Synthesis, structure and antiproliferative and optical activities of two new biphenyl-derived Schiff bases

Article abstract of DOI:10.1107/S2053229618017989

Two novel Schiff bases derived from indole and biphenyl have been designed and synthesized, namely 3-((E)-{(E)-[1-(biphenyl-4-yl)ethylidene]hydrazinylidene}methyl)-1-methyl-1H-indole (3-BEHMI) acetonitrile monosolvate, C₂₄H₂₁N₃·CH₃CN, and 3-((E)-{(E)-[1-(biphenyl-4-yl)ethylidene]hydrazinylidene}methyl)-1-methyl-1H-indole (3-BEHEI) acetonitrile monosolvate, C₂₄H₂₁N₃·CH₃CN. Their structures were characterized by elemental analysis, quadrupole time-of-flight MS, NMR and UV–Vis spectroscopy. The single-crystal packing structure of 3-BEHMI is largely dominated by C—H…π interactions and weak van der Waals interactions. The in vitro cytotoxicity of the two title compounds have been evaluated against two tumour cell lines (A549 human lung cancer and 4T₁ mouse breast cancer) and two normal cell lines (MRC-5 normal lung cells and NIH 3T3 fibroblasts) by MTT assay. The results indicate that 3-BEHEI exhibits a slightly weaker antiproliferative capability (IC₅₀ = ～50 μM) than the previously reported similar Schiff base 3-BEHI (IC₅₀?= ～20 μM). This is in line with docking results. 3-BEHMI demonstrates a weak cytotoxic activity, with IC₅₀ values around 110 μM, which disagrees with its docking results. Overall, the tested compounds manifest relevant cytotoxicities on the selected cancer cell lines and normal cell lines. The UV–Vis and fluorescence spectra were recorded and reproduced through the TD-DFT method with four types of hybrid density functionals, including B3LYP, M062X, PBE1PBE and WB97XD.

Full text of DOI:10.1107/S2053229618017989

research papers
Synthesis, structure and antiproliferative and
optical activities of two new biphenyl-derived
Schiff bases
ISSN 2053-2296
a,b
a
a
a
Xue-Jie Tan, * Ling-Yao Zhang, Yuan-Kai Sun and Xue-Min Zhou
a
School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology (Shandong Academy of Sciences),
Received 7 July 2018
b
Jinan, Shandong Province 250353, People’s Republic of China, and State Key Laboratory of Biobased Material and Green
Accepted 19 December 2018
Papermaking, Qilu University of Technology (Shandong Academy of Sciences), Jinan, Shandong Province 250353,
People’s Republic of China. *Correspondence e-mail: tanxuejie@163.com
Edited by A. G. Oliver, University of Notre
Dame, USA
Two novel Schiff bases derived from indole and biphenyl have been designed
and synthesized, namely 3-((E)-{(E)-[1-(biphenyl-4-yl)ethylidene]hydrazinyl-
idene}methyl)-1-methyl-1H-indole (3-BEHMI) acetonitrile monosolvate,
C H N ÁCH CN, and 3-((E)-{(E)-[1-(biphenyl-4-yl)ethylidene]hydrazinyl-
Keywords: Schiff bases; biphenyl; indole; cyto-
toxicity; antiproliferative; crystal structure;
optical activities.
2
4
21
3
3
idene}methyl)-1-methyl-1H-indole (3-BEHEI) acetonitrile monosolvate,
C H N ÁCH CN. Their structures were characterized by elemental analysis,
CCDC reference: 1886316
24
21
3
3
quadrupole time-of-flight MS, NMR and UV–Vis spectroscopy. The single-
crystal packing structure of 3-BEHMI is largely dominated by C—HÁ Á Áꢀ
interactions and weak van der Waals interactions. The in vitro cytotoxicity of the
two title compounds have been evaluated against two tumour cell lines (A549
Supporting information: this article has
supporting information at journals.iucr.org/c
human lung cancer and 4T mouse breast cancer) and two normal cell lines
1
(MRC-5 normal lung cells and NIH 3T3 fibroblasts) by MTT assay. The results
indicate that 3-BEHEI exhibits a slightly weaker antiproliferative capability
(
IC = ꢀ50 mM) than the previously reported similar Schiff base 3-BEHI (IC =
5
0
50
ꢀ
20 mM). This is in line with docking results. 3-BEHMI demonstrates a weak
cytotoxic activity, with IC50 values around 110 mM, which disagrees with its
docking results. Overall, the tested compounds manifest relevant cytotoxicities
on the selected cancer cell lines and normal cell lines. The UV–Vis and
fluorescence spectra were recorded and reproduced through the TD-DFT
method with four types of hybrid density functionals, including B3LYP, M062X,
PBE1PBE and WB97XD.
1
. Introduction
Dihydroorotate dehydrogenase (DHODH) is an important
target for anticancer drug discovery since these enzymes share
a common role in the DNA replication and repair mechanisms
(Abdullah et al., 2015). As a well-known DHODH-specific
inhibitor, Brequinar sodium (see Scheme 1) can potentially
inhibit the growth of a broad spectrum of human solid
tumours (including human colon, breast, lung, head and neck,
and stomach xenografts in athymic mice) for both in vitro and
in vivo xenograft models (Chen et al., 1992; Dexter et al., 1985;
Braakhuis et al., 1990), but was withdrawn from development
because of its toxicity (Dell, 1998; Maroun et al., 1993). Since
then, various similar DHODH inhibitors have been designed
and synthesized (Batt, 1999; Vyas & Ghate, 2011, 2012; Zhu et
al., 2015; Afzal et al., 2015; Saini et al., 2015; Liu et al., 2016;
Narsimha et al., 2016; Janzer et al., 2017).
We have reported a novel Schiff base derived from indole
and biphenyl (see 3-BEHI in Scheme 1) (Bu et al., 2017).
Compared with Brequinar sodium, this Schiff base shows
weaker lung A549 and breast 4T1 cancer cell inhibitory
activities (IC50 = 20.5 and 18.5 mM, respectively). Structurally,
#
2019 International Union of Crystallography
Acta Cryst. (2019). C75
https://doi.org/10.1107/S2053229618017989 1 of 10

