted for recording

the low-resolution mass spectra (MS) of the synthesized compounds at electron impact

(EI⁺) mode. Regarding purity analysis, the elemental analyses were recorded on Vario^®

EL-CHNS Elemental Analyzer (GmbH^TM, Hanau, Germany). The results of elemental

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analyses (C, H, N) were found to be in good agreement (

All compounds were >95% pure.

±

0.45%) with the calculated values.

3.2. Chemical Synthesis

3.2.1. Synthesis of 2-(chloromethyl)-1H-Benzimidazole (2)

A mixture of ortho-phenylene diamine (1.0 g, 9.25 mmol) and chloroacetic acid (1.32 g,

13.97 mmol) in 4 N HCl (60 mL) was reﬂuxed for 24 h. The puriﬁed product was obtained by

re-crystallization from water after neutralization with 6 N NH₄OH, yield 0.4 g (40%). The

product was used in the following step without further puriﬁcation [63]. M.p. 149–151 ^◦C

(Lit. 147.8–148.2 ^◦C). ¹H NMR (DMSO-d₆):

and 7.57–7.59 (m, 2H, CH, Ar).

δ 4.91 (s, 2H, CH₂), 7.25–7.29 (m, 2H, CH, Ar),

3.2.2. Synthesis of 2-Aminomethyl-Benzimidazole Derivatives (3a–d)

A mixture of compound ( ) (1 mmol) and the appropriate aromatic amine (1 mmol) in

2

the presence of potassium iodide (1 mmol) were reﬂuxed in absolute ethanol for 6 h, then

potassium iodide (1 mmol) dissolved in 5 mL water was added and reﬂuxed overnight.

After cooling to room temperature, the solution was poured into ice, ﬁltered and crude prod-

uct was puriﬁed by ﬂash column chromatography using gradient EtOAc: n-hexane [26].

N-((1H-benzo[d]imidazol-2-yl)methyl)-3-methylaniline (3a).

Prepared from 3-methylaniline, yield 0.07 g (71%), as an off-white powder. M.p.

139–141 ^◦C. TLC (EtOAc: n-Hexane 2:3), R_f: 0.23. ¹H NMR (DMSO-d₆):

δ 2.39 (m, 3H,

CH₃), 4.46 (d, J = 5.38 Hz, 2H, CH₂), 6.17 (t, J = 7.2 Hz, 1H, NH), 6.50 (s, 1H, NH), 6.96

(s, 1H, CH, Ar), 7.28 (d, J = 7.2 Hz, 2H, CH, Ar), 7.49 (m, 1H, CH, Ar), 7.56 (t, J = 7.4 Hz,

2H, CH, Ar), 7.76 (d, J = 7.4 Hz, 2H, CH, Ar). ¹³C NMR (DMSO-d₆):

δ

21.79 (CH₃), 42.29

(CH₂), 110 (2

×

CH, Ar), 113.6 (2

×

CH, Ar), 117.8 (CH, Ar), 129.2 (2

×

CH, Ar), 129.75 (CH,

Ar), 138.34 (3C, Ar), 143.4 (C, Ar). MS (EI⁺) m/z: 237.38 [M⁺]. Anal. Calcd for C₁₅H₁₅N₃

(237.31): C, 75.92; H, 6.38; N, 17.72. Found: C, 75.81; H, 6.22; N, 17.60

N-((1H-benzo[d]imidazol-2-yl)methyl)-2,6-dimethylaniline (3b).

Pre_◦pared from 3,5-dimethylaniline, yield 0.06 g (61%), as an off-white powder. M.p.

159–161 C. TLC (EtOAc: n-Hexane 1:2), R_f: 0.5. ¹H NMR (DMSO-d₆):

δ

2.30 (m, 6H, 2 × CH₃)

,

4.36 (s, 2H, CH₂), 4.54 (s, 1H, NH), 6.81–6.89 (m, 1H, Ar), 6.94 (d, J = 7.34 Hz, 2H, Ar), 7.16–7.19

(m, 2H, Ar), 7.54 (d, J = 7.5 Hz, 2H, Ar). ¹³C NMR (DMSO-d₆):

18.9 (2 × CH₃), 46.2 (CH₂),

CH, Ar), 129.6 (2C, Ar), 138.8

δ

111.8 (2

×

CH, Ar), 118.8 (CH, Ar), 122 (2

×

CH, Ar), 129.3 (2

×

(2C, Ar), 146.1 (C, Ar), 154.4 (C, Ar). MS (EI⁺) m/z: 251.17 [M⁺]. Anal. Calcd for C₁₆H₁₇N₃

(251.33): C, 76.46; H, 6.82; N, 16.72. Found: C, 76.37; H, 6.89; N, 16.73.

