Synthesis, crystal structures, antiproliferative activities and reverse docking studies of eight novel Schiff bases derived from benzil

research paper s

Synthesis, crystal structures, antiproliferative

activities and reverse docking studies of eight

novel Schiff bases derived from benzil

ISSN 2053-2296

Xue-Jie Tan,^a,b* Di Wang,^aXiao-Ming Hei,^a,bFeng-Cun Yang,^aYa-Ling Zhu,^a

Dian-Xiang Xing^a,band Jian-Ping Ma^c

Received 5 June 2019

^aSchool of Chemistry and Pharmaceutical Engineering, Qilu University of Technology (Shandong Academy of Sciences),

Jinan, Shandong Province 250353, People’s Republic of China, ^bKey Laboratory of Pulp and Paper Science & Technology

of Ministry of Education/Shandong Province, Qilu University of Technology (Shandong Academy of Sciences), Jinan,

Shandong Province 250353, People’s Republic of China, and ^cCollege of Chemistry, Chemical Engineering and Materials

Science, Collaborative Innovation Centre of Functionalized Probes for Chemical Imaging, Key Laboratory of Molecular

and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, People’s Republic of China.

*Correspondence e-mail: tanxuejie@163.com

Accepted 19 November 2019

Edited by F. A. Almeida Paz, University of

Aveiro, Portugal

Keywords: antiproliferative activity; Schiff base;

benzil; indole; reverse docking; crystal structure;

quantum chemistry.

Eight novel Schiff bases derived from benzil dihydrazone (BDH) or benzil

monohydrazone (BMH) and four fused-ring carbonyl compounds (3-formyl-

indole, FI; 3-acetylindole, AI; 3-formyl-1-methylindole, MFI; 1-formylnaph-

thalene, FN) were synthesized and characterized by elemental analysis, ESI–

CCDC references: 1966764; 1966763;

1966762; 1966761; 1966760; 1966759;

1966758; 1018144

1

QTOF–MS, H and ¹³C NMR spectroscopy, as well as single-crystal X-ray

Supporting information: this article has

diffraction. They are (1Z,2Z)-1,2-bis{(E)-[(1H-indol-3-yl)methylidene]hydra-

zinylidene}-1,2-diphenylethane (BDHFI), C₃₂H₂₄N₆, (1Z,2Z)-1,2-bis{(E)-[1-

(1H-indol-3-yl)ethylidene]hydrazinylidene}-1,2-diphenylethane (BDHAI),

C₃₄H₂₈N₆, (1Z,2Z)-1,2-bis{(E)-[(1-methyl-1H-indol-3-yl)methylidene]hydrazin-

ylidene}-1,2-diphenylethane (BMHMFI) acetonitrile hemisolvate, C₃₄H₂₈N₆ꢀ-

0.5CH₃CN, (1Z,2Z)-1,2-bis{(E)-[(naphthalen-1-yl)methylidene]hydrazinylidene}-

1,2-diphenylethane (BDHFN), C₃₆H₂₆N₄, (Z)-2-{(E)-[(1H-indol-3-yl)methyl-

idene]hydrazinylidene}-1,2-diphenylethanone (BMHFI), C₂₃H₁₇N₃O, (Z)-2-{(E)-

[1-(1H-indol-3-yl)ethylidene]hydrazinylidene}-1,2-diphenylethanone (BMHAI),

C₂₄H₁₉N₃O, (Z)-2-{(E)-[(1-methyl-1H-indol-3-yl)methylidene]hydrazinylidene}-

1,2-diphenylethanone (BMHMFI), C₂₄H₁₉N₃O, and (Z)-2-{(E)-[(naphthalen-1-

yl)methylidene]hydrazinylidene}-1,2-diphenylethanone (BMHFN) C₂₅H₁₈N₂O.

Moreover, the in vitro cytotoxicity of the eight title compounds was evaluated

against two tumour cell lines (A549 human lung cancer and 4T₁mouse breast

cancer) and two normal cell lines (MRC-5 normal lung cells and NIH 3T3

ﬁbroblasts) by MTT assay. The results indicate that four (BDHMFI, BDHFN,

BMHMFI and BMHFN) are inactive and the other four (BDHFI, BDHAI,

BMHFI and BMHAI) show severe toxicities against human A549 and mouse

4T₁cells, similar to the standard cisplatin. All the compounds exhibited weaker

cytotoxicity against normal cells than cancer cells. The Swiss Target Prediction

web server was applied for the prediction of protein targets. After analyzing the

differences in frequency hits between these active and inactive Schiff bases, 18

probable targets were selected for reverse docking with the Surﬂex-dock

supporting information at journals.iucr.org/c

´

function in SYBYL-X 2.0 software. Three target proteins, i.e. human ether-a-go-

go-related (hERG) potassium channel, the inhibitor of apoptosis protein 3 and

serine/threonine-protein kinase PIM1, were chosen as the targets. Finally, the

ligand-based structure–activity relationships were analyzed based on the

putative protein target (hERG) docking results, which will be used to design

and synthesize novel hERG ion channel inhibitors.

1. Introduction

Schiff bases are important compounds in chemistry and

biochemistry due to their ﬂexibility (Mayans et al., 2018;

Pramanik et al., 2018), easy preparation (Erxleben, 2018;

# 2020 International Union of Crystallography

Acta Cryst. (2020). C76

https://doi.org/10.1107/S2053229619015687 1 of 20

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Ganguly et al., 2014), exceptional chelating ability (Malik et al.,

2018; Vardhan et al., 2015) and to their broad spectrum of

biological and pharmaceutical activities, such as antimicrobial

(Anush et al., 2018; Unver & Bektas, 2018; Bharathi et al.,

2018; Carren˜o et al., 2018), anti-inﬂammatory (Venkatesan et

al., 2018; Farag et al., 2017; Bano et al., 2017; Khayyat et al.,

2015) and anticancer properties (Unver & Bektas, 2018;

Santhosh Kumar et al., 2018; Kalaiarasi et al., 2018; Ariyaeifar

et al., 2018). Thus, the chemistry of Schiff bases has always

been a promising ﬁeld of research.

convulsant (Saini et al., 2016; Ndagijimana et al., 2013; Ma et

al., 2016; Ensch et al., 2018) and many other therapeutic and

pharmacological properties. Numerous pharmaceutical mol-

ecules with the indole group have been marketed. These

include indomethacin, zaﬁrlukast, sumatriptan, indole-3-acetic

acid (IAA), serotonin, delavirdine, atevirdine etc. There are

also large numbers of indole-containing drugs currently going

through different clinical phases (Naim et al., 2016). Moreover,

the indole nucleus is commonly found in several natural

products and displays an indispensable role in therapeutic

chemistry (Tzvetkov et al., 2014; Gurer-Orhan et al., 2016;

Kumar et al., 2016; Johansson et al., 2013; Blunt et al., 2011; Gul

& Hamann, 2005; Sugiyama et al., 2009; Bao et al., 2007;

Shaaban et al., 2002; Shaaban & Abdel-Aziz, 2007; Ali Khan et

al., 2018; Ndagijimana et al., 2013). Although indole deriva-

tives have been the target of synthetic exploration for many

years, more anticancer compounds incorporating the indole

scaffold with improved anticancer properties are desired for

the systematic study of structure–activity relationships

(Hassam et al., 2012; Dai et al., 2016; Singh et al., 2018).

As an organic helical molecule with a long history (De et al.,

2006; Fisher & Stoufer, 1966), benzil dihydrazone (BDH) is

often used to construct polydentate diazine Schiff bases (Tan

et al., 2015), helical coordination complexes (Bai et al., 2012;

Drew et al., 2007; Mukherjee et al., 2013) and, more impor-

tantly, a leading scaffold for the further design and synthesis of

potential anticancer agents (Ke et al., 2013). Here we aimed to

use the BDH scaffold to construct some novel Schiff bases,

especially those containing indole rings. Another set of novel

Schiff bases based on benzil monohydrazone (BMH) were

also constructed for comparison.

´

As a privileged structure scaffold (de Sa Alves et al., 2009;

Evans et al., 1988), indole derivatives play an important role in

medicinal chemistry since they frequently exhibit broad and

remarkable biological activities. Substituted indole rings have

revealed antibacterial (El-Sawy et al., 2010; George et al.,

2008), antitumour (Pedada et al., 2016; Fortes et al., 2016; El-

´

Sawy et al., 2012, 2013; Wu et al., 2009; Pojarova et al., 2007;

Kamath et al., 2016), antifungal (Bai et al., 2018; Wang et al.,

2018; Mishra et al., 2018; Yu et al., 2018), antiviral (Cihan-

¨

˘

Ustundag et al., 2016; Brigg et al., 2016; Zhao et al., 2006;

Sellitto et al., 2010; Pu et al., 2017; Atienza et al., 2018), anti-

inﬂammatory (Mandour et al., 2010; Lamie et al., 2016), anti-

oxidant (Esteva˜o et al., 2010; Suzen & Buyukbingol, 2000; Mor

et al., 2004), antituberculosis (Naidu et al., 2016; Zhao, 2018;

Hong et al., 2017; Akula et al., 2016), analgesic (Fantinati et al.,

2017; Bertamino et al., 2018; Ali Khan et al., 2018), anti-

In order to gain a deeper insight into the antitumour activity

of various functionalized indoles, we have been conducting a

systematic study on indole derivatives with different scaffolds,

for example, novel Schiff bases derived from indole and

biphenyl show lung A549 and breast 4T₁cancer cell inhibitory

activities (IC₅₀= 20.5 and 18.5 mM, respectively) (Bu et al.,

ꢁ

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Acta Cryst. (2020). C76

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2.2.1. BDHFI. M.p. 274.0–275.0 ^ꢂC. Elemental analysis

found (calculated) for C₃₂H₂₄N₆(%): C 78.15 (78.03), H 4.98

(4.91), N 17.12 (17.06). HRMS (ESI): m/z calculated for

C₃₂H₂₄N₆+ H⁺: 493.2141 [M + H⁺]; found: 493.2142. ¹H NMR

(DMSO-d₆): 11.637 (s, 2H, –NH), 8.757 (s, 2H, –N CH),

7.878–7.825 (m, 6H, J = 7.2 Hz, Ar-H), 7.587 (d, 2H, J = 8.0 Hz,

Ar-H), 7.450 (d, 6H, J = 8.0 Hz, Ar-H), 7.330 (d, 2H, J = 8.0 Hz,

Ar-H), 7.076 (t, 2H, J = 8.0 Hz, Ar-H), 6.851 (t, 2H, J = 7.2 Hz,

Ar-H). ¹³C NMR (DMSO-d₆): 163.575 (CH N), 156.485

(CH N), 136.992 (Ar-C), 135.007 (Ar-C), 132.951 (Ar-C),

130.035 (Ar-C), 128.589 (Ar-C), 126.989 (Ar-C), 124.433

(Ar-C), 122.596 (Ar-C), 122.267 (Ar-C), 120.478 (Ar-C),

112.165 (Ar-C), 111.629 (Ar-C).

2.2.2. BDHAI. M.p. 270.2–271.7 ^ꢂC. Elemental analysis

found (calculated) for C₃₄H₂₈N₆(%): C 78.49 (78.44), H 5.47

(5.42), N 16.21 (16.14). HRMS (ESI): m/z calculated for

C₃₄H₂₈N₆+ H⁺: 521.2454 [M + H⁺]; found: 521.2456. ¹H NMR

(DMSO-d₆): 11.473 (s, 2H, –NH), 7.877–7.754 (m, 8H, J =

6.8 Hz, Ar-H), 7.452–7.271 (m, 8H, J = 6.8 Hz, Ar-H), 7.020 (s,

2H, Ar-H), 6.713 (s, 2H, Ar-H), 2.418 (s, 6H –CH₃). ¹³C NMR

(DMSO-d₆): 161.007 (CH N), 136.915 (CH N), 135.117

(Ar-C), 129.884 (Ar-C), 129.587 (Ar-C), 128.598 (Ar-C),

128.292 (Ar-C), 127.405 (Ar-C), 126.942 (Ar-C), 124.815

(Ar-C), 124.730 (Ar-C), 123.487 (Ar-C), 122.003 (Ar-C),

119.916 (Ar-C), 115.206 (Ar-C), 111.184 (Ar-C), 14.792

(–CH₃).