research papers
3
indole), 8.31 (d, 1H, J
both 3-BEHI and Brequinar sodium have the biphenyl moiety.
3-BEHI bears one indole ring and a flexible linker, while
= 7.6 Hz, proton on the six-
H-H
3
membered ring of indole), 8.04 (d, 2H, JH-H = 8.4 Hz,
Brequinar sodium is a fluorinated quinolinecarboxylic acid
sodium salt. As has been proved, the imine N atoms and
biphenyl moiety recognize different regions of the protein and
achieve different kinds of interactions (hydrogen bonds and
aromatic hydrophobic interactions). Therefore, to obtain a
greater chemical diversity, the biphenyl group and the flexible
linker were both taken as starting points for the design of new
inhibitors.
In the current work, we first designed two similar candidate
structures (see 3-BEHMI and 3-BEHEI in Scheme 1) having a
similar skeleton to 3-BEHI. Their performance was assessed
with the docking program SYBYL-X (Tripos, 2012) by calcu-
lating the Consensus Scores (C-scores). The satisfactory
results prompted us to synthesize and assess the in vitro
cytotoxicity of these compounds. In addition, various theore-
tical methods were used to reproduce the absorption and
emission optical properties in order to give an insight into
these highly conjugated molecules.
biphenyl protons), 7.96 (s, 1H, imine proton, H—C N), 7.75
= 8.4 Hz, biphenyl protons), 7.62–7.24 (m, 6H,
3
(dd, 4H, J
3
H-H
JH-H = 7.6–8.4 Hz, protons on the six-membered ring of indole
and biphenyl rings), 3.87 (s, 3H, methyl protons linked to
indole N, N—CH ), 2.61 (s, 3H, methyl protons linked to imine
3
1
3
C, C—CH3). C NMR (DMSO-d ): ꢁ (ppm) 161.70 (imine
6
carbon linked to indole), 161.65, 155.27, 155.15, 141.15, 139.41,
137.80, 137.18, 136.33, 136.25, 128.97, 127.75, 127.12, 126.62,
126.50, 125.10, 125.07, 122.85, 122.11, 121.17, 111.37, 110.43
(biphenyl carbons and indole ring carbons), 32.93 (methyl
carbon linked to indole N, N—CH ), 14.59 (methyl carbon
3
linked to imine C, C—CH ). UV–Vis (CH CH OH) ꢂ (nm)
3
3
2
max
À1
5
À1
5
5
("/l mol cm ): 203.0 (1.2 Â 10 ), 222.0 (0.8 Â 10 ), 282.0
5
(0.4 Â 10 ), 348.0 (0.8 Â 10 ).
2
. Experimental
2.1. Materials and measurements
All chemicals were purchased from Aladdin-reagent
Chemicals and were used without further purification.
Elemental analyses (C, H and N) were carried out with a
PerkinElmer 2400 microanalyzer. Accurate-mass measure-
ments were acquired on an Agilent-6520 quadrupole time-of-
1
13
flight tandem mass spectrometer (Q-TOF). H and C NMR
spectra were measured on a Bruker Avance 400 MHz instru-
ment. The chemical shifts are reported in parts per million
(
ppm) relative to tetramethylsilane (SiMe , ꢁ = 0 ppm),
4
referenced to the chemical shifts of the residual solvent peak
deuterated dimethyl sulfoxide (DMSO-d ) or deuterated
chloroform (CDCl )]. UV–Vis absorption spectra were
[
6
3
recorded using a UV-1700 spectrophotometer (Shimadzu,
À5
À1
Japan) in 1 Â 10 mol l ethanol solution. Fluorescence
spectra were measured at room temperature on a RF5301 PC
fluorescence spectrophotometer (Shimadzu, Japan) in the
wavelength range between 250 and 550 nm, still in
À5
À1
1
0
mol l ethanol solution. Melting points were determined
on a WRS-2A electrothermal digital melting-point apparatus
Shanghai Precision & Scientific Instrument Co. Ltd, China).
(
2.2. Synthesis and crystallization
As stated in our previous article (Bu et al., 2017), all Schiff
bases were readily prepared by similar two-step condensation
reactions (see Fig. 1). The physicochemical characterization
results are listed below. The NMR spectra are available in the
supporting information (see Figs. S1–S4; all figures and tables
with the prefix ‘S’ are available in the supporting information).
2.2.2. 3-BEHEI. Elemental analysis found (calculated for
C H N ) (%): C 82.11 (82.02), H 6.07 (6.02), N 12.03 (11.96);
2
4
21
3
+
HRMS (ESI): m/z calculated for C H N + H : 352.1814
24
21
3
+
[M + H ]; found: 352.1815; m.p. 282.1–283.8 C, H NMR
ꢁ
1
2.2.1. 3-BEHMI. Elemental analysis found (calculated for
C H N ) (%): C 82.08 (82.02), H 6.06 (6.02), N 11.98 (11.96);
(CDCl ): ꢁ (ppm) 8.67 (s, 1H, indole proton linked to N,
2
4
21
3
3
+
HRMS (ESI): m/z calculated for C H N + H : 352.1814 [M +
–N—H), 8.40 (s, 1H, proton on the five-membered ring of
= 7.2 Hz, proton on the six-
24
21
3
+
ꢁ
1
H ]; found: 352.1814; m.p. 168.5–169.0 C, H NMR (DMSO-
3
indole), 8.03 (d, 4H, J
H-H
3
membered ring of indole), 7.70–7.36 (m, 9H, J
d6): ꢁ (ppm) 8.78 (s, 1H, proton on the five-membered ring of
= 8.4–
H-H
ꢂ
2
of 10 Tan et al. Two new biphenyl-derived Schiff bases
Acta Cryst. (2019). C75

research papers
6
.8 Hz, biphenyl protons), 2.42 (s, 6H, methyl protons linked
Table 1
Crystallographic data and structure refinement summary for 3-BEHMI.
13
to imine C, C—CH3). C NMR (CDCl ): ꢁ (ppm) 168.78
3
(
imine carbon linked to indole), 162.65, 155.20, 155.15, 142.11,
Compound
3-BEHMI
ÁC
392.49
Block/yellow
Monoclinic
1
1
1
39.39, 137.36, 137.08, 136.13, 136.05, 128.67, 127.43, 127.20,
26.71, 126.49, 125.09, 125.05, 123.15, 122.91, 121.86, 110.92,
09.99 (biphenyl carbons and indole ring carbons), 15.86
Chemical formula
C
24
H
21
N
3
2 3
H N
M
r
Crystal habit
Crystal system
Space group
(methyl carbon linked to indole N, N—CH ), 14.59 (methyl
carbon linked to imine C, C—CH ). UV–Vis (CH CH OH)
ꢂ
3
1
P2 /c
˚
3
3
2
a (A)
8.1280 (2)
8.21470 (10)
32.9809 (6)
90.00
90.252 (2)
90.00
2202.08 (7)
4
1.184
0.071
298
832
À1
À1
5
˚
(nm) ("/l mol cm ): 204.0 (1.8 Â 10 ), 301.0 (1.1 Â
b (A)
max
5
0 ).
˚
c (A)
ꢃ ( )
1
ꢁ
ꢁ
ꢄ ( )
ꢁ
ꢅ ( )
2
.3. X-ray crystallographic analysis
˚
V /A
3
3-BEHEI has a tendency to crystallize as fine yellow
Z
À3
D
calc (Mg m
)
powders. No crystal of sufficient thickness and quality could be
obtained to perform a single-crystal analysis. Hence only
3
data, data collection and structure refinement details are
summarized in Table 1. All methyl H atoms of the acetonitrile
solvent and 3-BEHMI were included in calculated positions
À1
ꢆ (mm
T (K)
F(000)
)
-BEHMI was characterized by X-ray diffraction. Crystal
R
R
wR
S
int
0.0395
0.0525
0.151/4300
1.024
1
[I > 2ꢇ(I)]
/reflections
2
and were allowed to ride on the parent atoms, with C—H =
˚
Computer programs: SMART (Bruker, 2000), SAINT (Bruker, 2000), SHELXS2016
(Sheldrick, 2015a), SHELXL2016 (Sheldrick, 2015b), DIAMOND (Brandenburg & Putz,
1999), SHELXTL (Bruker, 2000), ORTEP-3 (Farrugia, 2012) and PLATON (Spek,
2009).
0
.96 A and with U (H) values refined freely. All other H
iso
atoms were found in difference Fourier maps; both positional
and isotropic displacement parameters were refined.
Bernardi, 1970). All geometries are extracted from the inter-
acting pairs in the crystal of 3-BEHMI without any structural
relaxation.
2
.4. Computational studies
In this work, theoretical calculations were mainly used in
two parts.
Firstly, Møller–Plesset theory (MP2; Møller & Plesset, 1934)
was employed to explore the intermolecular interactions in
Secondly, the absorption and emission spectra of 3-BEHMI
and 3-BEHEI were investigated. In preliminary optimizations,
the geometry of 3-BEHMI was first extracted from the single-
crystal X-ray structure and then optimized by employing
density functional theory (DFT) with four different density
functionals, including B3LYP (Becke, 1993; Lee et al., 1988),
M062X (Zhao & Truhlar, 2008), PBE1PBE (Perdew et al.,
1996) and WB97XD (Chai & Head-Gordon, 2008) with the
6-311+G(d,p) basis set (McLean & Chandler, 1980; Krishnan
et al., 1980). The original structure of 3-BEHEI is from the
3-BEHMI. This method requires time- and resource-
demanding simulations because of large basis sets due to
correlation effects, but it is much more reliable. A medium
basis set 6-31G(d,p) (Rassolov et al., 2001; Frisch et al., 1984)
was employed throughout the calculations. The standard
counterpoise method was applied to correct interaction
energies for the basis set superposition error (BSSE) (Boys &
Figure 1
The preparation of the title Schiff bases by similar two-step condensation reactions.
ꢂ
Acta Cryst. (2019). C75
Tan et al.
Two new biphenyl-derived Schiff bases 3 of 10