N-((1H-benzo[d]imidazol-2-yl)methyl)-3-bromoaniline (3c) [64].

Prepared from 3-bromoaniline, yield 0.07 g (73%), as a light brown powder. M.p.

1

139–141 ^◦C. TLC (EtOAc: n-hexane 1:2), R_f0.27. H NMR (DMSO-d₆): δ 4.16 (s, 2H, CH₂),

7.21–7.24 (m, 2H, Ar), 7.57 (d, J = 7.4 Hz, 2H, Ar), 7.59 (s, 1H, NH), 7.75 (d, J = 7.5 Hz, 1H,

Ar), 7.91–8.12 (m, 3H, Ar). ¹³C NMR (DMSO-d₆):

δ

41.9 (CH₂), 111.7 (2

×

CH, Ar), 115.1

(

2 × CH, Ar), 119.1 (CH, Ar), 121.8 (CH, Ar), 122.7 (2 ×

CH, Ar), 131.1 (1C, Ar), 150.5 (2C,

Ar), 153.5 (1C, Ar), 169. (1C, Ar). MS (EI⁺) m/z: 302.72 [M⁺]. Anal. Calcd for C₁₄H₁₂BrN₃

(302.18): C, 55.65; H, 4.01; N, 13.92. Found: C, 55.62; H, 3.93; N, 13.51.

4-(((1H-benzo[d]imidazol-2-yl)methyl)amino)phenol (3d).

Prepared from 4-hydroxyaniline, yield 0.05 g (51%), as a brown powder. M.p.139–141 ^◦C

.

TLC (EtOAc: n-hexane 1:2), R_f0.4. ¹H NMR (DMSO-d₆):

2H, Ar), 7.32 (d, J = 7.4 Hz, 2H, Ar), 7.47–7.49 (m, 1H, CH, Ar), 7.57 (d, J = 7.5 Hz, 2H, Ar),

7.59 (s, 1H, NH), 7.75 (d, J = 7.5 Hz, 1H, Ar), 9.45 (s, 1H, OH), 12.18 (s, 1H, NH-indole). ¹³

NMR (DMSO-d₆): 43.6 (CH₂), 115.2 (2 CH, Ar), 116.3 (2 CH, Ar), 116.7 (2 CH, Ar),

124.1 (2

δ 4.14 (s, 2H, CH₂), 7.22 (d, J = 7.5 Hz,

C

δ

×

CH, Ar), 139.1 (2C, Ar), 141.5 (1C, Ar), 142.1 (1C, Ar), 146.9 (1C, Ar). MS (EI⁺) m/z:

239.11 [M⁺]. Anal. Calcd for C₁₄H₁₃N₃O (239.28): C, 70.28; H, 5.48; N, 17.56. Found: C, 70.29;

H, 5.49; N, 17.45.

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3.2.3. Synthesis of Ethyl, 1H-Indole-2-Carboxylate (5)

To a stirred solution of 1H-indole-2-carboxylic acid (

4) (1.5 g, 9.31 mmol) in dry ethanol

(25 mL), sulfuric acid (0.5 mL) was added as catalyst. The reaction mixture was reﬂuxed

for 1.5 h. After the reaction was complete, water (25 mL) was added, and the mixture

was extracted with ethyl acetate (3

with brine, water, and dried over anhydrous Na₂SO₄. The solvent was evaporated under

×

50 mL). The combined organic phases were washed

◦

vacuum to give ethyl-1H-indole-2-carboxylate (

6

) as a white powder [65]. M.p. 126–127 C

(Lit. M.p. 123–124 ^◦C). ¹H NMR (CDCl₃)

δ

1.43 (t, J = 7.4 Hz, 3H, CH₃), 4.51 (q, J = 7.2 Hz,

2H, CH₂), 6.96–6.99 (m, 1H, Ar), 7.12 (d, J = 7.0 Hz, 2H, Ar), 7.35 (d, J = 7.2 Hz, 2H, Ar),

9.31 (s, 1H, NH).

3.2.4. Synthesis of 1H-Indole-2-Carbohydrazide (6)

To a stirred solution of ethyl 1H-indole-2-carboxylate (5) (0.46 g, 7.93 mmol) was added

hydrazine monohydrate (4 mL, 79.90 mmol) in the presence of 15 mL of absolute ethanol.