2.2.3. BDHMFI. M.p. 214.6–215.3 ^ꢂC. Elemental analysis

found (calculated) for C₃₄H₂₈N₆(%): C 78.51 (78.44), H 5.46

(5.42), N 16.19 (16.14). HRMS (ESI): m/z calculated for

C₃₄H₂₈N₆+ H⁺: 521.2454 [M + H⁺]; found: 521.2454. ¹H NMR

(DMSO-d₆): 8.731 (s, 2H, –N CH), 7.874 (d, 4H, J = 7.2 Hz,

Ar-H), 7.789 (s, 2H, Ar-H), 7.602 (d, 2H, J = 7.2 Hz, Ar-H),

7.474–7.373 (m, 8H, Ar-H), 7.151 (t, 2H, J = 7.2 Hz, Ar-H),

6.908 (t, 2H, J = 7.2 Hz, Ar-H), 3.733 (s, 6H –N—CH₃). ¹³C

NMR (DMSO-d₆): 163.813 (CH N), 155.643 (CH N),

137.807 (Ar-C), 137.575 (Ar-C), 136.220 (Ar-C), 136.059

(Ar-C), 129.753 (Ar-C), 128.178 (Ar-C), 127.936 (Ar-C),

127.608 (Ar-C), 124.885 (Ar-C), 122.636 (Ar-C), 122.121

(Ar-C), 120.644 (Ar-C), 111.325 (Ar-C), 110.007 (Ar-C),

32.770 (–N—CH₃).

2.2.4. BDHFN. M.p. 203.0–205.0 ^ꢂC. Elemental analysis

found (calculated) for C₃₆H₂₆N₄(%): C 84.08 (84.02), H 5.13

(5.09), N 10.94 (10.89). HRMS (ESI): m/z calculated for

C₃₆H₂₆N₄+ H⁺: 515.2236 [M + H⁺]; found: 515.2236. ¹H NMR

(DMSO-d₆): 8.770 (s, 2H, –N CH), 8.277 (t, 4H, J = 7.6 Hz,

Ar-H), 7.883–7.766 (m, 6H, Ar-H), 7.585 (d, 2H, J = 8.0 Hz,

Ar-H), 7.482–7.326 (m, 8H, Ar-H), 7.082 (t, 2H, J = 7.6 Hz, Ar-

H), 6.857 (t, 2H, J = 7.6 Hz, Ar-H). ¹³C NMR (DMSO-d₆):

163.655 (CH N), 156.624 (CH N), 139.670 (Ar-C), 137.877

(Ar-C), 136.970 (Ar-C), 134.977 (Ar-C), 133.068 (Ar-C),

130.076 (Ar-C), 128.623 (Ar-C), 126.980 (Ar-C), 124.391

(Ar-C), 122.620 (Ar-C), 122.270 (Ar-C), 120.504 (Ar-C),

112.123 (Ar-C), 111.655 (Ar-C).

2017; Tan et al., 2019). We report here the synthesis, crystal

structure and antiproliferative activities of six Schiff bases

derived from indole and BDH/BMH, as well as two com-

parative compounds derived from naphthalene and BDH/

BMH (see Scheme 1).

2. Experimental

2.1. Materials and measurements

The starting material benzil and related chemicals were

purchased from Aladdin Reagent Chemicals and were used

without further puriﬁcation. Elemental (C, H and N) analyses

were carried out with a PerkinElmer 2400 microanalyzer.

Accurate mass measurements were acquired on an Agilent-

6520 quadrupole time-of-ﬂight tandem mass spectrometer.

Melting points were determined on a WRS-2A electrothermal

digital melting point apparatus (Shanghai Precision & Scien-

1

tiﬁc Instrument Co. Ltd, China). H and ¹³C NMR spectra

were recorded on a Bruker Avance 400 MHz instrument. The

chemical shifts are reported in parts per million (ppm) relative

to tetramethylsilane (SiMe₄, ꢀ = 0 ppm), referenced to the

chemical shifts of the residual solvent peak [deuterated

dimethyl sulfoxide (DMSO-d₆)].

2.2. Synthesis and crystallization

As indicated in Scheme 2, a two-step process was used to

synthesize all eight title compounds. In the ﬁrst step, a mixture

of benzil (0.42 g or 2 mmol) and hydrazine hydrate (80%,

0.20 ml or 4 mmol) was added to dry ethanol (20 ml) and the

resulting solution reﬂuxed for 3 h. Most of the ethanol was

removed by distillation and the resulting solution was cooled

to room temperature. Crude benzil dihydrazone (abbreviated

as BDH) was ﬁltered off, recrystallized from ethanol and dried

in a vacuum. Pure BDH was obtained as colourless needle-

shaped crystals {yield 85%, 0.41 g; m.p. 151.4–152.0 ^ꢂC

[literature m.p. 151–152 (Bach et al., 1982), 150–151.5 (Kim &

Yoon, 2004), 149 (Salavati-Niasari & Hassani-Kabutarkhani,

2005), 152 (Chandra et al., 2007), 172 (Singh et al., 2008) and

172 ^ꢂC (Chauhan et al., 2008)]}. Elemental analysis found

(calculated) for C₁₄H₁₄N₄(%): C 70.64 (70.57), H 5.97 (5.92),

N 23.63 (23.51). On the other hand, if the molar ratio of benzil

and hydrazine hydrate was set at 1:1, benzil monohydrazone

(BMH) will be obtained in a similar yield. Pure BMH was

obtained as a colourless crystal (m.p. 138.4–139.2 ^ꢂC).

In the second step, all eight Schiff bases were readily

prepared by a similar method (see Scheme 2). BDH/BMH and

the required carbonyl compounds (FI, AI, MFI and FN) were

stirred under reﬂux for 5 h in dry ethanol in a 1:2 (BDH versus

carbonyl compounds) or 1:1 (BMH versus carbonyl com-

pounds) molar ratio. After the reaction, the solvent was

reduced in volume by slow evaporation and crystalline

products were usually obtained. Recrystallization can purify

the products. Their physical and spectroscopic properties are

listed in the following sections. All NMR spectra are available

in Figs. S1–S16 of the supporting information.

2.2.5. BMHFI. M.p. 197–197.5 ^ꢂC. Elemental analysis found

(calculated) for C₂₃H₁₇N₃O (%): C 77.59 (78.61), H 5.13

(4.88), N 12.22 (11.96). HRMS (ESI): m/z calculated for

1

C₂₃H₁₇N₃O + H⁺: 352.1450 [M + H⁺]; found: 352.1450. H

ꢁ

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Tan et al.

Eight novel Schiff bases derived from benzil 3 of 20

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Table 1

Experimental details.

Experiments were carried out with Mo Kꢁ radiation using a Bruker SMART CCD area detector. Absorption was corrected by multi-scan methods (SADABS;

Bruker, 2000). Only H atoms were constrained to ride on their bonding partners, but with freely reﬁned U_isovalues.

BDHFI

BDHAI

BDHMFI

BDHFN

Crystal data

Chemical formula

M_r

C₃₂H₂₄N₆

492.57

C₃₄H₂₈N₆

520.62

2C₃₄H₂₈N₆ꢀC₂H₃N

1082.30

C₃₆H₂₆N₄

514.61

Crystal system, space group

Temperature (K)

Monoclinic, P2₁/c

293

13.023 (4), 7.340 (2), 27.762 (9)

Triclinic, P1

293

10.2585 (4), 11.9610 (5),

12.8232 (6)

64.941 (4), 79.573 (3),

76.829 (4)

1381.45 (11)

2

0.08

0.35 ꢄ 0.15 ꢄ 0.12

Triclinic, P1

293

11.2710 (6), 11.6944 (7),

12.5164 (6)

79.875 (4), 87.701 (4),

64.941 (6)

1470.10 (15)

1

0.08

0.35 ꢄ 0.20 ꢄ 0.15

Monoclinic, C2/c

298

26.195 (8), 9.809 (3),

11.806 (4)

90, 115.230 (5), 90

˚

a, b, c (A)

ꢁ, ꢂ, ꢃ (^ꢂ)

90, 97.693 (5), 90

3

˚

V (A )

2630.0 (14)

4

0.08

0.30 ꢄ 0.18 ꢄ 0.15

2744.2 (15)

4

0.07

0.30 ꢄ 0.16 ꢄ 0.10

Z

ꢄ (mm^ꢃ1

Crystal size (mm)

)

Data collection

T_min, T_max

No. of measured, independent

and observed [I > 2ꢅ(I)]

reﬂections

0.903, 0.939

13632, 5145, 2852

0.903, 0.939

15195, 5328, 4490

0.905, 0.943

13418, 5640, 4445

0.986, 0.993

6631, 2413, 1197

R_int

0.046

0.617

0.026

0.618

0.029

0.617

0.058

0.595

ꢃ1

˚

(sin ꢆ/ꢇ)_max(A

)

Reﬁnement

R[F²> 2ꢅ(F²)], wR(F²), S

No. of reﬂections

0.049, 0.103, 0.88

5145

0.044, 0.129, 1.05

5328

0.049, 0.151, 1.04

5640

0.062, 0.130, 0.95

2413

No. of parameters

No. of restraints

Áꢈ_max, Áꢈ_min(e A

367

0

0.15, ꢃ0.22

392

0

0.19, ꢃ0.19

424

2

0.22, ꢃ0.17

194

0

0.11, ꢃ0.10

ꢃ3

˚

)

BMHFI

BMHAI

BMHMFI

BMHFN

Crystal data

Chemical formula

M_r

C₂₃H₁₇N₃O

351.39

C₂₄H₁₉N₃O

365.42

C₂₄H₁₉N₃O

365.42

C₂₅H₁₈N₂O

362.41

Crystal system, space group

Temperature (K)

Orthorhombic, P2₁2₁2₁

293

6.8767 (1), 8.3698 (2),

32.6317 (6)

90, 90, 90

1878.17 (6)

4

0.08

0.35 ꢄ 0.1 ꢄ 0.09

Tetragonal, P4₃2₁2

293

Monoclinic, P2₁/c

293

Monoclinic, P2₁/c

293

17.2081 (13), 9.4075 (8),

11.9703 (9)

90, 94.814 (7), 90

1931.0 (3)

˚

a, b, c (A)

8.3580 (1), 8.3580 (1),

54.6705 (7)

90, 90, 90

3819.07 (10)

8

0.08

0.3 ꢄ 0.1 ꢄ 0.1

18.6779 (6), 8.6694 (3),

12.7956 (4)

90, 106.910 (4), 90

1982.36 (12)

4

0.08

0.33 ꢄ 0.28 ꢄ 0.25

ꢁ, ꢂ, ꢃ (^ꢂ)

3

˚

V (A )

Z

4

0.08

ꢄ (mm^ꢃ1

Crystal size (mm)

)

0.40 ꢄ 0.40 ꢄ 0.16

Data collection

T_min, T_max

0.903, 0.939

10418, 3641, 3444

0.983, 0.998

21746, 3759, 3565

0.971, 0.987

9410, 3837, 2964

0.970, 0.989

10556, 3396, 1411

No. of measured, indepen-

dent and observed

[I > 2ꢅ(I)] reﬂections

R_int

0.023

0.618

0.030

0.618

0.021

0.618

0.078

0.595

ꢃ1

˚

(sin ꢆ/ꢇ)_max(A

)

Reﬁnement

R[F²> 2ꢅ(F²)], wR(F²), S

No. of reﬂections

0.035, 0.096, 1.02

3641

262

0.041, 0.113, 1.06

3759

274

0.045, 0.140, 1.03

3837

274

0

0.052, 0.083, 0.87

3396

272

0

No. of parameters

No. of restraints

Áꢈ_max, Áꢈ_min(e A

Absolute structure

0

ꢃ3

˚

)

0.15, ꢃ0.13

Not determined

because of

low anomalous

signal

0.16, ꢃ0.12

Not determined

because of

low anomalous

signal

0.16, ꢃ0.15

0.15, ꢃ0.15

–

Absolute structure para-

meter

ꢃ1.4 (6)

ꢃ1.5 (6)

–

Computer programs: SMART (Bruker, 2000), SAINT (Bruker, 2000), SHELXS2016 (Sheldrick, 2015a), SHELXTL (Bruker, 2000), PLATON (Spek, 2009), DIAMOND (Brandenburg

& Putz, 1999), SHELXL2016 (Sheldrick, 2015b) and WinGX (Farrugia, 2012).