research papers
Figure 2
The predicted binding mode of (a) 3-BEHMI and (b) 3-BEHEI with DHODH (PDB ID: 4igh). Two hydrogen bonds in (a) are shown with orange dotted
lines. No hydrogen bond is formed in (b) and 3-BEHEI is docked into the active site with aromatic hydrophobic interactions.
modified structure of 3-BEHMI since no crystal structure
could be used. Geometry optimizations were computed in
both the gas phase and an ethanol solution (with the same
solvents as those in the experiments), using PCM (polarizable
continuum model) (Tomasi et al., 2005). The absence of an
imaginary frequency in the calculated IR frequencies ensures
that the optimized geometries correspond to true energy
minima. For the UV–Vis spectrum, four TD-DFT methods
Gorelsky & Lever, 2001) using the Gaussian distribution
À1
model with a half-bandwidth of 500 cm based on TD-DFT
results.
To identify the type of frontier molecular orbitals (ꢇ, ꢀ or
n), relative atomicorbital contributions were calculated by
employing natural bond orbital (NBO) analyses. NBO
analyses were carried out on the file generated from the
Gaussian option ‘pop=NBOread’ using the NBO 5.0 program
(Glendening et al., 2001) at the same level of theory as the TD-
DFT calculations.
All calculations were carried out using the GAUSSIAN03
program package (Frisch et al., 2004) on a Sunway BlueLight
MPPsupercomputer housed at the National Supercomputer
Center in Jinan, China.
(Bauernschmitt & Ahlrichs, 1996; Stratmann et al., 1998;
Casida et al., 1998; Frisch et al., 2004), including the afore-
mentioned B3LYP, M062X, PBE1PBE and WB97XD, were
used, all employing the 6-311+g(d,p) basis set, based on the
optimized ground-state geometry (S state). For the fluores-
0
cence emission, the same four TD-DFT methods were used,
based on the optimized first excited singlet state (S ). UV–Vis
1
and fluorescence spectra were determined through the
GaussView package (Version 5.0; Nielsen & Holder, 1998).
The major contributions of the transitions were designated
with the aid of the SWizard program (Gorelsky, 2010;
2
.5. Molecular docking and cytotoxicity assays
.5.1. Molecular docking. Molecular docking studies of
-BEHMI and 3-BEHEI with DHODH (PDB code: 4igh)
Das et al., 2013) were performed using Sybyl/Surflex-dock in
2
3
(
order to evaluate the potential inhibitory activity. All solvent
molecules were removed from the protein structure. The
molecular structures of the ligands were designed and opti-
mized at the B3LYP/6-311+G(d,p) level. The default para-
meters were used with respect to charges, bond orders and
geometrical flexibility with no other constraints.
2.5.2. Cell culture. Human lung carcinoma A549 cells,
mouse breast cancer 4T cells, human MRC-5 lung normal
1
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 supplemented with 10% heat-
inactivated fetal bovine serum (FBS), 2 mM glutamine,
À1
À1
1
00 U ml
penicillin and 100 mg ml
maintained at 37 C in a humidified atmosphere of 5% CO .
streptomycin, and
Figure 3
ꢁ
2
The molecular structure of 3-BEHMI, showing the atom-numbering
scheme and displacement ellipsoids for non-H atoms drawn at the 30%
probability level at 298 K.
2
.5.3. MTT assay for cell proliferation. The MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay
ꢂ
4
of 10 Tan et al. Two new biphenyl-derived Schiff bases
Acta Cryst. (2019). C75

research papers
Table 2
Selected C—HÁ Á Áꢀ interaction geometry for 3-BEHEI (A, ).
˚
ꢁ
The table lists the H–centroid distance (HÁ Á ÁCg), the perpendicular distance of H to plane I (H-Perp), slippage angles between HÁ Á ÁCg and the normal to plane I
(ꢅ), the C—HÁ Á ÁCg angles (C—HÁ Á ÁCg), the C–centroid distance (CÁ Á ÁCg), and the angles between the C—H bond line and plane I (C—HÁ Á Áꢀ).
Notation
C—HÁ Á Áꢀ (plane I)
HÁ Á ÁCg
H-Perp
ꢅ
C—HÁ Á ÁCg
CÁ Á ÁCg
C—HÁ Á Áꢀ
i
a
b
C2—H2Á Á ÁCg1
2.80 (2)
3.0934
2.679
2.713
16.69
28.71
159.7 (13)
146.48
3.729 (2)
3.930 (3)
60.31
60.80
ii
C24—H24CÁ Á ÁCg1
1
2
1
2
Ring 1 consists of atoms C17–C22 Symmetry codes: (i) Àx + 1, y À , Àz + ; (ii) Àx + 2, Ày + 1, -.
˚
is only 0.31 (4) A]. Interestingly, the propeller twist of the
biphenyl ring system in 3-BEHMI is 29.2 (3) , the same as in
was conducted and modified as described in the literature
(Zhao et al., 2010; Cheng et al., 2016; Bu et al., 2017). The cells
ꢁ
(
8000 cells) were seeded on 96-well microtitre plates in F12K
3-BEHI. As we know, the degree of twist of the biphenyl ring
system is an important factor in determining its toxicity, which
might come from DNA intercalation by interacting with
nucleobases (Johar et al., 2008). A decrease in cytotoxicity
should therefore be achieved by disrupting the planarity of the
biphenyl ring system and/or avoiding the biphenyl ring system
(Gargano et al., 2016). From this point, 3-BEHMI should have
a similar toxicity to 3-BEHI. But our antiproliferative
experiments proved that this is not the case (see Section 3.3,
Cytotoxicity assays).
In the packing structure of 3-BEHMI, no classic inter-
molecular hydrogen bonds and ꢀ–ꢀ stacking can be found and
the interplay of C—HÁ Á Áꢀ interactions and van der Waals
interactions play crucial roles in the formation of the final
supramolecular frameworks (Table 2). For the convenience of
description, different notations are introduced in order to
denote these different kinds of C—HÁ Á Áꢀ interactions (the
first column in Table 2).
medium with 10% FBS and incubated overnight. 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-
À1
tration of 0.5 mg ml of MTT. After 3 h, the cell culture
medium was removed. Formazan crystals in adherent cells
were dissolved in 200 ml dimethyl sulfoxide (DMSO) and the
absorbance of the formazan solution was measured. Each
compound was tested in triplicate and the experiments were
repeated three times.
3
. Results and discussion
3
.1. Design and docking
As reported previously (Bu et al., 2017), the potent anti-
proliferative capabilities and structural novelty of 3-BEHI
deserves further structural modifications. Therefore, two new
compounds were designed with the replacement of two
different H atoms by methyl groups. In 3-BEHMI, the indole
N—H atom was replaced, while in 3-BEHEI, one imine C—H
atom was replaced.
The Hirshfeld surfaces (Fig. 4) and fingerprint plots (Fig. 5)
were generated based on the crystallographic information file
(CIF) using CrystalExplorer17 (Hirshfeld, 1977; Turner et al.,
According to our docking simulation, shown in Fig. 2,
3
(
-BEHMI and 3-BEHEI have different binding modes
hydrogen bonds versus aromatic hydrophobic interactions),
but both have high Consensus Scores (C-scores), the former is
8.522 and the latter 7.339. As reported, the C-scores of
3-BEHI and Brequinar sodium are 8.884 and 8.198, respec-
tively (Bu et al., 2017). Thus, it is reasonable to deduce that
-BEHMI and 3-BEHEI may have rather good DHODH
inhibitory activity and potential pharmacological properties.
3
3.2. Single-crystal structure of 3-BEHMI and Hirshfeld
surface analyses
In the unit cell of 3-BEHMI, the asymmetric unit consists of
one 3-BEHMI molecule and one acetonitrile solvent mol-
ecule. Similar to 3-BEHI (Bu et al., 2017), the 3-BEHMI
molecule is flat, except for the distal ring of the biphenyl ring
system (atoms C1–C6 in Fig. 3). The proximal ring of the
biphenyl ring system, the central H C—C N—N C– motif
3
Figure 4
and the indole skeleton, including the methyl C atom linking
with the N atom, are essentially coplanar, with an average
˚
r.m.s. deviation of only 0.014 (2) A [the largest r.m.s. deviation
˚
Hirshfeld surfaces for 3-BEHMI, mapped over dnorm (À0.10 to 1.40 A).
Tr ews op e rc et di v es lpy o. ts correspond to C—HÁ Á Áꢀ interactions ‘a’ (a) and ‘b’ (b),
ꢂ
Acta Cryst. (2019). C75
Tan et al.
Two new biphenyl-derived Schiff bases 5 of 10