The reaction mixture was refluxed for 6 h. After the reaction was complete, the solution was

evaporated under vacuum, the remaining residue after evaporation (0.50 g) was washed with

dichloromethane (0.5 mL

directly in the following reaction without any further purifications [66 ]. M.p. 245–246 ^◦C (Lit.

M.p. 247–248 ^◦C), ¹H-NMR (DMSO-d₆):

4.49 (s, 2H, NH₂), 7.13 (d, J = 4.6 Hz, 1H, Ar), 7.19

×

3). The product was obtained as colorless crystals and was used

δ

(s, 1H, Ar), 7.25 (d, J = 7.4 Hz, 1H, Ar), 7.49 (d, J = 7.4 Hz, 1H, Ar), 7.65 (d, J = 7.5 Hz, 1H, Ar),

9.79 (s, 1H, NH), 11.68 (s, 1H, NH, indole).

3.2.5. Synthesis of Different Carbohydrazide Derivatives (7, 8)

Equimolar amount of appropriate aromatic aldehyde was added to a solution of the

hydrazide compound (6) (10 mmol) in absolute ethanol (5 mL), in presence of catalytic

amount of glacial acetic acid (0.4 mL). Reaction mixture was allowed to reﬂux with continu-

ous stirring for about 1.5 h and poured into ice/water mixture. The precipitate was ﬁltered,

washed with cold water and puriﬁed by ﬂash column chromatography using gradient

elution of EtOAc: n-hexane to give the corresponding carbohydrazide [67–69].

N⁰-benzylidene-1H-indole-2-carbohydrazide (7a) [67].

Prepared from benzaldehyde, yield 0.045 g (45%), as an off-white powder. M.p. 111–

1

113 ^◦C. TLC (EtOAc: n-Hexane 1:2), R_f: 0.40. H NMR (DMSO-d₆):

δ 7.08 (d, J = 7.34 Hz,

2H, CH, Ar), 7.28 (s, 1H, CH, indole), 7.54–7.59 (m, 5H, CH, Ar), 7.78 (d, J = 6.11 Hz, 2H,

CH, Ar), 8.47 (s, 1H, CH=N), 11.76 (s, 1H, NH, indole), 11.92 (s, 1H, NH, amide). ¹³C

NMR (DMSO-d₆):

(CH, Ar), 128.8 (2

139.8 (C, Ar), 146.8 (2

δ

×

111.1 (CH, Ar), 114.9 (CH, Ar), 119.8 (CH, Ar), 120.7 (CH, Ar), 121.7

CH, Ar), 129.3 (2 CH, Ar), 131.3 (C, Ar), 133.7 (C, Ar), 138.5 (C, Ar),

CH, Ar), 157.6 (C=O). MS (EI⁺) m/z: 263.55 [M⁺]. Anal. Calcd for

×

C₁₆H₁₃N₃O (263.30): C, 72.99; H, 4.98; N, 15.96. Found: C, 72.92; H, 5.03; N, 16.14

N⁰-(4-methoxybenzylidene)-1H-indole-2-carbohydrazide (7b) [69].

Prepared from 4-methoxybenzaldehyde, yield 0.03 g (30%), as an off-white powder.

◦

M.p. 104–106 C (Lit. M.p. 98.9 C). TLC (EtOAc: n-Hexane 1:2), R_f: 0.35. ¹H NMR

(DMSO-d₆):

7.56–7.63 (m, 5H, CH, Ar), 7.82 (d, J = 6.15 Hz, 2H, CH, Ar), 8.49 (s, 1H, CH=N), 11.79 (s,

1H, NH, indole), 11.97 (s, 1H, NH, amide). ¹³C NMR (DMSO-d₆):

55.3 (CH₃), 111.3 (CH,

Ar), 115.2 (CH, Ar), 120.1 (CH, Ar), 120.8 (CH, Ar), 121.6 (CH, Ar), 129.1 (2 CH, Ar), 129.5

δ 4.33 (s, 3H, CH₃), 7.18 (d, J = 7.33 Hz, 2H, CH, Ar), 7.29 (s, 1H, CH, indole),

δ

×

(2

× CH, Ar), 131.7 (C, Ar), 133.5 (C, Ar), 138.4 (C, Ar), 140.1 (C, Ar), 146.5 (2 × CH, Ar),

157.4 (C=O). MS (EI⁺) m/z: 293.87 [M⁺]. Anal. Calcd for C₁₇H₁₅N₃O₂(293.30): C, 69.61; H,

5.15; N, 14.30. Found: C, 69.9; H, 5.11; N, 14.35

N⁰-(4-(dimethylamino)benzylidene)-1H-indole-2-carbohydrazide (7c) [69].