ꢁ

4 of 20 Tan et al. Eight novel Schiff bases derived from benzil

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2.3. Refinement

NMR (DMSO-d₆): 11.800 (s, 1H, –NH), 8.800 (s, 1H,

–N CH), 7.961–7.928 (m, 3H, J = 7.2 Hz, Ar-H), 7.767–7.218

(m, 10H, Ar-H), 7.087 (t, 1H, J= 7.6 Hz, Ar-H), 6.789 (t, 1H, J=

7.6 Hz, Ar-H). ¹³C NMR (DMSO-d₆): 197.921(C O), 163.669

(CH N), 158.076 (CH N), 137.065 (Ar-C), 135.223 (Ar-C),

134.228 (Ar-C), 134.056 (Ar-C), 132.698 (Ar-C), 131.033

(Ar-C), 130.507 (Ar-C), 129.266 (Ar-C), 129.180 (Ar-C),

128.936 (Ar-C), 128.554 (Ar-C), 128.369 (Ar-C), 127.445

(Ar-C), 126.900 (Ar-C), 124.230 (Ar-C), 122.793 (Ar-C),

121.724 (Ar-C), 120.659 (Ar-C), 111.860 (Ar-C), 111.605

(Ar-C).

Crystal data, data collection and structure reﬁnement

details are summarized in Table 1. All H atoms in the eight

compounds (except for the H atoms on C35 in BDHMFI)

were found in difference Fourier maps and then allowed to

ride on their parent atoms. The location and isotropic

displacement parameters of the H atoms on C35 in BDHMFI

were reﬁned freely.

2.4. Cytotoxicity assays

Human lung carcinoma A549 cells, mouse breast cancer 4T₁

cells, human MRC-5 lung normal cells and normal mouse NIH

3T3 ﬁbroblasts were purchased from the Shanghai Cell Bank,

Type Culture Collection Committee, Chinese Academy of

Sciences. The cells were cultured in F12K medium supple-

mented with 10% heat-inactivated fetal bovine serum (FBS),

2 mM glutamine, 100 U ml^ꢃ1penicillin and 100 mg ml^ꢃ1

streptomycin, and maintained at 310 K in a humidiﬁed

atmosphere of 5% CO₂.

The cells (8000 cells) were seeded on 96-well microtiter

plates in F12K medium with 10% FBS and incubated over-

night. The cell culture medium was replaced by different doses

of the compound solution, i.e. 1, 5, 10, 30, 50, 100 and 150 mM,

and then the cells were cultured for another 72 h. The MTT

reagent was added to the cell supernatant for a ﬁnal concen-

tration of 0.5 mg ml^ꢃ1of MTT. After 3 h, the cell culture

medium was removed. Formazan crystals in adherent cells

were dissolved in dimethyl sulfoxide (DMSO, 200 ml) and the

absorbance of the formazan solution was measured. Each

compound was tested in triplicate and the experiments were

repeated three times.

2.2.6. BMHAI. M.p. 223.1–224.4 ^ꢂC. Elemental analysis

found (calculated) for C₂₄H₁₉N₃O (%): C 79.24 (78.88), H 6.11

(5.24), N 12.03 (11.50). HRMS (ESI): m/z calculated for

1

C₂₄H₁₉N₃O + H⁺: 366.1606 [M + H⁺]; found: 366.1606. H

NMR (DMSO-d₆): 11.644 (s, 1H, –NH), 8.028–7.503 (m, 11H,

J = 6.8 Hz, Ar-H), 7.318–7.266 (q, 2H, J = 8.4 Hz, Ar-H), 7.034–

6.998 (t, 1H, J = 7.2 Hz, Ar-H), 6.649–6.612 (t, 1H, J = 7.2 Hz,

Ar-H), 2.584 (s, 3H –CH₃). ¹³C NMR (DMSO-d₆): 198.911

(C O), 163.948 (CH N), 161.192 (CH N), 136.957 (Ar-C),

134.953 (Ar-C), 134.166 (Ar-C), 133.209 (Ar-C), 131.003

(Ar-C), 130.813 (Ar-C), 129.467 (Ar-C), 129.226 (Ar-C),

129.056 (Ar-C), 128.868 (Ar-C), 126.831 (Ar-C), 124.468

(Ar-C), 122.699 (Ar-C), 122.632 (Ar-C), 122.147 (Ar-C),

121.559 (Ar-C), 121.275 (Ar-C), 120.132 (Ar-C), 114.671

(Ar-C), 111.414 (Ar-C), 14.688 (–CH₃).

2.2.7. BMHMFI. M.p. 220.1–221.3 ^ꢂC. Elemental analysis

found (calculated) for C₂₄H₁₉N₃O (%): C 79.25 (78.88), H 5.65

(5.24), N 11.89 (11.50). HRMS (ESI): m/z calculated for

1

C₂₄H₁₉N₃O + H⁺: 366.1606 [M + H⁺]; found: 366.1606. H

NMR (DMSO-d₆): 8.764 (s, 1H, –N CH), 7.927 (d, 3H, J =

8.4 Hz, Ar-H), 7.749 (d, 2H, J = 7.6 Hz, Ar-H), 7.665–7.421 (m,

8H, Ar-H), 7.239–7.138 (m, 1H, Ar-H), 6.834 (t, 1H, J = 7.2 Hz,

Ar-H), 3.799 (s, 3H –N—CH₃). ¹³C NMR (DMSO-d₆): 197.918

(C O), 163.667 (CH N), 157.569 (CH N), 137.675 (Ar-C),

137.549 (Ar-C), 135.209 (Ar-C), 134.066 (Ar-C), 132.680

(Ar-C), 131.044 (Ar-C), 130.126 (Ar-C), 129.975 (Ar-C),

129.298 (Ar-C), 129.261 (Ar-C), 129.179 (Ar-C), 129.055

(Ar-C), 128.564 (Ar-C), 126.840 (Ar-C), 124.658 (Ar-C),

122.873 (Ar-C), 121.838 (Ar-C), 120.960 (Ar-C), 110.576

(Ar-C), 110.261 (Ar-C), 32.950 (–N—CH₃).

Generally, the operations from cell culture to MTTassay for

cell proliferation are the same as we reported before (Cheng et

al., 2016; Bu et al., 2017; Tan et al., 2019).

2.5. Molecular docking and quantum chemistry calculations

Molecular docking studies of all eight Schiff bases with 18

potential target proteins were performed using the SYBYL/

Surﬂex-dock (Tripos, 2012) in order to screen the potential

targets and illustrate the binding modes between proteins and

2.2.8. BMHFN. M.p. 135.2–136.7 ^ꢂC. Elemental analysis

found (calculated) for C₂₅H₁₈N₂O (%): C 82.18 (82.85), H 5.11

(5.01), N 7.84 (7.73). HRMS (ESI): m/z calculated for

ligands. All solvent molecules except those within 5 A around

˚

the natural ligand were removed from the protein structure.

The molecular structures of the ligands were ﬁrst extracted

from the single-crystal X-ray structure and then optimized by

employing density functional theory (DFT) at the B3LYP/6-

311+G(d,p) level (Becke, 1993; Lee et al., 1988; McLean &

Chandler, 1980; Krishnan et al., 1980). The default parameters

were used regarding charges, bonds order and geometrical

ﬂexibility with no other constraints.

Except for the aforementioned structural optimizations,

quantum chemistry calculations were also used to explore

intermolecular interactions in the BMHFI crystal (see x3.1.5).

Two methods, i.e. MP2/6-31G(d,p) and DFT/B3LYP/6-311G+

(d,p), were used (Møller & Plesset, 1934; Rassolov et al., 2001;

Frisch et al., 1984). The standard counterpoise method was

applied to correct interaction energies for the basis set

1

C₂₅H₁₈N₂O + H⁺: 363.1497 [M + H⁺]; found: 363.1497. H

NMR (DMSO-d₆): 8.770 (s, 1H, –N CH), 8.275 (t, 1H, J =

7.6 Hz, Ar-H), 8.167 (t, 3H, J = 7.6 Hz, Ar-H), 7.852 (q, 4H, J =

7.6 Hz, Ar-H), 7.589–7.320 (m, 6H, Ar-H), 7.080 (t, 2H, J =

7.2 Hz, Ar-H), 6.850 (t, 1H, J =7.6 Hz, Ar-H). ¹³C NMR

(DMSO-d₆): 196.873 (C O), 162.531 (CH N), 156.980

(CH N), 139.701 (Ar-C), 137.101 (Ar-C), 136.981 (Ar-C),

135.238 (Ar-C), 134.231 (Ar-C), 134.107 (Ar-C), 132.702

(Ar-C), 131.051 (Ar-C), 130.515 (Ar-C), 129.277 (Ar-C),

129.176 (Ar-C), 128.907 (Ar-C), 128.499 (Ar-C), 128.402

(Ar-C), 127.391 (Ar-C), 126.879 (Ar-C), 124.121 (Ar-C),

122.741 (Ar-C), 121.698 (Ar-C), 120.571 (Ar-C), 111.761

(Ar-C), 109.432 (Ar-C).

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superposition error (BSSE) (Boys & Bernardi, 1970). All

geometries are extracted from the interacting pairs in the

crystal of BMHFI without any structural relaxation.

All calculations were carried out using the GAUSSIAN03

program package (Frisch et al., 2004) on a Sunway BlueLight

MPP supercomputer housed at the National Supercomputer

Center in Jinan, China.

C

N double bonds adopt the same Z,E conformations in

both arms. In fact, the other three di-Schiff bases (BDHAI,

BDHMFI and BDHFN) reported in this article are X-shaped

molecules and the two X-shaped arms in BDHFN are exactly

the same (see x3.1.4).

Both arms in BDHFI are rather ﬂat. One arm consists of

atoms C1–C7/N1/N2/C24–C31/N6/C32 and all 19 non-H atoms

are essentially coplanar, with an average r.m.s. deviation of

˚

only 0.0811 A and the largest r.m.s. deviation of ꢃ0.1435 A

occurring for atom N6. In another arm, all 19 non-H atoms are

slightly less coplanar, with an average r.m.s. deviation of

3. Results and discussion

3.1. X-ray structures of the eight title compounds

˚

0.1742 A (the largest r.m.s. deviation of 0.3027 A occurs for

atom N5). The dihedral angle between the planes of these two

arms is about 89.3^ꢂ.

The identities of the conformations of the eight title com-

pounds have been established through X-ray structure deter-

minations, which clearly show the similarities and differences.

3.1.1. BDHFI. The monomeric structure of BDHFI is shown

in Fig. 1(a), which indicates that the molecule contains a pair

of linkage arms (the connection point is the C1—C8 bond

axis). The pair of linkage arms crosses each other in an

X-shape; each arm has a similar conformation, i.e. the two

In the packing structure of BDHFI, three kinds of dominant

noncovalent interactions (NCIs), including hydrogen bond a

(Fig. 1b), C—Hꢀ ꢀ ꢀꢉ interaction b (Fig. 1b) and ꢉ–ꢉ packing c

(Fig. 1c), can be found with the help of PLATON (Tables S1–

S3 in the supporting information). The CrystalExplorer17

program (Hirshfeld, 1977; Turner et al., 2017) was used to

Figure 1

(a) The molecular structure of BDHFI, showing the atom-labelling scheme, with displacement ellipsoids for non-H atoms drawn at the 30% probability

˚

level at 293 K. (b) The Hirshfeld surface for BDHFI, mapped over d_norm(ꢃ0.10 to 1.40 A, same throughout this article). Four red spots correspond to

1

N5—H35ꢀ ꢀ ꢀN1(ꢃx + 2, y + , ꢃz + ) (marked a) and N6—H36ꢀ ꢀ ꢀꢉ (marked b) interactions, respectively. (c) The shape-index surface of BDHFI,

2

identifying ꢉ–ꢉ stacking interactions (marked c). Only red and blue triangles on the surface are shown for clarity. (d) Hydrogen bonds a (green dashed

lines) and b (red dashed lines) link molecules into inﬁnite 1D chains parallel to the crystallographic b and a axes, respectively, viewed along the

crystallographic [8, 1, 17] direction. Eight molecules are illustrated by the simpliﬁed structure (centre of gravity) for clarity and the two colours (red and

green) represent two different orientations.