research papers
Table 3
Inhibition of A549, 4T
1
, MRC-5 and NIH 3T3 cell growth by the title compounds, compared with 3-BEHEI and Brequinar sodium.
Compound
A549 cells, IC50
4T
1
cells, IC50
MRC-5 cells, IC50
NIH 3T3 fibroblasts, IC50
3
3
3
-BEHMI
-BEHEI
-BEHI
108.5Æ0.9 mM
50.5Æ0.7 mM
21.5Æ0.6 mM
12.6Æ0.5 mM
112.0Æ1.0 mM
52.0Æ0.9 mM
18.0Æ0.6 mM
9.9Æ0.4 mM
145.0Æ1.5 mM
78.5Æ0.9 mM
28.0Æ0.7 mM
18.0Æ0.3 mM
159.0Æ1.5 mM
79.0Æ1.2 mM
22.0Æ0.8 mM
15.5Æ0.5 mM
Brequinar sodium
2
017). They can provide information not only about areas of
2004), followed by CÁ Á ÁH/HÁ Á ÁC interactions, which contri-
bute 38.5% and are dominated by C—HÁ Á Áꢀ interactions
(these look like ‘wings’ in the figure) (McKinnon et al., 2004).
The next major contribution (12.7%) is due to NÁ Á ÁH/HÁ Á ÁN
interactions, which are clearly classified as van der Waals
interactions, because the shortest NÁ Á ÁH distance is over
close contacts but also about the strength of intermolecular
interactions by different colours and colour intensity. In the
dnorm map (Fig. 4), the vivid red spots in the Hirshfeld surface
are due to short normalized CÁ Á ÁH distances corresponding to
C—HÁ Á Áꢀ interactions. The two regions labelled ‘a’ connect
with one another through C—HÁ Á Áꢀ interaction ‘a’ and the
two regions labelled ‘b’ connect with each other through C—
HÁ Á Áꢀ interaction ‘b’. The colour intensity indicates that they
are rather strong interactions, which is supported by Quantum
Chemistry calculations in the following section.
The two-dimensional fingerprint plots (Fig. 5) quantify the
contribution of each type of noncovalent interaction to the
Hirshfeld surface (McKinnon et al., 2007). The major contri-
bution with 47.5% of the surface is due to HÁ Á ÁH contacts,
which represent van der Waals interactions (McKinnon et al.,
˚
2.709 A (between acetonitrile N and indole H atoms), while
the sum of the van der Waals radii of the contacting NÁ Á ÁH is
˚
around 2.57 A (Bondi, 1964). Finally, the presence of CÁ Á ÁC
(1.0%) and CÁ Á ÁN/NÁ Á ÁC (0.3%) contacts corresponding to
the ꢀ–ꢀ interactions contributes a small part of the surface
and may suggests a less ideal packing arrangement for the
molecule in the crystal (Hammouri et al., 2018).
Both C—HÁ Á Áꢀ interactions ‘a’ and ‘b’ come from the
benzene ring of indole skeleton. The former links 3-BEHMI
into S-shaped one-dimensional (1D) chains stretching along
Figure 5
Two-dimensional fingerprint plots and the corresponding surface area of 3-BEHMI, showing the individual contribution of each interaction.
ꢂ
6
of 10 Tan et al. Two new biphenyl-derived Schiff bases
Acta Cryst. (2019). C75

research papers
the crystallographic b axis, and the latter links two adjacent
molecules into a dimer (Fig. 6). Additional interactions of ‘b’
make these 1D chains spread out into a two-dimensinal (2D)
layer structure, which extends parallel to the crystallographic
As we can see, the replacement of an H atom by a methyl
group will decrease the cytotoxic activity, whether the
replaced H atom is on the indole N atom or on the imine C
atom. The most incredible occurrence is 3-BEHMI, which
shows rather weak cytotoxicities (IC50 values are 108.5 and
(104) plane (Fig. 6). There are square holes in the 2D layer
structure formed by four adjacent molecules, in which aceto-
nitrile solvent molecules are embedded. The linking force is
van der Waals interactions. It is still van der Waals interactions
that pack 2D layers into a three-dimensional (3D) structure.
The intermolecular interaction energies with the BSSE
correction have been calculated using the MP2/6-31G(d,p)
method on the basis of dimer geometries extracted from the
crystal structure. The calculated interaction energies for ‘a’
112.0 mM for A549 and 4T , respectively), much less potent
1
than 3-BEHI, and even less than 3-BEHEI. But the docking
result of 3-BEHMI is better than that of 3-BEHEI. The
situation is similar to 3-BEHI and Brequinar sodium, in which
the former has a better docking result than the latter, but their
experimental activities were the opposite. We have analysed
two possibilities: the first lies in the deviation between docking
predictions and experimental activities, and the second origi-
nates from the target difference, i.e. DHODH is not the most
important determinant of the antiproliferative capability
(Peters et al., 1989). However, we believe that the more
compounds we investigate, the closer we will get to under-
standing to the nature of the structure–activity relationships
concerning indole/biphenyl compounds, thus increasing the
opportunities for obtaining promising drugs.
À1
and ‘b’ are À32.5 and À26.9 kJ mol , respectively, indicating
rather strong favourable binding energy, which agrees with the
colour intensity in the dnorm map (Fig. 4).
3.3. Cytotoxicity assays
To study the growth inhibitory effects of 3-BEHMI and
-BEHEI on lung/breast cancer cells, human A549 and mouse
3
It was observed in our experiments that A549 cell lines
responded very fast with 3-BEHMI and 3-BEHEI compared
with 4T cell lines when the concentrations were 100 and
4T1 cells were treated and the growth of cells were examined
by MTT assay. Meanwhile, MRC-5 normal lung cells and NIH
T3 fibroblasts were also tested using the same method in
1
3
50 mM, respectively, indicating that they are more selective
against lung cancer cells, while 3-BEHI and Brequinar sodium
have different selectivity. When the concentration decreased
to 30 mM, 3-BEHMI and 3-BEHEI are likewise inactive
against both cell lines.
order to evaluate the selective cytotoxicity of these new
compounds. To gain further evidence for the antitumour
activities and docking results, the antiproliferative activities of
Brequinar sodium and 3-BEHI were also determined for
comparison. The cytotoxic activities as 50% inhibitory
concentration (IC ) values are shown in Table 3.
As for the selective cytotoxicity between normal and
malignant cells, we can see that all these four compounds can
5
0
Figure 6
The 2D structure of 3-BEHMI formed by C—HÁ Á Áꢀ interactions ‘a’ and ‘b’ (shown as yellow and red dotted lines, respectively), viewed perpendicular to
the spreading plane, i.e. the crystallographic (104) plane. Among them, ‘a’ link molecules into S-type 1D chains stretching along the b axis. All
acetonitrile solvent molecules have been omitted for clarity.
ꢂ
Acta Cryst. (2019). C75
Tan et al.
Two new biphenyl-derived Schiff bases 7 of 10

research papers
Figure 7
(a) UV–Vis and (b) fluorescence spectra of 3-BEHMI.
more effectively inhibit cancer cells than normal cells, but they
have relevant inhibitory concentrations. The higher IC value
different optical properties were detected for these two similar
title compounds (Figs. 7 and 8), which attracts our attention to
the mechanism. DFT calculations may provide a theoretical
basis to interpret the experimental results. Thus, different
functionals (B3LYP, M062X, PBE1PBE and WB97XD) were
performed to determine the most reliable theoretical predic-
tion. Besides different functionals, comparisons of the
absorption/emission spectra of all molecules in the gas phase
and ethanol solution (using polarizable continuum model)
were also carried out. The simulated spectra are illustrated in
Figs. S5–S20.
50
for selected cancer cell lines, the higher the value for
responding normal cell lines. This phenomenon may be
explained by their mechanism of action, i.e. these drugs may
block DNA synthesis through modulating pyrimidine bio-
synthesis (Norman, 2013; Munier-Lehmann et al., 2013). High
antiproliferative capacity is presumably attributed to the
enhanced cellular uptake.
3.4. Absorption/emission spectra and explanations
As shown in Figs. S5–S8, most of the calculated UV–Vis
spectra of 3-BEHMI have four bands above 185 nm (the only
exception is the three-bands-spectra calculated with the
WB97XD functional, see Fig. S8), which corresponds well with
UV–Vis and fluorescence spectra are useful tools in dedu-
cing the secondary structure and estimating the optical prop-
erties of ꢀ-conjugated molecules. Interesting and dramatically
Figure 8
(a) UV–Vis and (b) fluorescence spectra of 3-BEHEI.
ꢂ
8
of 10 Tan et al. Two new biphenyl-derived Schiff bases
Acta Cryst. (2019). C75