Prepared from 4-(dimethylamino)benzaldehyde, yield 0.04 g (35%), as a yellow pow-

der. M.p. 90–92 C (Lit. M.p. 82 C). TLC (EtOAc: n-Hexane 1:2), R_f: 0.35. ¹H NMR

◦

(DMSO-d₆):

dole), 7.19 (d, J = 7.34 Hz, 2H, CH, Ar), 7.24 (d, J = 7.34 Hz, 2H, CH, Ar), 7.55 (d, J = 7.35 Hz

2H, CH, Ar), 7.63–7.65 (m, 1H, CH, Ar), 8.31 (s, 1H, CH=N), 11.64 (s, 1H, NH, indole), 11.82

δ

3.05 (m, 6H, 2

×

CH₃), 6.77 (t, J = 7.33 Hz, 1H, CH, Ar), 7.07 (s, 1H, CH, in-

,

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(s, 1H, NH, amide). ¹³C NMR (DMSO-d₆):

δ

41.3 (2

×

CH₃), 103.1 (CH, Ar), 111.8 (2

×

CH,

Ar), 112.3 (CH, Ar), 119.9 (CH, Ar), 121.6 (CH, Ar), 121.8 (CH, Ar), 123.6 (C, Ar), 127.1 (CH,

Ar), 128.4 (CH, Ar), 130.4 (C, Ar), 136.8 (2C, Ar), 148.1 (CH, Ar), 151.4 (C, Ar), 157.2 (C=O).

MS (EI⁺) m/z: 306.82 [M⁺]. Anal. Calcd for C₁₈H₁₈N₄O (306.37): C, 70.57; H, 5.92; N, 18.29.

Found: C, 70.51; H, 5.94; N, 17.91.

N⁰-(pyridin-3-ylmethylene)-1H-indole-2-carbohydrazide (7d) [68].

Prepared from nicotinaldehyde, yield 0.03 g (29%), as an off-white powder. M.p.

1

252–254 ^◦C (Lit. M.p. 250–251 ^◦C). TLC (EtOAc: n-Hexane 1:2), R_f: 0.29. H NMR (DMSO-

d₆):

CH, Ar), 7.66 (s, 1H, CH, Ar), 7.78 (d, J = 7.44 Hz, 2H), 8.34 (s, 1H, CH=N), 11.66 (s, 1H,

NH, indole), 11.83 (s, 1H, NH, amide). ¹³C NMR (DMSO-d₆):

103.8 (CH, Ar), 112.4 (CH,

Ar), 120.1 (CH, Ar), 121.9 (CH, Ar), 124.1 (CH, Ar), 126.9 (CH, Ar), 129.8 (C, Ar), 130.2 (C,

δ 6.23 (t, J = 7.56 Hz, 2H, CH, Ar), 7.44–7.64 (m, 4H, CH, Ar), 7.33 (d, J = 7.56 Hz, 1H,

δ

Ar), 133.4 (CH, Ar), 136.9 (C, Ar), 144.3 (C, Ar), 148.7 (CH, Ar), 150.7 (2

×

CH, Ar), 157.7

(C=O). MS (EI⁺) m/z: 264.75 [M⁺]. Anal. Calcd for C₁₅H₁₂N₄O (264.29): C, 68.17; H, 4.58;

N, 21.2. Found: C, 68.21; H, 4.55; N, 20.90.

N⁰-((3-(4-ethylphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1H-indole-2- carbohy-

drazide (8a).

Prepared from 3-(4-ethylphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (12a), yield

0.04 g (41%), as an off-white powder. M.p. 254–256 ^◦C. TLC (EtOAc: n-Hexane 1:1), R_f:

1

0.42. H NMR (DMSO-d₆):

δ 1.24 (t, J = 7.56 Hz, 3H, CH₃), 2.69 (q, J = 7.56 Hz, 2H, CH₂),

6.23 (t, J = 7.22 Hz, 1H, Ar), 6.35 (t, 1H, Ar), 6.43 (s, 1H, Ar), 6.51–6.59 (m, 3H, CH, Ar), 6.71

(d, J = 8.25 Hz, 2H, CH, Ar), 6.89 (d, J = 7.56 Hz, 2H, CH, Ar), 6.95 (d, J = 8.25 Hz, 2H, CH,

Ar), 7.20 (d, J = 7.56 Hz, 2H, CH, Ar), 7.71 (s, 1H, CH, Ar), 8.33 (s, 1H, CH=N), 11.76 (s,