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verify the strength of these NCIs. It is worth noting that the

geometries of all NCIs, including these very weak ones,

calculated by PLATON are listed in the supporting informa-

tion for all eight compounds reported in this article (Tables

S1–S21 in the supporting information). But only those strong

NCIs that can be veriﬁed by CrystalExplorer17 will be

analysed. For the convenience of description, different kinds

of strong NCIs are simpliﬁed into various notations (the ﬁrst

column in Tables S1–S21 in the supporting information).

These weak intermolecular NCIs and all intramolecular NCIs

are not coded because they make less contribution to the

packing structure. For example, the C3—H3ꢀ ꢀ ꢀN3(x, y ꢃ 1, z)

hydrogen bond (Table S1 in the supporting information) is not

coded because its role is not apparent in the Hirshfeld surface

analysis (Fig. 1b). In rare cases, some ‘strong’ interactions

veriﬁed by CrystalExplorer17 have not been suggested by

PLATON, for example, N3—H25ꢀ ꢀ ꢀN2(x + ¹₂, ꢃy + ₂¹, ꢃz + ₄¹)

and C24—H24Aꢀ ꢀ ꢀO1(x + 1, y, z) in Fig. 6(b) (see x3.1.6), and

1

2

1

2

C11—H11ꢀ ꢀ ꢀN1(x, ꢃy ꢃ , z ꢃ ) in Fig. 7(b) (see x3.1.7).

Their roles will not be analysed.

Hydrogen bonds a and b link BDHFI molecules into one-

dimensional (1D) chains parallel to the b and a axes, respec-

tively (Fig. 1d), while ꢉ–ꢉ interaction c links two molecules

into a dimer. These chains and dimers serve as the main

building blocks for the three-dimensional (3D) structures with

the aid of other kinds of NCIs, such as van der Waals.

It should be mentioned that all the molecules in BDHFI

adopt two relative orientations, which are highlighted with red

and green colours, respectively (Fig. 1d). If these two relatively

small arene rings in one molecule are omitted, the X-shaped

molecule can be regarded as a V-shaped one. These V-shaped

molecules open towards entirely opposite directions: the red

Figure 2

(a) The molecular structure of BDHAI, showing the atom-labelling scheme, with displacement ellipsoids for non-H atoms drawn at the 30% probability

level at 293 K. (b) The Hirshfeld surface for BDHAI, mapped over d_norm. Four red spots correspond to N6—H36ꢀ ꢀ ꢀN4(ꢃx, ꢃy + 1, ꢃz + 1) (marked a)

and N5—H35ꢀ ꢀ ꢀꢉ (marked b) interactions, respectively. (c) The shape-index surface of BDHAI, identifying ꢉ–ꢉ stacking interactions (marked c and d).

Only red and blue triangles on the surface are shown for clarity. (d) Four kinds of NCIs, i.e. a, b, c and d (shown with red, green, purple and yellow dashed

lines, respectively), link BDHAI molecules into an inﬁnite 1D chain extending roughly along the [111] direction, viewed along the [7, 3, 7] direction.

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ones are directed toward the positive half of the c axis, while

the green ones are directed toward the negative half (both

roughly). Interestingly, the molecules in the other three di-

Schiff bases have two similar orientations, so there also exist

red and green highlighted molecules in Figs. 2, 3 and 4. While

molecules in the four mono-Schiff bases (BMHFI, BMHAI,

BMHMFI and BMHFN) have four or eight orientations,

mostly because of the two apparently asymmetrical arms (see

x3.1.5 to x3.1.8).

S4–S6 in the supporting information). Hydrogen bonds a link

two molecules into a dimer (Figs. 2b and 2d), while b, c and d

link two molecules into another dimer (Figs. 2b, 2c and 2d),

whether they are used separately or collectively. But a, b, c and

d will crosslink molecules into inﬁnite 1D chains if working

together (Fig. 2d).

3.1.3. BDHMFI. The monomeric structure of BDHMFI is

depicted in Fig. 3(a), which contains one complete BDHMFI

molecule and a half acetonitrile molecule in the asymmetric

unit. The half acetonitrile molecule lies on an inversion centre

(atom C35, which resides on a centre of inversion at the

origin) and was reﬁned disordered into two parts with the site-

occupancy factors being ﬁxed at 0.5. The inversion operator

will create two BDHMFI molecules and one complete

acetonitrile molecule, which are linked together by hydrogen

bonds a (Fig. 3b). By the way, a only occurs between BDHMFI

and acetonitrile (Figs. 3b and 3d).

3.1.2. BDHAI. Similar to BDHFI, BDHAI is an X-shaped

molecule with both arms being somewhat ﬂat. But the

coplanarity is worse than in BDHFI. In one arm, consisting of

atoms C1–C7/N1/N2/C24–C31/N6/C32/C34, the average r.m.s.

˚

deviation is 0.3698 A and the largest r.m.s. deviation of

˚

0.8308 A occurs for atom C7. In another arm, all 20 non-H

˚

atoms have an average r.m.s. deviation of 0.2542 A and the

˚

largest r.m.s. deviation of 0.4662 A occurs for atom N5. The

dihedral angle between the planes of the two arms is about

98.0^ꢂ(Fig. 2a).

In the packing structure of BDHAI, the most important

four NCIs link molecules into dimers independently (Tables

These two arms of X-shaped BDHMFI have a similar

coplanarity to that in BDHFI. In one arm, consisting of atoms

C1–C7/N1/N2/C24–C31/N6/C32/C34, the average r.m.s. devi-

˚

ation is 0.1063 A and the largest r.m.s. deviation of 0.1870 A

Figure 3

(a) The molecular structure of BDHMFI, showing the atom-labelling scheme, with displacement ellipsoids for non-H atoms drawn at the 30%

probability level at 293 K [symmetry code: (A) ꢃx + 2, ꢃy, ꢃz]. (b) The dimer structure formed by two BDHMFI molecules and one disordered

acetonitrile molecule through hydrogen bonds a (two red spots on the d_normHirshfeld surface). (c) The shape-index surface of BDHMFI, identifying ꢉ–ꢉ

stacking interaction b. Only red and blue triangles on the surface are shown for clarity. (d) NCIs a and b (shown with red and green dashed lines,

respectively) link BDHMFI molecules into an inﬁnite 1D chain extending roughly along the [103] direction, viewed along the [1, 9, 5] direction.

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occurs for atom C6. In another arm, all 20 non-H atoms have

˚

an average r.m.s. deviation of 0.1964 A and the largest r.m.s.

The only kind of hydrogen bond is intramolecular inter-

actions in the packing structure of BDHFN (Table S10 in the

supporting information). Besides van der Waals, only one kind

of intermolecular ꢉ–ꢉ interaction a plays an important role

(Table S11 in the supporting information), which accounts for

merely 6.1% of the Hirshfeld surface (Fig. 4b). That is to say, it

is van der Waals interactions that play a critical role in the

crystal molecular assembly. As for ꢉ–ꢉ interactions a (Fig. 4c),

they help to link BDHFN molecules into 1D lines parallel to

the crystallographic c axis (Fig. 4d).

3.1.5. BMHFI. The molecular structure of BMHFI is shown

in Fig. 5(a), which indicates that the molecule still contains a

pair of linkage arms, but one arm is shorter than the other

because one C O group has not been converted into a Schiff

base. The longer arm has a similar conformation to that in its

˚

deviation of 0.4540 A occurs for atom C33. The dihedral angle

between the planes of these two arms is about 90.6^ꢂ.

Either hydrogen bond a or ꢉ–ꢉ interaction b links two

adjacent BDHMFI molecules into dimers, but they are not the

same dimer (Figs. 3b and 3c). If a and b collaborate together,

1D chains extending along the crystallographic [24, 1, 63]

direction will be formed (Fig. 3d).

3.1.4. BDHFN. The molecular structure of BDHFN is

shown in Fig. 4(a), which is the result of a glide reﬂection along

the crystallographic c axis. That is to say, the asymmetric unit

contains only one half BDHMFI molecule, which happened to

be one arm of the X-shaped structure. The dihedral angle

between the planes of the naphthalene and arene rings is

22.6 (3)^ꢂand the dihedral angle between the two symmetry-

related arms is 85.1 (4)^ꢂ.

corresponding di-Schiff base BDHFI, i.e. these two C

double bonds adopt the same Z,E conformations. In fact, all

N

Figure 4

(a) The molecular structure of BDHFN, showing the atom-labelling scheme, with displacement ellipsoids for non-H atoms drawn at the 30% probability

level at 293 K [symmetry code: (A) ꢃx, y, ꢃz + ¹₂]. (b) The contribution of Cꢀ ꢀ ꢀC contacts in the 2D ﬁngerprints of BDHFN, indicating ꢉ–ꢉ interaction a.

(c) The shape-index surface of BDHFN, identifying ꢉ–ꢉ stacking interaction a (not marked as there is only one type of interaction). (d) 1D chains

formed by ꢉ–ꢉ stacking interaction a (shown with red dashed lines), viewed along the [501] direction. The upper one is illustrated by the simpliﬁed

structure (centre of gravity).

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eight Schiff bases reported in this paper adopt similar Z,E

conformations.

As a whole, both arms in BMHFI are ﬂat. The long arm has

In the packing structure of BMHFI, two kinds of inter-

molecular hydrogen bonds (a and b) (Table S12 in the

supporting information) link molecules into 1D chains parallel

to the crystallographic a axis, whether they are used separately

or collectively. Unlike its corresponding di-Schiff base

BDHFI, BMHFI adopts four relative orientations, which are

highlighted in red, green, purple and blue (Figs. 5c and 5d). It

should be noted that the colour intensity of the vivid spot

˚

an average r.m.s. deviation of 0.2242 A and the largest r.m.s.

˚

deviation of ꢃ0.3858 A occurs for N3. The short arm has much

better coplanarity, with an average r.m.s. deviation of only

˚

0.0160 A (the largest r.m.s. deviation of ꢃ0.0647 A occurs for

O1). The dihedral angle between the two arms is about 93.8^ꢂ.

Figure 5

(a) The molecular structure of BMHFI, showing the atom-labelling scheme, with displacement ellipsoids for non-H atoms drawn at the 30% probability

level at 293 K. (b) The Hirshfeld surface for BMHFI, mapped over d_norm. Three red spots correspond to N3—H24ꢀ ꢀ ꢀO1(x + ¹₂, ꢃy + ₂¹, ꢃz) (marked a) and

C3—H3ꢀ ꢀ ꢀO1(x + 1, y, z) (marked b) interactions. (c) 1D chains formed by NCIs a and b (shown with green and red dashed lines, respectively), viewed

perpendicular to the extending direction, i.e. the a axis. The upper two lines are illustrated by the simpliﬁed structure (centre of gravity). (d) 1D chains

formed by the aforementioned NCIs a and b, viewed along the extending direction, i.e. the a axis, aiming to show the packing mode of four different

orientations (simpliﬁed structure with four different colours).

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representing hydrogen bond a is very high, indicating a strong

hydrogen bond. Thus, the intermolecular interaction energy

with the BSSE correction has been calculated using the MP2/

6-31G(d,p) and B3LYP/6-311G+(d,p) methods on the basis of

dimer geometries extracted from the crystal structure. The

calculated interaction energies for a and b are ꢃ50.97 and

ꢃ19.92 kJ mol^ꢃ1(MP2), and ꢃ19.71 and ꢃ4.25 kJ mol^ꢃ1

(DFT), respectively, indicating a much stronger favourable

binding energy for a than that for b, which agrees with the

colour intensity in the d_normmap (Fig. 5b).

value is 20.4 (4)^ꢂ, while in BDHAI, the dihedral angles are

23.6 (4) and 39.9 (5)^ꢂ, but in BMHAI, this value is increased to

54.5 (3)^ꢂ.

There are eight kinds of molecular orientations and one

kind of intermolecular hydrogen bond, i.e. a, in the crystal

structure of BMHAI (Figs. 6b and 6c) (Tables S15–S17 in the

supporting information). Hydrogen bonds a link molecules

into two kinds of 1D chains, one is parallel to the crystal-

lographic a axis (these molecules are simpliﬁed into green,

blue, yellow and indigo skeletons in Fig. 6c) and the other is

parallel to the b axis (these molecules are simpliﬁed into

orange, red, purple and violet skeletons in Fig. 6c).