research papers
the experimental spectrum (Fig. 7a). But the band maximum
positions of the simulated spectra differ from the experimental
results, more or less. It is found that the theoretical results
determined by the M062X method in PCM are in good
agreement with the experimental data (Fig. S6). All peaks are
blue shifted by 10–20 nm relative to the experimental spec-
trum (Fig. 7a). The second coincident predictions are obtained
from the PBE1PBE method, but two peaks are blue shifted
and the other two are red shifted, so molecular orbital (MO)
composition analysis was performed on the basis of TD-DFT/
M062X/PCM calculations.
4. Conclusion
This work stems from our interest in the design, synthesis,
characterization and antiproliferative activities of novel Schiff
bases targeting DHODH.
The present results add two new examples (3-BEHMI and
-BEHEI) of Schiff bases containing indole and biphenyl
3
groups. They are methyl replacement derivatives of one of our
reported compounds, i.e. 3-BEHI. According to our docking
simulations, the introduction of a methyl group at different
sites will affect the binding modes between the donor and
acceptor, one still adopting hydrogen bonds and the other with
aromatic hydrophobic interactions. Both 3-BEHMI and
The predictions of the UV–Vis spectrum of 3-BEHEI are
not as good as that of 3-BEHMI. There are two absorption
bands in Fig. 8(a), one is around 204 nm and the other around
3-BEHI form similar hydrogen bonds to DHODH and have
similar C-scores, but MTT results suggest that they have very
different cytotoxicities (IC50 = ꢀ110 versus ꢀ20 mM). Mean-
while, 3-BEHEI displays a slightly reduced cytotoxicity (IC =
301 nm. However, all calculated spectra (Figs. S9–S12) have
three or four bands above 185 nm. No satisfactory result can
be obtained if considering all peaks. Meanwhile, the band
around 204 nm is almost extended into the vacuum-UV (VUV,
5
0
ꢀ
50 mM) compared with 3-BEHI, which is in line with their
docking prediction. These results give another proof that
Schiff bases derived from indole and biphenyl could poten-
tially serve as antitumour agents. But it is still challenging to
design and synthesize more diverse compounds with more
efficacious cytotoxicity against cancer cell lines than normal
cells. Systematic investigations on structure–activity relation-
ships concerning these kinds of Schiff bases are ongoing.
The absorption and emission spectra of the title compounds
in ethanol have been analysed in detail by means of four TD-
DFT calculations. These results highlighted the complexity of
the fully delocalized ꢀ-bonding properties. Reported calcula-
tions also implicate that the optical spectra are usually so rich
that they could not be predicted exactly.
<
200 nm), so the judgment was made only according to their
agreement with the band around 301 nm. The agreement
between the calculated and experimental results follows the
order: WB97XD-PCM > WB97XD-GAS > M062X-GAS >
M062X-PCM > PBE1PBE-GAS > PBE1PBE-PCM > B3LYP-
GAS > B3LYP-PCM. Interestingly, we found that when the
spectra calculated in the solvent are closer to those computed
in the gas phase, the theoretical spectra fit the experimental
data more closely. For example, the theoretical results calcu-
lated by the WB97XD method in ethanol solution and in the
gas phase are 302 and 299 nm, respectively. Both are in good
agreement with the experimental data. On the other hand, the
theoretical results performed by the B3LYP method in the gas
phase and ethanol solution are 358 and 370 nm, respectively.
Both disagree with the experimental values. As a result, MO
analysis was performed on the basis of TD-DFT/WB97XD/
PCM calculations when interpreting the UV–Vis spectrum of
Acknowledgements
We are very grateful to Dr Yun-Xue Zhao (School of Medi-
cine, Shandong University) for the MTT assays and Dr Min-
Yong Li (School of Pharmacy, Shandong University) for the
molecular docking studies. Generous support with respect to
computing resources from the National Supercomputer
Center in Jinan, China, is greatly appreciated.
3-BEHEI.
For the fluorescence properties of 3-BEHMI and 3-BEHEI,
the agreements between theoretical and experimental transi-
tion energies are not as good as those in their absorption
properties, so corresponding explanations are consigned to the
supporting information (see the first two paragraphs).
Funding information
Kohn–Sham orbitals to describe the absorption and emis-
sion spectra are available in the supporting information
Funding for this research was provided by: Science and
Technology Development Projects of Shandong Province in
China (award Nos. 2011GGX10706 and 2012GGX10702);
National Natural Science Foundation of China (award No.
(Tables S1–S2 and Figs. S23–S25). Analysis of these molecular
orbitals reveals that most of the initial and final states are
related to the MOs with ꢀ-type character (the contributions of
P and P orbitals are over 80%; localized on the biphenyl/
21103100).
y
z
indole rings or C N bonds). Generally, these absorption
bands are dominant ꢀ–ꢀ* transitions, even though most of
them are not in the visible region.
It is necessary to calculate the absorption and emission
spectra with different methods or different functionals,
because the most reliable method is not consistent from one
system to another. In other words, it is not always clear what
the best choice of functional is for the system of interest and
the agreement between theoretical and experimental spectra
may be fortuitous.
References
Abdullah, I., Chee, C. F., Lee, Y., Thunuguntla, S. S. R., Satish Reddy,
K., Nellore, K., Antony, T., Verma, J., Mun, K. W., Othman, S.,
Subramanya, H. & Rahman, N. A. (2015). Bioorg. Med. Chem. 23,
4669–4680.
Afzal, O., Kumar, S., Haider, M. R., Ali, M. R., Kumar, R., Jaggi, M. &
Bawa, S. (2015). Eur. J. Med. Chem. 97, 871–910.
Batt, D. G. (1999). Expert Opin. Ther. Pat. 9, 41–54.
Bauernschmitt, R. & Ahlrichs, R. (1996). Chem. Phys. Lett. 256, 454–
464.
Becke, A. D. (1993). J. Chem. Phys. 98, 5648–5652.
ꢂ
Acta Cryst. (2019). C75
Tan et al.
Two new biphenyl-derived Schiff bases 9 of 10