1H, NH, indole), 11.85 (s, 1H, NH, amide). ¹³C NMR (DMSO-d₆):

δ

15.6 (CH₃), 28.2 (CH₂),

CH, Ar), 119.9 (CH, Ar), 121.7 (CH,

CH, Ar), 126.9 (CH, Ar), 128.2 (CH), 128.5 (CH, Ar), 129.4

103.2 (CH, Ar), 112.3 (C, Ar), 116.8 (CH, Ar), 118.8 (2

Ar), 123.7 (CH, Ar), 126.8 (2

×

(3 × CH, Ar), 129.6 (C, Ar), 130.2 (C, Ar), 136.7 (2C, Ar), 139.1 (CH, Ar), 140.4 (C, Ar), 144.3

(C, Ar), 152.1 (C, Ar), 157.3 (C=O). MS (EI⁺) m/z: 233.14 [M⁺]. Anal. Calcd for C₂₇H₂₃N₅O

(433.52): C, 74.81; H, 5.84; N, 15.35. Found: C, 74.85; H, 5.49; N, 15.24.

N⁰-((1-phenyl-3-(4-propylphenyl)-1H-pyrazol-4-yl)methylene)-1H-indole-2- carbohy-

drazide (8b).

Prepared from 1-phenyl-3-(4-propylphenyl)-1H-pyrazole-4-carbaldehyde (12b), yield

0.04 g (39%), as an off-white powder. M.p. 219–221 ^◦C. TLC (EtOAc: n-Hexane 1:2), R_f:

1

0.51. H NMR (DMSO-d₆):

δ 0.93 (t, J = 7.4 Hz, 3H, CH₃), 1.65 (sext, J = 7.3 Hz, 2H, CH₂),

2.63 (t, J = 7.4 Hz, 2H, CH₂), 6.22 (t, J = 7.56 Hz, 1H, CH, Ar), 6.33 (t, J = 7.56 Hz, 1H, CH,

Ar), 6.44 (s, 1H, CH, Ar), 6.55–6.61 (m, 3H, CH, Ar), 6.69 (d, J = 8.25 Hz, 2H, CH, Ar), 6.75

(t, J = 7.90 Hz, 2H, CH, Ar), 6.81 (d, J = 8.25 Hz, 2H, CH, Ar), 7.19 (d, J = 7.56Hz, 2H, CH,

Ar), 7.73 (s, 1H, CH, Ar), 8.33 (s, 1H, CH=N), 11.76 (s, 1H, NH, indole), 11.85 (s, 1H, NH,

amide). ¹³C NMR (DMSO-d₆):

(C, Ar), 116.8 (CH, Ar), 118.8 (2

126.8 (CH, Ar), 126.9 (CH, Ar), 127.1 (CH, Ar), 128.3 (CH), 128.7 (CH, Ar), 129.4 (2

δ

×

13.7 (CH₃), 24.1 (CH₂), 37.9 (CH₂), 103.4 (CH, Ar), 112.5

CH, Ar), 119.9 (CH, Ar), 121.7 (CH, Ar), 123.8 (CH, Ar),

CH,

×

Ar), 129.5 (2

×

CH, Ar), 130.2 (C, Ar), 137.8 (2C, Ar), 139.5 (C, Ar), 140.3 (C, Ar), 143.5 (C,

Ar), 152.5 (C, Ar), 156.5 (C=O). MS (EI⁺) m/z: 447.79 [M⁺]. Anal. Calcd for C₂₈H₂₅N₅O

(447.54): C, 75.15; H, 5.63; N, 15.65. Found: C, 75.11; H, 5.89; N, 15.64.

N⁰-((3-(4-isopropylphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1H-indole-2- carbo-

hydrazide (8c).

Prepared from 3-(4-isopropylphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (12c),

◦

yield 0.03 g (39%), as an off-white powder. M.p. 219–221 C. TLC (EtOAc: n-Hexane

1

1:2), R_f: 0.52. H NMR (DMSO-d₆):

δ

1.26 (d, J = 6.9 Hz, 6H, 2

×

CH₃), 2.97 (sept, J = 6.8 Hz,

1H, CH), 7.07 (t, J = 7.56 Hz, 1H, CH, Ar), 7.16 (t, J = 7.56 Hz, 1H, CH, Ar), 7.29 (s, 1H, CH,

Ar), 7.32–7.39 (m, 3H, CH, Ar), 7.43–7.52 (m, 3H, CH, Ar), 7.61 (d, J = 7.61 Hz, 2H, CH, Ar),