3.1.6. BMHAI. The ‘long arm’ in BMHAI assumes less

coplanarity than that in BMHFI, and even less than that in

BDHAI, which can be deduced from the dihedral angles

between the indole and arene rings (Fig. 6a). In BMHFI, the

3.1.7. BMHMFI. Both the ‘long arm’ and the ‘short arm’ in

BMHMFI have good coplanarity. The dihedral angle between

Figure 6

(a) The molecular structure of BMHAI, showing the atom-labelling scheme, with displacement ellipsoids for non-H atoms drawn at the 30% probability

level at 293 K [symmetry code: (A) ꢃx, y, ꢃz + ¹₂]. (b) The Hirshfeld surface for BMHAI, mapped over d_norm. Three kinds of intermolecular hydrogen

bonds and one kind of intramolecular hydrogen bond can be visualized as red spots, but only hydrogen bond a was conﬁrmed by the PLATON program

(Spek, 2009). (c) 1D chains along the a and b axes formed by the aforementioned hydrogen bonds a (shown with red dashed lines), viewed along the a

axis, aiming to show the packing mode of the eight different orientations as a simpliﬁed structure with eight different colours, i.e. orange, red, green, blue,

purple, violet, yellow and indigo.

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the planes of the indole and arene rings in the ‘long arm’ is

only 14.0 (2)^ꢂ. The long arm has an average r.m.s. deviation of

in the supporting information), and they link two adjacent

molecules into a dimer (Fig. 7d). The collaboration of a, b and

c links molecules into a 2D layer structure (Fig. 7d).

3.1.8. BMHFN. The dihedral angle between the planes of

the naphthalene and benzene rings in the ‘long arm’ is

24.2 (5)^ꢂ. The dihedral angle between the long and short arms

is about 81.4 (5)^ꢂ(Fig. 8a).

˚

0.1492 A and the largest r.m.s. deviation is 0.3816 A for atom

C24; the short arm has an average r.m.s. deviation of only

˚

0.0324 A and the largest r.m.s. deviation is 0.1135 A for atom

O1. These two arms are roughly perpendicular to each other,

with a dihedral angle of 91.1 (3)^ꢂ(Fig. 7a).

The most intense red spots represent a C23—H23ꢀ ꢀ ꢀ

There are four kinds of molecular orientations and four

kinds of intermolecular NCIs (a, b, c and d) in the crystal

structure of BMHFN (Figs. 8b, 8c and 8d) (Tables S20 and S21

in the supporting information). Hydrogen bond a links mol-

ecules into 1D chains parallel to the crystallographic b axis

(Figs. 8b and 8d). Among these three types of ꢉ–ꢉ stacking

interactions, b and d always co-exist and link molecules into

1D chains parallel to the crystallographic c axis (Figs. 8c and

8d), c joins two molecules (red and green, and blue and yellow;

1

2

1

2

ring(C17–C22)(ꢃx + 1, y + , ꢃz + ) interaction, coded as a

(Fig. 7b) (Table S18 in the supporting information), which

links molecules into a 1D chain parallel to the b axis (Fig. 7d).

The second intense spot represents a C11—H11ꢀ ꢀ ꢀN1(x,

1

ꢃy ꢃ , z ꢃ ) hydrogen bond (Fig. 7b), which was not

2

suggested by PLATON. Thus, its role in the packing structure

will not be analysed in this article. Two kinds of intermolecular

ꢉ–ꢉ interactions (b and c) always co-exist (Fig. 7c) (Table S19

Figure 7

(a) The molecular structure of BMHMFI, showing the atom-labelling scheme, with displacement ellipsoids for non-H atoms drawn at the 30%

probability level at 293 K. (b) The Hirshfeld surface for BMHMFI, mapped over d_norm. The intermolecular hydrogen bonds a calculated using PLATON,

1

but another kind of hydrogen bond [C11—H11ꢀ ꢀ ꢀN1(x, ꢃy ꢃ , z ꢃ ), which can be found as red spots] was not suggested by PLATON. (c) The shape-

2

index surface of BMHMFI, identifying ꢉ–ꢉ stacking interactions b and c. (d) 1D chains formed by C—Hꢀ ꢀ ꢀꢉ interactions a (shown with red dashed lines)

can be woven into a 2D layer structure by ꢉ–ꢉ interactions b and c (shown with green and blue dashed lines, respectively), viewed along the a axis.

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Fig. 8d) into dimers. The co-operation of all three types of ꢉ–ꢉ

stacking interactions (b, c and d) links molecules into a 2D

layer structure extending along the crystallographic (100)

plane (Fig. 8d). In fact, all four kinds of intermolecular

NCIs crosslink molecules into the same inﬁnite 2D layer

structure.

mouse 4T₁cells with the compounds and examined the growth

of cells with an MTT assay. Meanwhile, MRC-5 normal lung

cells and NIH 3T3 ﬁbroblasts were also tested using the same

method in order to evaluate the selective cytotoxicity of these

new compounds. All experiments were carried out with

cisplatin (a compound used as a chemotherapy drug to treat

many types of cancers) for comparison. The cytotoxic activ-

ities as 50% inhibitory concentration (IC₅₀) values are shown

in Table 2.

3.2. Cytotoxicity assays – inhibition of lung and breast cancer

cell growth

As we can see, both BDHFI and BDHAI have similar

cytotoxic activities to the standard cisplatin for both cancer

cell lines. All mono-Schiff bases show slightly weaker inhibi-

To study the growth inhibitory effects of these eight Schiff

bases on lung/breast cancer cells, we treated human A549 and

Figure 8

(a) The molecular structure of BMHFN, showing the atom-labelling scheme, with displacement ellipsoids for non-H atoms drawn at the 30% probability

1

level at 293 K. (b) The Hirshfeld surface for BMHFN, mapped over d_norm. Two red spots correspond to C5—H5ꢀ ꢀ ꢀO1(ꢃx + 1, y ꢃ , ꢃz + ³₂) (marked a).

2

(c) The shape-index surface of BMHFN, identifying ꢉ–ꢉ stacking interactions b, c and d. Only red and blue triangles on the surface are shown for clarity.

(d) 2D layer structure formed by NCIs a b, c and d (shown with red, green, blue and yellow dashed lines, respectively), viewed along the a axis. Four

discontinuous lines are shown, the left two (along the b axis) are formed by NCI a and the top two (along the c axis) are formed by NCIs b and d.

Molecules in the bottom right corner are illustrated by the simpliﬁed structure (centre of gravity), showing four kinds of orientations.

ꢁ

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Eight novel Schiff bases derived from benzil 13 of 20

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Table 2

Inhibition of A549, 4T₁, MRC-5 and NIH 3T3 cell growth by the title compounds compared with cisplatin (ꢄM).

Compound

A549 cells, IC₅₀

4T₁cells, IC₅₀

MRC-5 cells, IC₅₀

NIH 3T3 ﬁbroblasts, IC₅₀

BDHFI

8.0ꢅ0.5

8.5ꢅ0.6

7.5ꢅ0.5

7.0ꢅ0.6

24.5ꢅ1.5

36.5ꢅ1.5

>150.0

29.5ꢅ1.0

43.0ꢅ1.5

>150.0

BDHAI

BDHMFI

BDHFN

BMHFI

BMHAI

BMHMFI

BMHFN

Cisplatin

125.0ꢅ1.0

130.0ꢅ1.0

46.5ꢅ0.5

43.0ꢅ0.5

148.0ꢅ1.2

150.0ꢅ1.2

6.5 ꢅ 0.5

122.0ꢅ1.0

125.0ꢅ1.0

32.5ꢅ0.5

30.0ꢅ0.5

141.0ꢅ1.0

148.0ꢅ1.0

0.5 ꢅ 0.1

>150.0

88.0ꢅ1.5

83.0ꢅ1.5

>150.0

22.5 ꢅ 1.5

85.0ꢅ1.5

76.0ꢅ1.5

>150.0

21.0 ꢅ 1.0

tory activity of A549 and 4T₁compared with their corre-

sponding di-Schiff bases. Incorporating a CH₃group at the

N—H position in the indole ring proved detrimental for

anticancer activity (BDHMFI and BMHMFI), while substi-

tution on the imine C atom has little effect (BDHAI and

BMHAI). This might be due to the availability of enough

space to accommodate a methyl group at this particular site of

the inhibitor binding pocket, or the N—H group favours

combination with the hydrogen-bonding pocket. In one word,

for BDHFI, BDHAI, BMHFI and BMHAI, the normal indole

rings should contribute greatly to their cytotoxicities. When

indole rings are changed into 1-methylindole or naphthalene,

the cytotoxicity is greatly decreased.

As for the selective cytotoxicity between normal and

malignant cells, MTT assays reveal a similar pattern to what

were seen in our previous work (Tan et al., 2019). That is to

say, all Schiff bases possess selective cytotoxicity on cancer

cells over normal cells. But they have relevant inhibitory

concentrations. The higher the IC₅₀value for selected malig-

nant cells, the higher the value for responding normal cells.

Among them, BDHFI, BDHAI, BMHFI and BMHAI

displayed high potency and selectivity in all cell lines.

These experimental studies clearly predict cytotoxic activ-

ities of indole-containing Schiff bases. Such encouraging

preliminary results conﬁrm the feasibility and reliability of this

excellent framework in the discovery of potent antitumour

agents.

group 2, the targets in group 1 are probably the same, while

the ligands in group 2 should not hit these targets. Technically,

the most probable targets should be hit four times in group 1

and should not be hit in group 2. In our work, the targets that

were hit four or three times in group 1 were selected (total of

32 targets) on account of random error in the prediction

model. But if one target was also hit more than twice

(including twice) in group 2, it should be removed (14 targets

were removed). In the end, 18 targets remained for further

screening through the reverse docking method.

As mentioned above, the initially assessed Rounds com-

prised a total of 18 targets. Only two of these targets do not

have detailed 3D structural information, i.e. Neurokinin 2

receptor and Serotonin 6 (5-HT6) receptor. Thus, homology

models built using the SWISS-MODEL server (https://swiss-

model.expasy.org/) (Waterhouse et al., 2018; Bienert et al.,

2017) and optimized by SYBYL-X (Tripos, 2012) were used.

The structure of Neurokinin 2 receptor was modelled on the

bovine rhodopsin crystal structure (PDB ID: 1f88) as a

template (Chandrashekaran et al., 2009), while the structure of

the 5-HT6 receptor was modelled using the ꢂ2 adrenergic

˙

receptor template (PDB ID: 4lde) (Łazewska et al., 2017)

(Figs. S17 and S18 in the supporting information). Though the

obtained models exhibit only limited accuracy, it has been

demonstrated that the modelled receptor pocket conforma-

tions can be validated or improved via docking of known

ligands. For the Neurokinin 2 receptor, ligand 6-methylbenzo-

[b]thiophene-2-carboxylic acid (1-{(S)-1-benzyl-4-[4-(tetra-

hydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}cyclo-

pentyl)amide (abbreviated as 10i in the original article) was

used as the well-known antagonist with high afﬁnity (Fattori et

al., 2010). As for the 5-HT6 receptor, AVN-492, a new

promising ligand (now tested in phase I trials) was selected

3.3. Reverse docking studies for target fishing

In order to ﬁnd a few most possible molecular targets for

these Schiff bases, Swiss Target Prediction web server (http://

www.swisstargetprediction.ch/) (Daina et al., 2019; Gfeller et

al., 2013) was used for the initial selection of potential targets.

For each of these eight Schiff bases, 100 most potential targets

were selected for further evaluation. Then cross screenings

were carried out among these eight sets of potential targets

based on the following method. Firstly, eight sets of targets

were divided into two groups according to the anti-

proliferative activities of the Schiff bases. Four sets of poten-

tial targets related to BDHFI, BDHAI, BMHFI and BMHAI

belong to group 1, and another four sets of potential targets

related to BDHMFI, BDHFN, BMHMFI and BMHFN are

group 2. Considering that all the Schiff bases in group 1 have a

similar skeleton and have much higher activities than those in

˙

(Ivachtchenko et al., 2017; Łazewska et al., 2019).

Eventually, all eight Schiff bases were docked into these 18

possible targets with the program SYBYL-X (Tripos, 2012) by

calculating the Total Score of Surﬂex-dock (Table 3) (Rarey et

al., 1996). The docking results were then compared with MTT

results to gain insight into the most possible molecular targets.