research papers
Bondi, A. (1964). J. Phys. Chem. 68, 441–451.
Lee, C., Yang, W. & Parr, R. G. (1988). Phys. Rev. B, 37, 785–789.
Liu, Z., Tang, L., Zhu, H., Xu, T., Qiu, C., Zheng, S., Gu, Y., Feng, J.-P.,
Zhang, Y. & Liang, G. (2016). J. Med. Chem. 59, 4637–4650.
Maroun, J., Ruckdeschel, J., Natale, R., Morgan, R., Dallaire, B., Sisk,
R. & Gyves, J. (1993). Cancer Chemother. Pharmacol. 32, 64–66.
McKinnon, J. J., Jayatilaka, D. & Spackman, M. A. (2007). Chem.
Commun. pp. 3814–3816.
Boys, S. B. & Bernardi, F. (1970). Mol. Phys. 19, 553–566.
Braakhuis, B. J., van Dongen, G. A., Peters, G. J., van Walsum, M. &
Snow, G. B. (1990). Cancer Lett. 49, 133–137.
Brandenburg, K. & Putz, H. (1999). DIAMOND. Crystal Impact
GbR, Bonn, Germany.
Bruker (2000). SMART, SAINT, SADABS and SHELXTL. Bruker
AXS Inc., Madison, Wisconsin, USA.
Bu, F.-Z., Tan, X.-J., Xing, D.-X. & Wang, C. (2017). Acta Cryst. C73,
McKinnon, J. J., Spackman, M. A. & Mitchell, A. S. (2004). Acta
Cryst. B60, 627–668.
5
46–555.
McLean, A. D. & Chandler, G. S. (1980). J. Chem. Phys. 72, 5639–
5648.
Møller, C. & Plesset, M. S. (1934). Phys. Rev. 46, 618–622.
Munier-Lehmann, H., Vidalain, P.-O., Tangy, F. & Janin, Y. L. (2013).
J. Med. Chem. 56, 3148–3167.
Casida, M. E., Jamorski, C., Casida, K. C. & Salahub, D. R. (1998). J.
Chem. Phys. 108, 4439–4449.
Chai, J.-D. & Head-Gordon, M. (2008). Phys. Chem. Chem. Phys. 10,
6615–6620.
Chen, S. F., Perrella, F. W., Behrens, D. L. & Papp, L. M. (1992).
Cancer Res. 52, 3521–3527.
Cheng, S.-S., Shi, Y., Ma, X.-N., Xing, D.-X., Liu, L.-D., Liu, Y., Zhao,
Y.-X., Sui, Q.-C. & Tan, X.-J. (2016). J. Mol. Struct. 1115, 228–240.
Das, P., Deng, X., Zhang, L., Roth, M. G., Fontoura, B. M., Phillips,
M. A. & De Brabander, J. K. (2013). ACS Med. Chem. Lett. 4, 517–
Narsimha, S., Satheesh Kumar, N., Kumara Swamy, B., Vasudeva
Reddy, N., Althaf Hussain, S. K. & Srinivasa Rao, M. (2016).
Bioorg. Med. Chem. Lett. 26, 1639–1644.
Nielsen, A. B. & Holder, A. J. (1998). GaussView. Version 5.0.
Gaussian Inc., Pittsburgh, PA, USA.
Norman, P. (2013). Expert Opin. Ther. Pat. 23, 1391–1394.
Perdew, J. P., Burke, K. & Ernzerhof, M. (1996). Phys. Rev. Lett. 77,
3865–3868.
521.
Dell, C. P. (1998). Curr. Med. Chem. 5, 179–194.
Dexter, D. L., Hesson, D. P., Ardecky, R. J., Rao, G. V., Tippett, D. L.,
Dusak, B. A., Paull, K. D., Plowman, J., DeLarco, B. M., Narayanan,
V. L., et al. (1985). Cancer Res. 45, 5563–5568.
Farrugia, L. J. (2012). J. Appl. Cryst. 45, 849–854.
Frisch, M. J., Pople, J. A. & Binkley, J. S. (1984). J. Chem. Phys. 80,
Peters, G. J., Laurensse, E., de Kant, E., Nadal, J. C. & Pinedo, H. M.
(1989). Adv. Exp. Med. Biol. 253B, 375–382.
Rassolov, V. A., Ratner, M. A., Pople, J. A., Redfern, P. C. & Curtiss,
L. A. (2001). J. Comput. Chem. 22, 976–984.
Saini, T., Kumar, S. & Narasimhan, B. (2015). Cent. Nerv. Syst. Agents
Med. Chem. 16, 19–28.
3265–3269.
Frisch, M. J., et al. (2004). GAUSSIAN03. Revision C.02. Gaussian
Inc., Wallingford, CT, USA. http://www.gaussian.com.
Gargano, E. M., Perspicace, E., Carotti, A., Marchais-Oberwinkler, S.
Sheldrick, G. M. (2015a). Acta Cryst. A71, 3–8.
Sheldrick, G. M. (2015b). Acta Cryst. C71, 3–8.
Spek, A. L. (2009). Acta Cryst. D65, 148–155.
&
Hartmann, R. W. (2016). Bioorg. Med. Chem. Lett. 26, 21–24.
Stratmann, R. E., Scuseria, G. E. & Frisch, M. J. (1998). J. Chem.
Phys. 109, 8218–8224.
Tomasi, J., Mennucci, B. & Cammi, R. (2005). Chem. Rev. 105, 2999–
3093.
Tripos (2012). SYBYL-X. Version 2.0. Tripos International, St Louis,
MO, USA. http://www.tripos.com.
Turner, M. J., McKinnon, J. J., Wolff, S. K., Grimwood, D. J.,
Spackman, P. R., Jayatilaka, D. & Spackman, M. A. (2017).
CrystalExplorer17. University of Western Australia. http://hirsh-
feldsurface.net.
Glendening, E. D., Badenhoop, J. K., Reed, A. E., Carpenter, J. E.,
Bohmann, J. A., Morales, C. M. & Weinhold, F. (2001). NBO.
Version 5.0. Theoretical Chemical Institute, University of Wis-
consin, Madison, Wisconsin, USA.
Gorelsky, S. I. (2010). SWizard. Revision 4.6. University of Ottawa,
Canada. http://www.sg-chem.net.
Gorelsky, S. I. & Lever, A. B. P. (2001). J. Organomet. Chem. 635,
187–196.
Hammouri, M., Garcia, T. M., Cook, C., Monaco, S., Jezowski, S.,
Marom, N. & Schatschneider, B. (2018). J. Phys. Chem. C, 122,
Vyas, V. K. & Ghate, M. (2011). Mini Rev. Med. Chem. 11, 1039–
1055.
23828–23844.
Hirshfeld, F. L. (1977). Theor. Chim. Acta, 44, 129–138.
Vyas, V. K. & Ghate, M. (2012). Med. Chem. Res. 21, 3021–3034.
Zhao, Y., Liu, W., Zhou, Y., Zhang, X. & Murphy, P. V. (2010). Bioorg.
Med. Chem. Lett. 20, 7540–7543.
Zhao, Y. & Truhlar, D. G. (2008). Theor. Chem. Acc. 120, 215–241.
Zhu, J., Han, L., Diao, Y., Ren, X., Xu, M., Xu, L., Li, S., Li, Q., Dong,
D., Huang, J., Liu, X., Zhao, Z., Wang, R., Zhu, L., Xu, Y., Qian, X.
& Li, H. (2015). J. Med. Chem. 58, 1123–1139.
Janzer, A., Stoeckigt, D., Meyer, H., Sykes, D. B., Scadden, D. &
Lewis, T. A. (2017). PCT Int. Appl. WO 2017037022 A1 20170309.
Johar, Z., Zahn, A., Leumann, C. J. & Jaun, B. (2008). Chem. Eur. J.
14, 1080–1086.
Krishnan, R., Binkley, J. S., Seeger, R. & Pople, J. A. (1980). J. Chem.
Phys. 72, 650–654.
ꢂ
1
0 of 10 Tan et al. Two new biphenyl-derived Schiff bases
Acta Cryst. (2019). C75

supporting information
supporting information
Acta Cryst. (2019). C75 [https://doi.org/10.1107/S2053229618017989]
Synthesis, structure and antiproliferative and optical activities of two new bi-
phenyl-derived Schiff bases
Xue-Jie Tan, Ling-Yao Zhang, Yuan-Kai Sun and Xue-Min Zhou
Computing details
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) and PLATON (Spek, 2009); software used to prepare
material for publication: SHELXL2016 (Sheldrick, 2015b).
3-((E)-{(E)-[1-(Biphenyl-4-yl)ethylidene]hydrazinylidene}methyl)-1-methyl-1H-indole acetonitrile monosolvate
Crystal data
·C
= 392.49
Monoclinic, P2
a = 8.1280 (2) Å
b = 8.2147 (1) Å
c = 32.9809 (6) Å
β = 90.252 (2)°
V = 2202.08 (7) Å
Z = 4
C
M
24
H N
21 3
2
H
3
N
F(000) = 832
= 1.184 Mg m
−3
r
D
x
1
/c
Mo Kα radiation, λ = 0.71073 Å
Cell parameters from 380 reflections
θ = 2.5–26.0°
−1
µ = 0.07 mm
T = 298 K
3
Block, yellow
0.30 × 0.27 × 0.20 mm
Data collection
Bruker SMART CCD area detector
diffractometer
Radiation source: fine-focus sealed tube
Detector resolution: 10.13 pixels mm
phi and ω scans
26680 measured reflections
4300 independent reflections
3623 reflections with I > 2σ(I)
-1
R
int = 0.040
θ
max = 26.1°, θmin = 2.5°
Absorption correction: multi-scan
h = −9→10
k = −10→9
l = −34→40
(
SADABS; Bruker, 2000)
T
min = 0.971, Tmax = 0.993
Refinement
2
2
2
2
Refinement on F
w = 1/[σ (F
o
) + (0.0717P) + 0.4656P]
2
2
Least-squares matrix: full
R[F > 2σ(F )] = 0.053
where P = (F
(Δ/σ)max < 0.001
Δρmax = 0.16 e Å
o
+ 2F )/3
c
2
2
2
−3
wR(F ) = 0.151
−3
S = 1.02
Δρmin = −0.16 e Å
4
3
0
300 reflections
44 parameters
restraints
Extinction correction: SHELXL2014
(Sheldrick, 2015b),
Fc =kFc[1+0.001xFc λ /sin(2θ)]
*
2
3
-1/4
Hydrogen site location: mixed
Extinction coefficient: 0.021 (2)
Only H-atom displacement parameters refined
Acta Cryst. (2019). C75
sup-1