7.73 (d, J = 7.58 Hz, 2H, CH, Ar), 7.94 (d, J = 7.57 Hz, 2H, CH, Ar), 8.52 (s, 1H, CH, Ar), 8.64

(s, 1H, CH=N), 11.79 (s, 1H, NH, indole), 11.93 (s, 1H, NH, amide). ¹³C NMR (DMSO-d₆):

δ

23.8 (2

×

CH₃), 33.3 (CH), 103.7 (CH, Ar), 113.1 (C, Ar), 116.9 (CH, Ar), 118.8 (2

×

CH,

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Ar), 119.9 (CH, Ar), 121.1 (CH, Ar), 123.7 (CH, Ar), 126.7 (2

×

CH, Ar), 126.9 (2

×

CH, Ar),

127.0 (2

×

CH, Ar), 128.5 (2

×

CH, Ar), 129.6 (C, Ar), 130.1 (C, Ar), 136.8 (2C, Ar), 139.1

(CH, Ar), 140.4 (C, Ar), 149.9 (C, Ar), 153.3 (C, Ar), 157.8 (C=O). MS (EI⁺) m/z: 447.45 [M⁺].

Anal. Calcd for C₂₈H₂₅N₅O (447.54): C, 75.15; H, 5.63; N, 15.65. Found: C, 75.11; H, 5.89;

N, 15.85.

N⁰-((3-(4-isobutylphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1H-indole-2- carbo-

hydrazide (8d).

Prepared from 3-(4-isobutylphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (12d), yield

0.04 g (42%), as an off-white powder. M.p. 229–231 ^◦C. TLC (EtOAc: Hexane 1:2), R_f: 0.45.

¹H NMR (DMSO-d₆):

δ

0.88 (d, J = 6.7 Hz, 6H, 2

J = 6.8 Hz, 2H, CH₂), 7.07 (t, J = 6.6 Hz, 1H, CH, Ar), 7.21 (t, J = 7.5 Hz, 1H, CH, Ar), 7.29 (s,

1H, CH, Ar), 7.35 (t, J = 6.9 Hz, 2H, CH, Ar), 7.49–7.56 (m, 2H, CH, Ar), 7.63 (d, J = 7.4 Hz

×

CH₃) 1.88 (m, 1 H, CH), 2.51 (d,

,

2H, CH, Ar), 7.68–7.72 (m, 3H, CH, Ar), 8.03 (d, J = 7.5 Hz, 2H, CH, Ar), 8.53 (s, 1H, CH,

Ar), 8.69 (s, 1H, CH=N), 11.78 (s, 1H, NH, indole), 11.92 (s, 1H, NH, amide). ¹³C NMR

(DMSO-d₆):

(CH, Ar), 118.8 (CH, Ar), 119.9 (CH, Ar), 121.7 (CH, Ar), 123.7 (2

Ar), 126.9 (CH, Ar), 127 (CH, Ar), 128.2 (CH, Ar), 129.4 (C, Ar), 129.5 (2

δ

22.1 (2

×

CH₃), 29.6 (CH), 44.3 (CH₂), 103.2 (CH, Ar), 112.4 (C, Ar), 116.8

CH, Ar), 126.8 (CH,

CH, Ar), 129.6

×

(

2 × CH, Ar), 130.1 (C, Ar), 136.8 (C, Ar), 139.1 (C, Ar), 141.3 (C, Ar), 141.8 (CH, Ar), 151.8

(2C, Ar), 157.2 (C=O). MS (EI⁺) m/z: 461 [M⁺]. Anal. Calcd for C₂₉H₂₇N₅O (461.57): C,

75.46; H, 5.90; N, 15.17. Found: C, 75.30; H, 6.11; N, 15.20.

N⁰-((3-(benzo[d][

1 ,3 ]dioxol-5-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1H-indole-2- car-

bohydrazide (8e).

Prepared from 3-(benzo[d][1,3]dioxol-5-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (12e),

yield 0.03 g (33%), as an off-white powder. M.p. 239–241 ^◦C. TLC (EtOAc: n-Hexane 1:2),

1

R_f: 0.35. H NMR (DMSO-d₆):

δ 5.29 (s, 2H, CH₂), 6.31–6.37 (m, 2H, CH, Ar), 6.49 (s, 1H,

CH, Ar), 6.57–6.62 (m, 3H, CH, Ar), 6.62 (d, J = 8.25 Hz, 1H, CH, Ar), 6.73 (d, J = 7.90 Hz,