Spearman’s rank correlation coefﬁcient ꢈ (Fieller et al., 1957)

was introduced to assess the strength of each target’s mono-

tonic relationship (Table 3). The closer ꢈ is to 1, the stronger

the monotonic relationship. As we can see, ꢈ values of three

´

targets [i.e. human ether-a-go-go-related (hERG) potassium

channel, inhibitor of apoptosis protein 3 and serine/threonine-

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Table 3

Total scores and Spearman’s rank correlation coefﬁcients (ꢈ in the last two rows) of 18 possible targets (PDB IDs are in brackets; two targets in the last

two columns were docked using homology modules for the absence of detailed 3D information).

À

Á

2

6Æ Rank_iꢃ Rank_A549

ꢈ_A549¼ 1 ꢃ

ð1Þ

ð2Þ

nðn²ꢃ 1Þ

ꢀ

ꢁ

2

6Æ Rank_iꢃ Rank_4T

1

ꢈ_4T¼ 1 ꢃ

1

nðn²ꢃ 1Þ

Gonadotropin-

releasing

hormone

receptor

Probable

G-protein

coupled

receptor

88 (5xf1)

c-Jun

N-terminal CaM

Delta

Inhibitor of

apoptosis

protein 3

(5c3h)

opioid

receptor

(4n6h)

Kinesin-like Mu opioid

receptor

(4dkl)

kinase 3

(2r9s)

kinase II

(2vz6)

hERG

(3o0u)

protein 1

(3zcw)

(6nbf)

Ligandⁱ

BDHFI

BDHAI 8.16

BDHMFI 5.96

BDHFN 5.94

12.24

8.08

9.27

8.43

8.82

6.92

7.59

7.32

6.80

5.98

6.03

0.74

0.76

9.37

7.56

7.44

6.53

7.05

7.44

6.32

6.65

6.45

0.57

0.55

7.76

6.19

6.36

6.77

5.00

5.29

5.88

4.19

4.67

0.71

0.74

9.87

6.90

8.02

4.54

6.25

5.97

6.41

4.91

4.20

0.86

0.88

7.33

6.23

6.14

5.07

5.33

5.83

4.77

4.42

4.30

0.83

0.81

19.68

10.44

6.99

8.61

9.04

9.44

8.27

7.76

6.16

0.48

0.33

10.53

8.57

9.46

8.23

7.45

7.31

6.15

6.36

6.47

0.52

0.55

6.67

6.85

6.15

5.28

5.84

4.39

4.52

4.58

4.37

0.62

0.60

BMHFI

6.64

BMHAI 5.60

BMHMFI 6.39

BMHFN 5.59

ꢈA549ⁱⁱ

0.67

0.69

iii

ꢈ4T₁

Protein

Serine/threonine- Serine/threonine- Serine/threonine- Serine/threonine- Sigma

Tryptase

beta-1

(4mpu)

Neurokinin 2 5-HT6

receptor (by

homology)

kinase C

alpha

(4ra4)

protein

kinase

AKT2 (3d0e)

protein

kinase

PIM1 (1yxt)

protein

kinase

PIM2 (4x7q)

protein

kinase

PIM3 (5dwr)

opioid

receptor

(6dk0)

receptor (by

homology)

Ligandⁱ

BDHFI

BDHAI 6.79

BDHMFI 5.27

BDHFN 5.47

7.34

6.35

16.79

32.82

15.18

10.64

10.81

23.41

7.39

8.12

6.02

7.99

5.44

5.51

5.05

5.22

0.62

0.67

10.04

9.00

8.22

6.40

5.65

5.51

7.06

6.67

6.77

7.46

0.12

0.00

9.73

9.60

6.64

7.59

7.95

7.01

5.52

5.80

0.86

0.83

6.70

8.19

5.56

5.63

7.87

7.68

5.92

0.62

0.69

8.00

9.92

4.45

5.51

6.39

7.49

6.42

6.06

0.69

0.71

8.34

7.17

1.87

5.23

6.41

8.27

7.00

6.23

0.69

0.64

9.28

8.80

8.17

8.73

6.71

5.68

6.00

6.44

0.52

0.50

7.95

4.56

6.40

7.07

5.05

5.50

4.43

6.18

0.17

0.02

BMHFI

4.43

BMHAI 4.87

BMHMFI 5.06

BMHFN 4.30

ꢈA549ⁱⁱ

0.60

0.62

iii

ꢈ4T₁

Notes: (i) for all proteins with crystal structures, the ‘ligand’ means the natural ligand included in the protein structure; for the last two proteins built by homology modelling, the ‘ligand’

means the known ligand reported before, i.e. 10i and AVN-492 (see text). (ii) see equation (1) above; (iii) see equation (2) above, n = 8. Rank_iis the rank value of each Schiff base in the

virtual screening, which is determined according to its sequence listed in descending order of total score value (see Table S22 in the supporting information). Rank_A549/Rank_4T1is the

rank value of each Schiff base in the A549/4T₁cell growth MTTassays, which is determined according to its sequence listed in ascending order of IC₅₀value (see Table 2 and Table S23 in

the supporting information).

protein kinase PIM1] are above 0.80, their correlations can be

described as ‘very strong’ (bold in Table 3). Thus, they can be

regarded as the most possible targets.

both BDHFI and BDHAI, two hydrogen bonds were formed,

one is between the indole N—H group and GLN19/GLU59,

and the other is between an imine N atom and HOH267/

GLY66. But in both BDHMFI and BDHFN, no hydrogen

bond was formed and aromatic hydrophobic interactions

dominate the afﬁnity between the protein and the ligand. This

phenomenon can be conﬁrmed by the Polar values, which are

2.60 and 2.18 for BDHFI and BDHAI, respectively, but zero

for BDHMFI and BDHFN.

As for the BMH series of four mono-Schiff bases, all of

them exhibit one to ﬁve hydrogen bonds within the active site

(Fig. 10). Indole N—H groups and imine N atoms form one or

two hydrogen bonds in both BMHFI and BMHAI. A carboxyl

O atom forms an extra hydrogen bond in BMHAI, which is

3.4. Comparison of binding modes between active and less

active ligands

Among the three most possible targets, hERG (PDB ID

3o0u) has the highest Spearman’s rank correlation coefﬁcient

values and is ranked as the best, so the comparison of binding

modes is performed based on the docking results of eight

Schiff bases with hERG, in order to gain insight into the

nature of the structure–activity relationships.

According to our docking simulation, shown in Fig. 9, the

active and less active ligands have different binding modes. In

ꢁ

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Eight novel Schiff bases derived from benzil 15 of 20

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Figure 9

The calculated binding modes of BDHFI, BDHAI, BDHMFI and BDHFN with hERG (PDB ID: 3o0u). Hydrogen bonds are shown with orange dotted

lines. If no hydrogen bond is formed, surfaces that contain the active sites are displayed (red surface). Total score values and seven additional

contributing parts or scores are listed underneath for comparison.

the reason why it has a higher Total Score (also Polar value)

than BMHFI. BMHMFI and BMHFN can form only one

hydrogen bond with protein through a carboxyl O or imine N

atom, so their docking Total Scores (also Polar values) are

lower than those of BMHFI and BMHAI. Put simply, the

more hydrogen bonds formed, the higher the Total Score

values in the docking simulations, and the better the anti-

proliferative activities of the ligands.

There is one phenomenon that seems confusing, i.e.

BDHFN has a higher Total Score than BMHFI (6.25 versus

5.97), but the former has a lower cytotoxic activity than the

latter. Apparently, the deviation between docking predictions

and experimental activities may partly explain this result.

Here, we consider another class of scoring function based on a

potential of mean force, so-called PMF-based scores, which is

a knowledge-based scoring approach based on the work of

Muegge and Martin (Muegge & Martin, 1999; Muegge, 2006).

The PMF-based scoring functions are statistical, because it is

calculated as the sum of the overall atom-pair interaction

Helmholtz free energies between the protein and ligand,

neglecting other characteristics of the environment and

mutual orientation of the atom (Lizunov et al., 2015). As we

know, the higher the PMF value, the poorer the protein–ligand

binding afﬁnity (Sharma & Ghoshal, 2006). From the seventh

columns in Figs. 9 and 10, we can see that BDHFN has the

highest PMF score (68.14) and is ranked as the worst with

ꢁ

16 of 20 Tan et al. Eight novel Schiff bases derived from benzil

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Figure 10

The calculated binding modes of BMHFI, BMHAI, BMHMFI and BMHFN with hERG (PDB ID: 3o0u). Hydrogen bonds are shown with orange dotted

lines. Total score values and seven additional contributing parts or scores are listed underneath for comparison.

respect to the means of the distance-dependent Helmholtz

free energies. That is to say, a long distance between the

protein and ligand has a detrimental contribution to the afﬁ-

nity, which has not been reﬂected apparently in the Total

Score. On the other hand, this phenomenon conﬁrms the

conclusion that PMF has essential differences with all of the

other scoring functions. Its unique knowledge-based algorithm

parameterized using crystal complexes is distinctive (Liu et al.,

2012).

The above explanation is also suitable to BMHAI versus

BDHFI/BDHAI. The former exhibit ﬁve hydrogen bonds

within the active site, but it has a lower Total Score and weaker

cytotoxic activity than the latter two. The PMF score of

BMHAI is 6.08, revealing poorer binding afﬁnities compared

with the meager binding scores of BDHFI and BDHAI (ꢃ5.70

and ꢃ6.26, respectively).

In brief, all of the docking results were in good agreement

with the experimental results, indicating the most probable

target and reasonable structure–activity relationships.

4. Conclusion

Schiff bases have been used widely in the pharmaceutical

industry because of their antimicrobial, anti-inﬂammatory and

anticancer properties. We have described the synthesis and

structural characterization of eight novel Schiff bases derived

from BDH/BMH. These synthesized Schiff base compounds

have similar molecular structures despite having different

terminal fused two-ring aromatics and different substituents.

MTT assays proved that the BDH series of compounds show

higher inhibitory activity compared with the BMH series. The

biological screening results favour the activities of indole-

ꢁ

Acta Cryst. (2020). C76

Tan et al.

Eight novel Schiff bases derived from benzil 17 of 20

research papers

A., Novellino, E., T a glialatela, M., Campiglia, P . & G omez-

Monterrey, I. (2018). J. Med. Chem. 61 , 6140–6152.

Bharathi Dileepan, A. G., Daniel Prakash, T., Ganesh Kumar, A.,

Shameela Rajam, P ., Violet Dhayabaran, V . & R ajaram, R. (2018).

J. Photochem. Photobiol. B , 183 , 191–200.

Bienert, S., Waterhouse, A., de Beer, T. A. P ., T a uriello, G., Studer, G.,

Bordoli, L. & Schwede, T. (2017). Nucleic Acids Res. 45 , D313–

D319.

Blunt, J. W., Copp, B. R., Munro, M. H., Northcote, P . T. & P rinsep,

M. R. (2011). Nat. Prod. Rep. 28 , 196–268.

Boys, S. B. & Bernardi, F . (1970). Mol. Phys. 19 , 553–566.

Brandenburg, K. & Putz, H. (1999). DIAMOND . Crystal Impact

GbR, Bonn, Germany.

containing Schiff bases instead of naphthalene-containing

ones. The cytotoxic activity is also affected by the nature of the

substituents at the indole N atom; the replacement of

hydrogen by methyl will decrease the cytotoxic activities

greatly, while the same replacement on the imine C—H group

has little effect. Generally, our in vitro ﬁndings show that four

of these compounds have high antiproliferative activity

against human lung cancer cell line A549 and mouse breast

cancer cell line 4T₁; two show obvious and comparable

activities with cisplatin. All compounds exhibited weaker

cytotoxicity against normal cells than cancer cells.

Brigg, S., Pribut, N., Basson, A. E., Avgenikos, M., Venter, R., Blackie,

A., van Otterlo, W . A . L. & Pelly, S. C. (2016). Bioorg. Med. Chem.

Lett. 26 , 1580–1584.

Bruker (2000). SMART , SAINT , SADABS , XPREP and SHELXTL .

Bruker AXS Inc., Madison, Wisconsin, USA.

Swiss Target Prediction online servers were used to screen

compounds against large numbers of molecular targets. After

careful examination, 18 possible targets were generated for

further screening through the reverse docking approach.

Afterwards, the three most possible targets were chosen on

account of their correlation with experimental data. Bearing

the highest consistency, the docking results of hERG (PDB

ID: 3o0u) with eight Schiff bases can easily explain the

structure–activity relationships obtained experimentally.