supporting information
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.
2
2
Refinement. Refinement of F against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F ,
conventional R-factors R are based on F, with F set to zero for negative F . The threshold expression of F > 2sigma(F ) is
used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based
2
2
2
2
on F are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger.
2
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å )
x
y
z
Uiso*/Ueq
C1
C2
C3
C4
C5
C6
C7
C8
1.0305 (2)
1.1581 (2)
1.2654 (3)
1.2440 (3)
1.1167 (2)
1.00710 (19)
0.86788 (18)
0.87657 (19)
0.74443 (19)
0.59637 (18)
0.5885 (2)
0.7214 (2)
0.45344 (18)
0.2979 (2)
0.3158
−0.0658 (2)
−0.0543 (2)
0.0741 (3)
0.1923 (3)
0.1816 (2)
0.05118 (18)
0.04172 (17)
0.1149 (2)
0.1122 (2)
0.03798 (17)
−0.0368 (2)
−0.0350 (2)
0.04005 (18)
−0.0395 (3)
−0.1543
0.16179 (5)
0.13436 (6)
0.13619 (6)
0.16526 (6)
0.19267 (6)
0.19154 (4)
0.22050 (4)
0.25885 (5)
0.28499 (5)
0.27413 (4)
0.23637 (5)
0.21020 (5)
0.30195 (4)
0.28876 (6)
0.2857
0.0580 (4)
0.0695 (5)
0.0781 (6)
0.0819 (6)
0.0680 (5)
0.0510 (4)
0.0493 (3)
0.0537 (4)
0.0535 (4)
0.0482 (3)
0.0574 (4)
0.0570 (4)
0.0501 (3)
0.0825 (6)
0.179 (16)*
0.115 (9)*
0.094 (7)*
0.0636 (4)
0.0616 (4)
0.0599 (4)
0.0686 (5)
0.0835 (6)
0.0940 (8)
0.0852 (7)
0.0682 (5)
0.0735 (5)
0.0999 (9)
0.122 (9)*
0.21 (2)*
C9
C10
C11
C12
C13
C14
H14A
H14B
H14C
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
H24A
H24B
H24C
C25
H25A
H25B
H25C
C26
0.2629
0.2145
0.0060
−0.0214
0.2633
0.3088
0.3693 (2)
0.2504 (2)
0.0815 (2)
−0.0230 (2)
−0.1814 (3)
−0.2386 (3)
−0.1384 (3)
0.0210 (2)
0.2810 (3)
0.1338 (4)
0.0406
0.2323
0.1211
0.2584 (5)
0.2202
0.1659
0.1851 (2)
0.2018 (2)
0.1469 (2)
0.0630 (2)
0.0321 (3)
0.0810 (3)
0.1627 (3)
0.1956 (2)
0.2782 (3)
0.3442 (4)
0.4156
0.4040
0.2573
0.7005 (5)
0.8091
0.6307
0.39525 (5)
0.42686 (5)
0.42844 (5)
0.40208 (6)
0.41404 (8)
0.45228 (9)
0.47869 (7)
0.46654 (5)
0.46332 (6)
0.52775 (6)
0.5292
0.5339
0.5469
0.39055 (10)
0.3853
0.3956
0.140 (11)*
0.1204 (10)
0.32 (3)*
0.21 (2)*
0.198 (19)*
0.0950 (7)
0.3180
0.3626 (3)
0.6611
0.7014 (4)
0.3675
0.42481 (8)
Acta Cryst. (2019). C75
sup-2

supporting information
N1
N2
N3
N4
H1
H2
H3
H4
0.47564 (16)
0.33775 (17)
0.1452 (2)
0.4440 (3)
0.959 (2)
1.169 (2)
1.359 (3)
1.323 (3)
1.105 (3)
0.977 (2)
0.753 (2)
0.489 (2)
0.707 (2)
0.477 (3)
0.011 (2)
−0.255 (3)
−0.343 (3)
−0.178 (3)
0.382 (3)
0.11150 (19)
0.11183 (19)
0.2767 (2)
0.7039 (6)
−0.153 (2)
−0.140 (3)
0.077 (3)
0.283 (3)
0.266 (3)
0.176 (2)
0.159 (2)
−0.082 (2)
−0.081 (2)
0.236 (3)
0.029 (2)
−0.020 (3)
0.055 (3)
0.200 (3)
0.330 (3)
0.33626 (4)
0.36187 (4)
0.48697 (4)
0.45186 (9)
0.1602 (5)
0.1140 (6)
0.1186 (6)
0.1675 (6)
0.2116 (7)
0.2677 (5)
0.3112 (6)
0.2283 (5)
0.1825 (6)
0.3994 (6)
0.3748 (6)
0.3967 (8)
0.4605 (8)
0.5051 (7)
0.4728 (7)
0.0608 (4)
0.0608 (4)
0.0763 (5)
0.1703 (16)
0.066 (5)*
0.077 (6)*
0.086 (6)*
0.088 (6)*
0.092 (7)*
0.063 (5)*
0.067 (5)*
0.065 (5)*
0.075 (6)*
0.080 (6)*
0.061 (5)*
0.098 (8)*
0.110 (8)*
0.087 (6)*
0.085 (7)*
H5
H8
H9
H11
H12
H15
H18
H19
H20
H21
H23
2
Atomic displacement parameters (Å )
11
22
33
12
13
23
U
U
U
U
U
U
C1
C2
C3
C4
C5
C6
C7
C8
0.0615 (9)
0.0754 (12)
0.0750 (12)
0.0851 (14)
0.0772 (12)
0.0539 (8)
0.0534 (8)
0.0489 (8)
0.0513 (8)
0.0494 (8)
0.0564 (9)
0.0634 (9)
0.0493 (8)
0.0559 (10)
0.0576 (10)
0.0635 (10)
0.0630 (9)
0.0685 (11)
0.0694 (12)
0.0701 (14)
0.0844 (14)
0.0747 (11)
0.0758 (12)
0.135 (2)
0.0530 (8)
0.0713 (11)
0.0913 (14)
0.0897 (14)
0.0658 (10)
0.0511 (8)
0.0451 (7)
0.0601 (9)
0.0612 (9)
0.0467 (7)
0.0603 (9)
0.0587 (9)
0.0507 (8)
0.1263 (19)
0.0771 (11)
0.0715 (10)
0.0626 (9)
0.0664 (10)
0.0785 (13)
0.0964 (16)
0.0926 (15)
0.0717 (11)
0.0872 (13)
0.1115 (18)
0.129 (3)
0.0596 (9)
−0.0038 (7)
−0.0031 (9)
−0.0166 (10)
−0.0353 (11)
−0.0173 (9)
0.0003 (6)
0.0011 (6)
−0.0023 (7)
0.0003 (7)
0.0017 (6)
−0.0122 (7)
−0.0097 (7)
0.0022 (6)
−0.0202 (11)
0.0025 (8)
0.0101 (8)
0.0175 (7)
0.0105 (8)
0.0024 (10)
0.0116 (12)
0.0264 (12)
0.0230 (9)
0.0115 (10)
0.0424 (18)
0.014 (2)
0.0094 (7)
0.0181 (9)
0.0258 (9)
0.0204 (10)
0.0134 (8)
0.0034 (6)
0.0043 (6)
0.0017 (6)
0.0012 (6)
0.0025 (6)
0.0036 (7)
0.0067 (7)
0.0024 (6)
0.0096 (8)
0.0027 (7)
0.0038 (7)
0.0099 (7)
0.0068 (9)
0.0053 (12)
0.0287 (13)
0.0319 (12)
0.0146 (8)
0.0012 (9)
0.0092 (12)
−0.0211 (19)
−0.0029 (7)
−0.0093 (8)
−0.0039 (10)
−0.0111 (10)
−0.0101 (8)
0.0025 (6)
0.0619 (10)
0.0682 (11)
0.0711 (12)
0.0610 (10)
0.0482 (8)
0.0494 (8)
0.0521 (8)
0.0481 (8)
0.0485 (7)
0.0556 (9)
0.0490 (8)
0.0502 (8)
0.0655 (11)
0.0562 (9)
0.0498 (8)
0.0542 (8)
0.0709 (11)
0.1027 (16)
0.1156 (19)
0.0789 (13)
0.0583 (9)
0.0574 (10)
0.0537 (11)
0.107 (2)
0.0013 (6)
−0.0053 (7)
−0.0064 (7)
0.0025 (6)
−0.0050 (7)
−0.0066 (7)
0.0048 (6)
−0.0145 (11)
−0.0002 (8)
0.0021 (7)
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
0.0112 (7)
0.0065 (9)
0.0108 (12)
0.0255 (15)
0.0192 (11)
0.0125 (8)
−0.0036 (9)
−0.0068 (12)
−0.0079 (18)
0.125 (2)
Acta Cryst. (2019). C75
sup-3