4H, CH, Ar), 6.89 (d, J = 8.25 Hz, 1H, CH, Ar), 7.20 (s, 1H, CH, Ar), 7.71 (s, 1H, CH, Ar), 8.33

(s, 1H, CH=N), 11.76 (s, 1H, NH, indole), 11.85 (s, 1H, NH, amide). ¹³C NMR (DMSO-d₆):

δ

101.3 (CH₂), 103.3 (CH, Ar), 108.6 (CH, Ar), 108.7 (CH, Ar), 112.3 (C, Ar), 116.7 (2

×

CH,

Ar), 118.8 (C, Ar), 119.9 (CH, Ar), 121.7 (CH, Ar), 122.5 (CH, Ar), 123.7 (CH, Ar), 124.1 (CH,

Ar), 126.9 (2C, Ar), 127.2 (CH, Ar), 127.5 (CH, Ar), 130.2 (CH, Ar), 130.5 (CH, Ar), 136.8 (C,

Ar), 139.0 (C, Ar), 140.3 (CH, Ar), 147.6 (C, Ar), 147.7 (C, Ar), 151.6 (C, Ar), 157.2 (C=O).

MS (EI⁺) m/z: 449 [M⁺]. Anal. Calcd for C₂₉H₂₇N₅O (461.57): C, 69.48; H, 4.26; N, 15.58. It

was Found: H, 4.16; C, 69.03; N, 15.22.

0

N -((5-chloro-1-(3,4-dinitrophenyl)-3-methyl-1H-pyrazol-4-yl)methylene)-1H-indole-2-

carbohydrazide (8f).

Prepared from 5-chloro-1-(3,4-dinitrophenyl)-3-methyl-1H-pyrazole-4-carbaldehyde,

yield 0.04 g (37%), as an off-white powder. M.p. 204–206 ^◦C. TLC (EtOAc: n-Hexane 1:2),

1

R_f: 0.37. H NMR (DMSO-d₆):

δ 1.65 (s, 3H, CH₃), 6.24–6.26 (m, 2H, CH, Ar), 6.49 (s, 1H,

CH, Ar), 6.59 (d, J = 7.4 Hz, 2H, CH, Ar), 6.78 (d, J = 8.26 Hz, 2H, CH, Ar), 8.02 (s, 1H, CH,

Ar), 8.63 (s, 1H, CH=N), 11.76 (s, 1H, NH, indole), 11.85 (s, 1H, NH, amide). ¹³C NMR

(DMSO-d₆):

δ 18.4 (CH₃), 101.8 (CH, Ar), 103.1 (CH, Ar), 112.2 (CH, Ar), 112.3 (C, Ar), 119.6

(C, Ar), 119.8 (C, Ar), 121.3 (CH, Ar), 121.6 (CH, Ar), 123.0 (CH, Ar), 123.6 (CH, Ar), 127.0

(CH, Ar), 127.1 (CH, Ar), 130.1 (C, Ar), 130.4 (C, Ar), 136.2 (C, Ar), 136.6 (C, Ar), 147.7 (C,

Ar), 157.3 (C, Ar), 161.3 (C=O). MS (EI⁺) m/z: 467.79 [M⁺]. Anal. Calcd for C₂₀H₁₄ClN₇O₅

(467.83): C, 51.35; H, 3.02; N, 20.96. Found: C, 51.34; H, 3.04; N, 20.91

3.3. MTT-Cell Proliferation Assay and Morphological Evaluation

Evaluating the cytotoxicity of tested compounds on two cancer cell lines (A549/lung

and MDA-MB-231/breast adenocarcinoma) and non-cancerous (MDCK/kidney cells) was

performed while adopting the formerly described method with small alterations [70 ]. Cancer

cell lines were propagated within Dulbecco’s Modified Eagle Medium-High Glucose (Cat.#:

P0103; DMEM_High Glucose with Na.Pyruvate and stable Glutamine, Biowest^TM, Nuaillé,

France), while MDA-MB-231 cells were propagated within RPMI-1640 L-Glutamine medium

Pharmaceuticals 2021, 14, 113

30 of 37

(Cat.#:12-604F; Lonza-Verviers SPRL^TM, Verviers, Belgium). The culturing media were

supplemented via 1% antibiotic-antimycotic 100X (Cat.#: L0010; Biowest^TM, Nuaillé, France)

and 10% fetal bovine serum (FBS) (Cat.#: EU-000-H; Seralab^TM, West Sussex, UK). Cells

were seeded as triplicates within 96-well plate, at 1

×

10⁴cells per well density, after being

counted and viability checked using the trypan blue staining solution (Cat.# ab233465;