In conclusion, the present work indicates that introduction

of an indole ring on the terminal of this kind of X-shaped

Schiff base leads to the generation of potent anticancer agents.

Bu, F .-Z., T a n, X.-J., Xing, D.-X. & Wang, C. (2017). Acta Cryst. C 73 ,

546–555.

´

Carre n ˜o , A., Zu n ˜i ga, C., Paez-Hernandez, D., Gacitua, M., Polanco,

R., Otero, C., Arratia-Perez, R. & Fuentes, J. A. (2018). New J.

´

Chem. 42 , 8851–8863.

Chandra, S., Verma, S. & Gautam, A. (2007). J. Saudi Chem. Soc. 11 ,

483–488.

Chandrashekaran, I. R., Rao, G. S. & Cowsik, S. M. (2009). J. Chem.

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Chauhan, S., Swami, M., Malik, S. & Singh, R. V . (2008). Main Group

Met. Chem. 31 , 263–272.

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Y .-X., Sui, Q.-C. & Tan, X.-J. (2016). J. Mol. Struct. 1115 , 228–

Funding information

Funding for this research was provided by: Key Laboratory of

Pulp and Paper Science and Technology of Ministry of

Education/Shandong Province of China (award Nos.

KF201712 and KF201821); State Key Laboratory of Biobased

Material and Green Papermaking, Qilu University of Tech-

nology, Shandong Academy of Sciences (grant No.

ZZ20190115).

Cihan-Ustundag, G., Gursoy, E., Naesens, L., Ulusoy-Guzeldemirci,

N. & C ¸ apan, G. (2016). Bioorg. Med. Chem. 24 , 240–246.

Dai, W ., Jiang, X. L., T a o, J. Y. & Shi, F. (2016). J. Org. Chem. 81 , 185–

192.

Daina, A., Michielin, O. & Zoete, V . (2019). Nucleic Acids Res. 47 ,

W357–W364.

De, S., Chowdhury, S., Tocher, D. A. & Datta, D. (2006).

CrystEngComm , 8 , 670–673.

Drew, M. G. B., Parui, D., De, S., Chowdhury, S. & Datta, D. (2007).

New J. Chem. 31 , 1763–1768.

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supporting information

Acta Cryst. (2020). C76 [https://doi.org/10.1107/S2053229619015687]

Synthesis, crystal structures, antiproliferative activities and reverse docking

studies of eight novel Schiff bases derived from benzil

Xue-Jie Tan, Di Wang, Xiao-Ming Hei, Feng-Cun Yang, Ya-Ling Zhu, Dian-Xiang Xing and Jian-

Ping Ma

Computing details

For all structures, data collection: SMART (Bruker, 2000); cell refinement: SMART (Bruker, 2000); data reduction: SAINT

(Bruker, 2000); program(s) used to solve structure: SHELXS2016 (Sheldrick, 2015a); program(s) used to refine structure:

SHELXL2016 (Sheldrick, 2015b); molecular graphics: SHELXTL (Bruker, 2000), PLATON (Spek, 2009) and DIAMOND

(Brandenburg & Putz, 1999); software used to prepare material for publication: SHELXL2016 (Sheldrick, 2015b) and

WinGX (Farrugia, 2012).

(1Z,2Z)-1,2-Bis{(E)-[(1H-indol-3-yl)methylidene]hydrazinylidene}-1,2-diphenylethane (1-BDHFI)

Crystal data

C₃₂H₂₄N₆

M_r= 492.57

F(000) = 1032

D_x= 1.244 Mg m⁻³

Mo Kα radiation, λ = 0.71073 Å

Cell parameters from 380 reflections

θ = 2.5–26.0°

Monoclinic, P2₁/c

a = 13.023 (4) Å

b = 7.340 (2) Å

c = 27.762 (9) Å

β = 97.693 (5)°

V = 2630.0 (14) Å³

Z = 4

µ = 0.08 mm⁻¹

T = 293 K

Block, yellow

0.30 × 0.18 × 0.15 mm

Data collection

Bruker SMART CCD area detector

diffractometer

Radiation source: fine-focus sealed tube

Detector resolution: 10.13 pixels mm^-1

phi and ω scans

13632 measured reflections

5145 independent reflections

2852 reflections with I > 2σ(I)

R_int= 0.046

θ

_max= 26.0°, θ_min= 1.5°

Absorption correction: multi-scan

(SADABS; Bruker, 2000)

h = −16→13

k = −8→9

T

_min= 0.903, T_max= 0.939

l = −33→34

Refinement

Refinement on F²

Least-squares matrix: full

R[F²> 2σ(F²)] = 0.049

wR(F²) = 0.103

367 parameters

0 restraints

Hydrogen site location: inferred from

neighbouring sites

S = 0.88

Only H-atom displacement parameters refined

5145 reflections

Acta Cryst. (2020). C76

sup-1

supporting information

w = 1/[σ²(F_o²) + (0.0408P)²]

where P = (F_o²+ 2F_c²)/3

(Δ/σ)_max< 0.001

Δρ_max= 0.15 e Å⁻³

Δρ_min= −0.22 e Å⁻³

Special details

Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance

matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles;

correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate

(isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.

Refinement. Refinement of F²against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F²,

conventional R-factors R are based on F, with F set to zero for negative F². The threshold expression of F²> 2sigma(F²) is

used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based

on F²are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å²)

x

y

z

U_iso*/U_eq

C1

C2

C3

H3

C4

H4

C5

H5

C6

H6

C7

H7

C8

C9

C10

H10

C11

H11

C12

H12

C13

H13

C14

H14

C15

H15

C16

C17

C18

H18

C19

H19

0.83514 (13)

0.92588 (13)

0.94898 (15)

0.904843

1.03531 (16)

1.049033

1.10143 (19)

1.160256

1.08036 (19)

1.125208

0.99353 (16)

0.980068

0.82392 (13)

0.74742 (14)

0.70293 (18)

0.720298

0.6329 (2)

0.603863

0.6059 (2)

0.558179

0.64870 (18)

0.630452

0.71892 (15)

0.747607

0.99888 (13)

0.984927

1.07404 (13)

1.12335 (12)

1.12274 (14)

1.084885

0.3197 (2)

0.2300 (2)

0.0495 (3)

−0.018427

−0.0314 (3)

−0.153332

0.0661 (3)

0.011204

0.2454 (4)

0.312545

0.3268 (3)

0.448615

0.5230 (2)

0.5987 (3)

0.4886 (4)

0.365746

0.5586 (5)

0.483089

0.7378 (5)

0.784282

0.8485 (4)

0.970999

0.7801 (3)

0.856790

0.6384 (3)

0.762714

0.5654 (2)

0.3906 (3)

0.2432 (3)

0.248467

0.0908 (3)

−0.008483

0.09431 (6)

0.07841 (6)

0.08906 (7)

0.105749

0.07558 (8)

0.082980

0.05130 (8)

0.042183

0.04047 (9)

0.023980

0.05380 (7)

0.046176

0.08899 (6)

0.05028 (7)

0.01317 (8)

0.013003

−0.02378 (10)

−0.048881

−0.02370 (11)

−0.048581

0.01274 (10)

0.012709

0.04968 (8)

0.074567

0.18379 (7)

0.183158

0.22132 (6)

0.22344 (6)

0.19159 (7)

0.160739

0.20652 (8)

0.185639

0.0363 (4)

0.0378 (5)

0.0474 (5)

0.060 (6)*

0.0600 (6)

0.069 (7)*

0.0713 (7)

0.084 (8)*

0.0768 (7)

0.089 (8)*

0.0592 (6)

0.051 (6)*

0.0377 (4)

0.0437 (5)

0.0734 (7)

0.079 (8)*

0.1124 (11)

0.153 (13)*

0.1102 (11)

0.119 (10)*

0.0823 (8)

0.077 (8)*

0.0580 (6)

0.066 (7)*

0.0411 (5)

0.046 (5)*

0.0385 (5)

0.0369 (4)

0.0439 (5)

0.042 (5)*

0.0539 (6)

0.058 (6)*

1.17878 (15)

1.177756

Acta Cryst. (2020). C76

sup-2

supporting information

C20

H20

C21

H21

C22

C23

H23

C24

H24

C25

C26

C27

H27

C28

H28

C29

H29

C30

H30

C31

C32

H32

N1

1.23696 (15)

1.274298

1.24053 (15)

1.280095

1.18325 (13)

1.10613 (14)

1.086467

0.62285 (14)

0.634232

0.53425 (14)

0.49308 (14)

0.52005 (16)

0.579267

0.45807 (19)

0.476153

0.3689 (2)

0.328037

0.3405 (2)

0.281159

0.40318 (18)

0.46970 (18)

0.477661

0.76775 (11)

0.68695 (11)

0.88012 (11)

0.95074 (11)

1.17037 (12)

1.198934

0.0810 (3)

−0.024387

0.2222 (3)

0.215710

0.3765 (3)

0.6476 (3)

0.763553

0.2475 (3)

0.123968

0.3321 (3)

0.5105 (3)

0.6557 (3)

0.650487

0.8070 (3)

0.905300

0.8173 (4)

0.921466

0.6771 (4)

0.683543

0.5254 (4)

0.2500 (4)

0.132346

0.2260 (2)

0.3368 (2)

0.6319 (2)

0.5349 (2)

0.5365 (2)

0.561788

0.3652 (3)

0.341855

0.25202 (8)

0.261014

0.28373 (8)

0.314160

0.26927 (7)

0.26490 (7)

0.273594

0.14849 (7)

0.154452

0.16411 (7)

0.15329 (7)

0.12573 (8)

0.110411

0.12143 (10)

0.103174

0.14363 (12)

0.139702

0.17108 (12)

0.186279

0.17543 (10)

0.19247 (9)

0.204873

0.11353 (5)

0.12654 (5)

0.11766 (5)

0.15116 (5)

0.29348 (6)

0.322448

0.0580 (6)

0.061 (6)*

0.0544 (6)

0.055 (6)*

0.0416 (5)

0.0486 (5)

0.043 (5)*

0.0484 (5)

0.048 (6)*

0.0505 (5)

0.0562 (6)

0.052 (6)*

0.0786 (7)

0.076 (8)*

0.1049 (10)

0.122 (10)*

0.1036 (10)

0.101 (9)*

0.0733 (7)

0.0773 (7)

0.071 (7)*

0.0405 (4)

0.0433 (4)

0.0419 (4)

0.0432 (4)

0.0519 (5)

0.065 (7)*

0.0954 (8)

0.103 (9)*

N2

N3

N4

N5

H35

N6

H36

0.39279 (16)

0.344338

0.19973 (9)

0.217039

Atomic displacement parameters (Å²)

U¹¹

U²²

U³³

U¹²

U¹³

U²³

C1

C2

C3

C4

C5

C6

C7

C8

0.0382 (11)

0.0403 (11)

0.0471 (12)

0.0624 (15)

0.0667 (17)

0.0735 (17)

0.0676 (15)

0.0348 (10)

0.0402 (11)

0.0896 (19)

0.139 (3)

0.0372 (11)

0.0408 (12)

0.0435 (13)

0.0486 (15)

0.0754 (18)

0.0847 (19)

0.0539 (15)

0.0393 (12)

0.0441 (12)

0.0590 (17)

0.095 (2)

0.0317 (10)

−0.0033 (9)

−0.0045 (9)

−0.0016 (10)

0.0082 (12)

0.0164 (15)

0.0034 (15)

0.0078 (12)

−0.0019 (9)

−0.0054 (10)

−0.0068 (14)

−0.016 (2)

−0.0014 (9)

0.0029 (9)

0.0022 (9)

0.0017 (9)

0.0317 (10)

0.0517 (12)

0.0702 (16)

0.0768 (17)

0.0810 (18)

0.0602 (14)

0.0395 (11)

0.0460 (12)

0.0626 (15)

0.084 (2)

0.0076 (10)

0.0130 (12)

0.0276 (14)

0.0430 (15)

0.0244 (12)

0.0063 (9)

0.0029 (9)

−0.0226 (14)

−0.059 (2)

−0.0016 (11)

−0.0046 (12)

−0.0022 (15)

0.0145 (16)

0.0141 (12)

0.0061 (9)

0.0133 (10)

0.0078 (13)

0.0148 (19)

0.045 (2)

C9

C10

C11

C12

C13

C14

0.100 (2)

0.0655 (16)