supporting information
C26
N1
N2
N3
N4
0.0777 (14)
0.0506 (7)
0.0523 (7)
0.0914 (12)
0.1039 (19)
0.124 (2)
0.0833 (15)
0.0530 (7)
0.0529 (7)
0.0487 (8)
0.1065 (19)
0.0088 (13)
−0.0036 (6)
−0.0003 (6)
0.0250 (9)
0.023 (2)
0.0163 (12)
0.0094 (6)
0.0087 (6)
0.0101 (7)
−0.0050 (16)
−0.0233 (14)
−0.0055 (6)
−0.0022 (6)
−0.0006 (7)
−0.055 (2)
0.0789 (9)
0.0771 (9)
0.0889 (11)
0.301 (5)
Geometric parameters (Å, º)
C1—C2
C1—C6
C1—H1
C2—C3
C2—H2
C3—C4
C3—H3
C4—C5
C4—H4
C5—C6
C5—H5
C6—C7
C7—C12
C7—C8
C8—C9
C8—H8
C9—C10
C9—H9
C10—C11
C10—C13
C11—C12
C11—H11
C12—H12
C13—N1
C13—C14
C14—H14A
C14—H14B
C14—H14C
1.383 (2)
1.387 (2)
0.924 (19)
1.370 (3)
0.98 (2)
1.376 (3)
0.96 (2)
1.380 (3)
0.99 (2)
C15—N2
1.280 (2)
C15—C16
C15—H15
C16—C23
C16—C17
C17—C18
C17—C22
C18—C19
C18—H18
C19—C20
C19—H19
C20—C21
C20—H20
C21—C22
C21—H21
C22—N3
1.431 (2)
0.98 (2)
1.378 (3)
1.446 (2)
1.395 (3)
1.409 (2)
1.372 (3)
0.982 (18)
1.405 (4)
0.93 (3)
1.366 (4)
0.92 (3)
1.384 (3)
0.98 (2)
1.382 (3)
1.354 (2)
0.98 (2)
1.458 (3)
0.9600
0.9600
0.9600
1.410 (4)
0.9600
0.9600
0.9600
1.108 (3)
1.4063 (17)
1.394 (2)
0.94 (2)
1.486 (2)
1.388 (2)
1.402 (2)
1.380 (2)
1.000 (19)
1.395 (2)
0.949 (19)
1.390 (2)
1.4836 (19)
1.386 (2)
0.927 (18)
0.99 (2)
1.287 (2)
1.487 (2)
0.9600
0.9600
0.9600
C23—N3
C23—H23
C24—N3
C24—H24A
C24—H24B
C24—H24C
C25—C26
C25—H25A
C25—H25B
C25—H25C
C26—N4
N1—N2
C2—C1—C6
C2—C1—H1
C6—C1—H1
C3—C2—C1
C3—C2—H2
C1—C2—H2
C2—C3—C4
C2—C3—H3
C4—C3—H3
C3—C4—C5
C3—C4—H4
121.41 (16)
119.2 (11)
119.4 (11)
120.21 (17)
121.6 (12)
118.2 (12)
119.46 (17)
120.0 (13)
120.4 (13)
120.57 (18)
120.0 (13)
N2—C15—C16
N2—C15—H15
C16—C15—H15
C23—C16—C15
C23—C16—C17
C15—C16—C17
C18—C17—C22
C18—C17—C16
C22—C17—C16
C19—C18—C17
C19—C18—H18
122.58 (17)
119.7 (12)
117.7 (12)
124.00 (18)
106.18 (16)
129.82 (16)
118.83 (18)
135.01 (16)
106.16 (17)
118.8 (2)
118.4 (11)
Acta Cryst. (2019). C75
sup-4

supporting information
C5—C4—H4
C4—C5—C6
C4—C5—H5
C6—C5—H5
C1—C6—C5
C1—C6—C7
C5—C6—C7
C12—C7—C8
C12—C7—C6
C8—C7—C6
C9—C8—C7
C9—C8—H8
C7—C8—H8
C8—C9—C10
C8—C9—H9
C10—C9—H9
C11—C10—C9
C11—C10—C13
C9—C10—C13
C12—C11—C10
C12—C11—H11
C10—C11—H11
C11—C12—C7
C11—C12—H12
C7—C12—H12
N1—C13—C10
N1—C13—C14
C10—C13—C14
C13—C14—H14A
C13—C14—H14B
H14A—C14—H14B
C13—C14—H14C
H14A—C14—H14C
H14B—C14—H14C
119.4 (13)
120.84 (17)
117.6 (14)
121.5 (14)
117.51 (15)
121.71 (14)
120.76 (14)
117.10 (14)
121.40 (13)
121.46 (13)
121.35 (14)
117.7 (10)
120.9 (10)
121.31 (14)
120.3 (11)
118.4 (11)
117.35 (14)
121.76 (14)
120.89 (13)
121.38 (15)
120.4 (11)
118.0 (11)
121.49 (14)
118.4 (11)
119.8 (11)
116.23 (13)
125.00 (15)
118.77 (14)
109.5
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
N3—C22—C21
N3—C22—C17
C21—C22—C17
N3—C23—C16
N3—C23—H23
C16—C23—H23
N3—C24—H24A
N3—C24—H24B
H24A—C24—H24B
N3—C24—H24C
H24A—C24—H24C
H24B—C24—H24C
C26—C25—H25A
C26—C25—H25B
H25A—C25—H25B
C26—C25—H25C
H25A—C25—H25C
H25B—C25—H25C
N4—C26—C25
C13—N1—N2
122.7 (11)
121.3 (3)
120.7 (15)
117.9 (15)
120.9 (2)
118.4 (17)
120.7 (17)
117.9 (2)
121.7 (13)
120.4 (13)
129.5 (2)
108.34 (17)
122.2 (2)
110.70 (19)
120.4 (13)
128.9 (13)
109.5
109.5
109.5
109.5
109.5
109.5
109.5
109.5
109.5
109.5
109.5
109.5
179.2 (4)
114.83 (13)
111.13 (14)
108.62 (16)
125.6 (2)
125.7 (2)
109.5
109.5
109.5
109.5
C15—N2—N1
C23—N3—C22
C23—N3—C24
C22—N3—C24
109.5
Hydrogen-bond geometry (Å, º)
D—H···A
D—H
H···A
D···A
D—H···A
C9—H9···N1
C14—H14C···N2
0.949 (19)
0.96
2.440 (18)
2.29
2.769 (2)
2.731 (2)
100.1 (13)
107
Acta Cryst. (2019). C75
sup-5