Abcam^TM, Cambridge, MA, USA). Seeded cells were permitted to adhere for 24 h under

5% CO₂and at 37 ^◦C incubating conditions. The assigned compounds were dissolved in

500

µL DMSO affording the stock solution (100 mM) being ready for more diluting within

the whole medium to obtain the compound’s final concentrations; 0.1, 1, 10, 100

µM for

cell treatments. Notably, the final DMSO-culture medium concentration was not allowed

to exceed 0.2% (v/v) [71 ]. Following 24 h compound-cell treatment, the medium was

substituted by fresh one and cells were permitted to develop for 48 h. At four hours

prior the end of incubation, 10

magnesium and calcium; Cat.# 17-516F, Lonza-Verviers^TM, Basel, Switzerland) were added

to all wells. Following the complete 48 h incubation, 100 L DMSO was all added to all wells

µ

L MTT Sigma-Aldrich^TM(5 mg/mL in PBS 1X without

µ

where they were subsequently centrifuged at 4000 rpm for 5 min allowing the formazan

crystals of formazan to precipitate. Color was established and intensities were recorded at

490 nm using Synergy-Neo2^®Hybrid MultiMode Plate Reader (BioTek^TM, Winooski, VT,

USA), while subtracting the multi-well plates background absorbance at 690 nm. Percentage

cell viability was estimated using the subsequent formula: % cell-viability = (average

absorbance of treated wells/average absorbance of controls) × 100.

3.4. SRB Cytotoxicity Assay

MDA-MB-231: Breast Cancer cell line was obtained from Nawah^TMScientiﬁc Inc.,

(Cairo, Egypt). Cells were maintained within DMEM provided by 100 units/mL of peni-

cillin, 100 mg/mL streptomycin, and 10% of heat-inactivated FBS within 5% (v/v) CO₂

humidiﬁed atmosphere (37 ^◦C). The validity of cell line was evaluated adopting the previ-

ously reported approach [72]. Brieﬂy, an accurate volume (100 µL) of cell line suspension

(

5 × 10³cells) was placed in 96-well plates and incubated for 24 h within complete me-

dia [73]. Cell line suspensions were then treated with another 100 µL media spiked with

various drug concentrations (0.01, 0.1, 1, 10, 100

µM/mL) and kept for 72 h. Then the

media were replaced with 150

subsequent 1 h incubation at 4 C [74]. Following incubation, TCA was removed, and cells

µ

◦

L of 10% trichloroacetic acid (TCA) for ﬁxing cells through

were washed via distilled water 5 times. Aliquots of 70 µL SRB solution (0.4% w/v; Cat.#

S1402; Sigma-Aldrich^TM, Taufkirchen, Germany) were added and cell were then incubated

in dark for 10 min at 25 ^◦C. Plates were subjected to triplicate washing via 1% acetic acid

and then permitted to be air-dried overnight [62]. Protein-bound SRB stain was extracted

by addition of tris base (150 µL, 10 mM) and the absorbance was measured at 540 nm using

FLUOstar-Omega^®microplate reader (BMG-Labtech^TMGmbH, Ortenberg, Germany) [75].

3.5. Morphological Evaluation

The impact of this tested ﬁnal compounds on the morphology of treated MDA-MB-

231/breast cancer cell line was investigated, through planting cells within 6-well plates

and subsequently incubated with 0.1, 1, 10, 100 µM of 8b for 24 h. Variations within

the cells’ morphology were identiﬁed via Olympus^®-CKX53 Inverted Metallurgical light

microscope (Olympus^TM, Center Valley, PA, USA), snapped by Olympus^®Digital Camera,

and analyzed by OLYMPUS^®Stream image analysis software [76].

3.6. Flow Cytometer Analysis

Apoptotic assay and cell cycle analysis were proceeded according to previous litera-

tures [77]. In brief, cells were planted in 1.0

×

10⁶cells/ﬂask density for 24 h. Subsequently,

8b was added at its IC₅₀value and incubated for 48 h. Following incubation, MDA-MB-231

cells were trypsinized, harvested, and ﬁxed according to information cited within Annexin^®

V-FITC Detection Kit (Cat.#: K101-25, BioVision^TM, Milpitas, CA, USA), for quantifying

Article Doi

Design, synthesis, anticancer activity, and solid lipid nanoparticle formulation of indole-and benzimidazole-based compounds as pro-apoptotic agents targeting bcl-2 protein

DOI: 10.3390/ph14020113

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Authors:

Article abstract of DOI:10.3390/ph14020113

Full text of DOI:10.3390/ph14020113

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