0.0476 (13)

0.109 (3)

0.0687 (19)

0.0526 (14)

0.104 (2)

0.107 (2)

0.0711 (15)

−0.008 (2)

0.0087 (15)

−0.0012 (11)

−0.0505 (19)

−0.0107 (15)

−0.0019 (12)

0.0384 (17)

0.0162 (13)

Acta Cryst. (2020). C76

sup-3

supporting information

C15

C16

C17

C18

C19

C20

C21

C22

C23

C24

C25

C26

C27

C28

C29

C30

C31

C32

N1

0.0357 (11)

0.0303 (10)

0.0279 (10)

0.0381 (11)

0.0461 (13)

0.0451 (13)

0.0378 (12)

0.0323 (10)

0.0365 (11)

0.0394 (12)

0.0363 (12)

0.0360 (12)

0.0464 (13)

0.0728 (18)

0.078 (2)

0.0711 (19)

0.0542 (15)

0.0602 (16)

0.0377 (9)

0.0371 (9)

0.0391 (9)

0.0424 (9)

0.0405 (10)

0.0672 (14)

0.0341 (12)

0.0425 (12)

0.0447 (12)

0.0480 (13)

0.0457 (13)

0.0558 (15)

0.0779 (17)

0.0540 (13)

0.0500 (14)

0.0478 (14)

0.0561 (14)

0.0565 (14)

0.0570 (15)

0.0533 (16)

0.070 (2)

0.0541 (13)

0.0421 (11)

0.0384 (11)

0.0445 (12)

0.0700 (15)

0.0730 (16)

0.0457 (13)

0.0388 (11)

0.0592 (13)

0.0568 (13)

0.0599 (14)

0.0593 (14)

0.0650 (15)

0.112 (2)

0.173 (3)

0.169 (3)

0.105 (2)

0.109 (2)

0.0432 (9)

0.0486 (10)

0.0472 (9)

0.0471 (10)

0.0397 (10)

0.141 (2)

0.0003 (9)

0.0086 (10)

0.0028 (9)

0.0052 (8)

0.0016 (9)

0.0084 (11)

0.0069 (12)

−0.0010 (10)

0.0059 (9)

0.0054 (10)

0.0017 (10)

0.0097 (10)

0.0076 (10)

0.0071 (11)

0.0201 (16)

0.040 (2)

−0.0037 (10)

−0.0082 (10)

−0.0024 (9)

−0.0051 (10)

−0.0045 (13)

0.0171 (13)

0.0176 (13)

0.0001 (10)

−0.0159 (11)

0.0080 (11)

0.0034 (11)

−0.0086 (12)

−0.0049 (12)

−0.0005 (16)

−0.009 (2)

−0.0002 (9)

−0.0037 (9)

0.0010 (10)

0.0040 (11)

0.0089 (12)

0.0011 (12)

−0.0034 (10)

−0.0020 (10)

−0.0046 (10)

−0.0085 (11)

−0.0070 (10)

−0.0012 (12)

0.0033 (14)

0.0164 (18)

0.0079 (17)

−0.0063 (14)

−0.0021 (14)

0.0002 (8)

0.083 (2)

0.061 (2)

−0.018 (2)

0.0665 (17)

0.0682 (18)

0.0403 (9)

0.0443 (10)

0.0377 (10)

0.0375 (9)

0.0736 (13)

0.0917 (18)

0.0320 (14)

0.0310 (15)

0.0047 (8)

0.0064 (8)

−0.0012 (8)

−0.0034 (8)

−0.0013 (8)

0.0629 (15)

−0.0091 (16)

0.0156 (16)

0.0053 (8)

0.0054 (8)

0.0063 (8)

0.0011 (8)

−0.0112 (10)

0.0055 (16)

N2

N3

N4

N5

−0.0012 (8)

0.0001 (8)

0.0013 (8)

−0.0041 (9)

−0.0066 (13)

N6

Geometric parameters (Å, º)

C1—N1

C1—C2

C1—C8

C2—C7

C2—C3

C3—C4

C3—H3

C4—C5

C4—H4

C5—C6

C5—H5

C6—C7

C6—H6

C7—H7

C8—N3

C8—C9

C9—C10

C9—C14

C10—C11

C10—H10

C11—C12

1.286 (2)

C18—C19

1.370 (2)

0.9300

1.385 (3)

0.9300

1.357 (3)

0.9300

1.386 (3)

0.9300

1.374 (2)

1.348 (2)

0.9300

1.278 (2)

1.428 (3)

0.9300

1.367 (3)

1.431 (3)

1.384 (3)

1.398 (3)

1.369 (3)

0.9300

1.471 (2)

1.505 (2)

1.382 (2)

1.367 (3)

0.9300

1.365 (3)

0.9300

1.370 (3)

0.9300

1.373 (3)

0.9300

1.286 (2)

1.473 (2)

1.375 (3)

1.382 (3)

1.378 (3)

0.9300

C18—H18

C19—C20

C19—H19

C20—C21

C20—H20

C21—C22

C21—H21

C22—N5

C23—N5

C23—H23

C24—N2

C24—C25

C24—H24

C25—C32

C25—C26

C26—C27

C26—C31

C27—C28

C27—H27

C28—C29

1.361 (4)

1.387 (3)

Acta Cryst. (2020). C76

sup-4

supporting information

C11—H11

C12—C13

C12—H12

C13—C14

C13—H13

C14—H14

C15—N4

C15—C16

C15—H15

C16—C23

C16—C17

C17—C18

C17—C22

0.9300

1.358 (3)

0.9300

1.374 (3)

0.9300

1.280 (2)

1.435 (2)

0.9300

1.367 (2)

1.432 (2)

1.397 (2)

1.404 (2)

C28—H28

C29—C30

C29—H29

C30—C31

C30—H30

C31—N6

C32—N6

C32—H32

N1—N2

0.9300

1.361 (4)

0.9300

1.376 (3)

0.9300

1.371 (3)

1.347 (3)

0.9300

1.4146 (19)

1.4104 (18)

0.8600

N3—N4

N5—H35

N6—H36

0.8600

N1—C1—C2

N1—C1—C8

C2—C1—C8

C7—C2—C3

C7—C2—C1

C3—C2—C1

C4—C3—C2

C4—C3—H3

C2—C3—H3

C5—C4—C3

C5—C4—H4

C3—C4—H4

C4—C5—C6

C4—C5—H5

C6—C5—H5

C5—C6—C7

C5—C6—H6

C7—C6—H6

C6—C7—C2

C6—C7—H7

C2—C7—H7

N3—C8—C9

N3—C8—C1

C9—C8—C1

C10—C9—C14

C10—C9—C8

C14—C9—C8

C9—C10—C11

C9—C10—H10

C11—C10—H10

C12—C11—C10

C12—C11—H11

C10—C11—H11

C13—C12—C11

120.37 (17)

120.56 (16)

119.05 (16)

117.65 (19)

120.61 (18)

121.69 (17)

121.4 (2)

119.3

120.2 (2)

119.9

119.4 (2)

120.3

120.5 (2)

119.8

120.8 (2)

119.6

C17—C18—H18

C18—C19—C20

C18—C19—H19

C20—C19—H19

C21—C20—C19

C21—C20—H20

C19—C20—H20

C20—C21—C22

C20—C21—H21

C22—C21—H21

N5—C22—C21

N5—C22—C17

C21—C22—C17

N5—C23—C16

N5—C23—H23

C16—C23—H23

N2—C24—C25

N2—C24—H24

C25—C24—H24

C32—C25—C24

C32—C25—C26

C24—C25—C26

C27—C26—C31

C27—C26—C25

C31—C26—C25

C28—C27—C26

C28—C27—H27

C26—C27—H27

C27—C28—C29

C27—C28—H28

C29—C28—H28

C30—C29—C28

C30—C29—H29

C28—C29—H29

120.5

121.5 (2)

119.2

121.3 (2)

119.4

117.78 (19)

121.1

130.74 (18)

106.91 (17)

122.34 (19)

110.24 (18)

124.9

121.8 (2)

119.1

124.6 (2)

106.42 (19)

129.00 (19)

118.2 (2)

134.79 (19)

107.0 (2)

118.9 (2)

120.6

121.6 (3)

119.2

120.9 (3)

119.5

119.6

119.37 (17)

121.09 (16)

119.54 (16)

118.1 (2)

120.07 (19)

121.78 (19)

120.6 (3)

119.7

120.3 (3)

119.9

119.9 (3)

119.5

Acta Cryst. (2020). C76

sup-5

supporting information

C13—C12—H12

C11—C12—H12

C12—C13—C14

C12—C13—H13

C14—C13—H13

C13—C14—C9

C13—C14—H14

C9—C14—H14

N4—C15—C16

N4—C15—H15

C16—C15—H15

C23—C16—C17

C23—C16—C15

C17—C16—C15

C18—C17—C22

C18—C17—C16

C22—C17—C16

C19—C18—C17

C19—C18—H18

120.0

120.2 (3)

119.9

120.8 (2)

119.6

120.91 (17)

119.5

106.07 (16)

125.16 (17)

128.56 (16)

118.16 (17)

134.69 (17)

107.15 (16)

118.92 (18)

120.5

C29—C30—C31

C29—C30—H30

C31—C30—H30

N6—C31—C30

N6—C31—C26

C30—C31—C26

N6—C32—C25

N6—C32—H32

C25—C32—H32

C1—N1—N2

117.3 (3)

121.4

129.8 (2)

107.0 (2)

123.2 (3)

109.6 (2)

125.2

111.77 (15)

112.49 (16)

111.16 (15)

112.39 (15)

109.62 (16)

125.2

109.9 (2)

125.0

C24—N2—N1

C8—N3—N4

C15—N4—N3

C23—N5—C22

C23—N5—H35

C22—N5—H35

C32—N6—C31

C32—N6—H36

C31—N6—H36

125.0

Hydrogen-bond geometry (Å, º)

D—H···A

D—H

H···A

D···A

D—H···A

N5—H35···N1ⁱ

C3—H3···N3ⁱⁱ

0.86

0.93

2.14

2.61

2.948 (2)

3.320 (3)

156

133

Symmetry codes: (i) −x+2, y+1/2, −z+1/2; (ii) x, y−1, z.

(1Z,2Z)-1,2-Bis{(E)-[1-(1H-indol-3-yl)ethylidene]hydrazinylidene}-1,2-diphenylethane (2-BDHAI)

Crystal data

C₃₄H₂₈N₆

Z = 2

M_r= 520.62

Triclinic, P1

F(000) = 548

D_x= 1.252 Mg m⁻³

Mo Kα radiation, λ = 0.71073 Å

Cell parameters from 380 reflections

θ = 2.5–26.0°

a = 10.2585 (4) Å

b = 11.9610 (5) Å

c = 12.8232 (6) Å

α = 64.941 (4)°

β = 79.573 (3)°

γ = 76.829 (4)°

V = 1381.45 (11) Å³

µ = 0.08 mm⁻¹

T = 293 K

Bar, yellow

0.35 × 0.15 × 0.12 mm

Data collection

Bruker SMART CCD area detector

diffractometer

Radiation source: fine-focus sealed tube

Detector resolution: 10.13 pixels mm^-1

phi and ω scans

15195 measured reflections

5328 independent reflections

4490 reflections with I > 2σ(I)

R_int= 0.026

θ

_max= 26.0°, θ_min= 1.8°

Absorption correction: multi-scan

(SADABS; Bruker, 2000)

h = −12→12

k = −14→14

l = −13→15

T

_min= 0.903, T_max= 0.939

Acta Cryst. (2020). C76

sup-6

supporting information

Refinement

Refinement on F²

Least-squares matrix: full

R[F²> 2σ(F²)] = 0.044

wR(F²) = 0.129

S = 1.05

5328 reflections

Only H-atom displacement parameters refined

w = 1/[σ²(F_o²) + (0.0694P)²+ 0.1725P]

where P = (F_o²+ 2F_c²)/3

(Δ/σ)_max< 0.001

Δρ_max= 0.19 e Å⁻³

Δρ_min= −0.18 e Å⁻³

392 parameters

0 restraints

Hydrogen site location: inferred from

neighbouring sites

Extinction correction: SHELXL2016

(Sheldrick, 2015b),

Fc^*=kFc[1+0.001xFc²λ³/sin(2θ)]^-1/4

Extinction coefficient: 0.083 (4)