Unsymmetrically-Substituted 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione Scaffold—A Useful Tool for Bioactive Molecules Design

Article abstract of DOI:10.3390/molecules25122855

Unsymmetrically N-substituted and N,N’-disubstituted 5,12-dihydrodibenzo [b,f][1,4]diazocine-6,11-diones were synthesized in the new protocol. The desired modifications of the dibenzodiazocine scaffold were introduced at the stages of proper selection of building blocks as well as post-cyclization modifications with alkylation or acylation agents, expanding the structural diversity and possible applications of synthesized molecules. The extension of developed method resulted in the synthesis of novel: tricyclic 5,10-dihydrobenzo[b]thieno[3,4-f][1,4]diazocine-4,11-dione scaffold and fused pentacyclic framework possessing two benzodiazocine rings within its structure. Additionally, the unprecedented rearrangement of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones to 2-(2-aminophenyl)isoindoline-1,3-diones was observed under the basic conditions in the presence of sodium hydride for secondary dilactams. The structures of nine synthesized products have been established by single-crystal X-ray diffraction analysis. Detailed crystallographic analysis of the investigated tri- and pentacyclic systems has shed more light on their structural features. One cell line derived from non-cancerous cells (EUFA30—human fibroblasts) and three tumor cells (U87—human primary glioblastoma, HeLa—cervix adenocarcinoma, BICR18—laryngeal squamous cell carcinoma) were used to determine the cytotoxic effect of the newly synthesized compounds. Although these compounds showed a relatively weak cytotoxic effect, the framework obtained for 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione could serve as a convenient privilege structure for the design and development of novel bioactive molecules suitable for drug design, development and optimization programs.

Full text of DOI:10.3390/molecules25122855

molecules
Article
Unsymmetrically-Substituted 5,12-dihydrodibenzo[b,f]
[1,4]diazocine-6,11-dione Scaffold—A Useful Tool for
Bioactive Molecules Design
Bartosz Bieszczad 1 , Damian Garbicz 1, Damian Trzybin´ski 2, Marta K. Dudek 3
,
Krzysztof Woz´niak 2 , Elz˙bieta Grzesiuk 1 and Adam Mieczkowski 1,
*
1
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warszawa, Poland;
b.bieszczad@ibb.waw.pl (B.B.); dgarbicz@ibb.waw.pl (D.G.); elag@ibb.waw.pl (E.G.)
Biological and Chemical Research Centre, University of Warsaw, 02-089 Warszawa, Poland;
dtrzybinski@cnbc.uw.edu.pl (D.T.); kwozniak@chem.uw.edu.pl (K.W.)
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, 90-363 Łódz´, Poland;
mdudek@cbmm.lodz.pl
2
3
*
Correspondence: amiecz@ibb.waw.pl
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 28 May 2020; Accepted: 16 June 2020; Published: 20 June 2020
Abstract: Unsymmetrically N-substituted and N,N’-disubstituted 5,12-dihydrodibenzo [b,f][1,4]
diazocine-6,11-diones were synthesized in the new protocol. The desired modifications of the
dibenzodiazocine scaffold were introduced at the stages of proper selection of building blocks as
well as post-cyclization modifications with alkylation or acylation agents, expanding the structural
diversity and possible applications of synthesized molecules. The extension of developed method
resulted in the synthesis of novel: tricyclic 5,10-dihydrobenzo[b]thieno[3,4-f][1,4]diazocine-4,11-dione
scaffold and fused pentacyclic framework possessing two benzodiazocine rings within its structure.
Additionally, the unprecedented rearrangement of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones
to 2-(2-aminophenyl)isoindoline-1,3-diones was observed under the basic conditions in the presence
of sodium hydride for secondary dilactams. The structures of nine synthesized products have
been established by single-crystal X-ray diffraction analysis. Detailed crystallographic analysis
of the investigated tri- and pentacyclic systems has shed more light on their structural features.
One cell line derived from non-cancerous cells (EUFA30—human fibroblasts) and three tumor cells
(U87—human primary glioblastoma, HeLa—cervix adenocarcinoma, BICR18—laryngeal squamous
cell carcinoma) were used to determine the cytotoxic effect of the newly synthesized compounds.
Although these compounds showed a relatively weak cytotoxic effect, the framework obtained for
5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione could serve as a convenient privilege structure for
the design and development of novel bioactive molecules suitable for drug design, development and
optimization programs.
Keywords: unsymmetrical dibenzo[b,f][1,4]diazocines; X-ray diffraction analysis; cytotoxic effect;
privilege structure; nitrogen heterocycles
1. Introduction
Modern medicinal chemistry is mainly based on the design of novel, biologically active compounds
containing diverse heterocyclic scaffolds, including privileged structures. Its further development is
largely dependent on the availability of new synthetic methods and approaches leading to heterocyclic
derivatives [
by a seven-membered heterocycle, and their structural analogues, are challenging synthetic targets
for organic chemists [ ]. The development of a wide range of novel synthetic methods has led to
1]. Tricyclic heterocyclic scaffolds, possessing two non-polar phenyl rings separated
2–5
Molecules 2020, 25, 2855; doi:10.3390/molecules25122855
www.mdpi.com/journal/molecules

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the synthesis of a broad variety of derivatives with significant biological activity, where the tricyclic
antipsychotic drugs focused the most attention [ ]. Our own research on the development of novel
therapeutic agents, based on tricyclic benzodiazepine derivatives, resulted in the development of
6
unique antileucemic agents
CCRF-7 cell lines [7,8].
1 (Figure 1), exhibiting a selective cytotoxic effect against MV4-11 and
Contrary to dibenzoazepines, azolobenzodiazepines and their structural analogues, tricyclic
heterocyclic scaffolds, possessing two non-polar phenyl rings separated by an eight-membered
heterocycle (Figure 1), such as dibenzodiazocines
2 [9] and 3, are rarely recognized and considered to
be potential privileged structures for design and development of new drugs, despite great structural
similarity to their seven-membered heterocyclic analogues. The sparse reports in the literature of
application of such molecules include compound
a chemosensitizer [10 11] or compound
(R1 = R2 = R3 = R4 = H) developed as a ligand for GABAAR
receptors exhibiting anticonvulsant activity [12].
2
(R1 = R2 = 2,8-di-OMe, R3 = R4 = Bn) developed as
,
3
Figure 1. Selective anti-leukemic agents
1 [7] and their structural analogues 2 [9] and 3—potential
scaffolds for the design of bioactive molecules.
While searching for novel tricyclic heterocyclic derivatives of medicinal importance [
7–9],
we turned our attention to 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione as a potential privileged
3
scaffold for design and development of novel bioactive compounds. A brief literature survey revealed,
that such compounds could be obtained from a condensation reaction of benzene-1,2-diamine and
diethyl phthalate [13], benzene-1,2-diamine, and benzocyclobutene-l,2-dione [14
,15] or Beckmann
rearrangement of appropriate tricyclic dibenzoazepine oximes [16 17]. The parent compound
,
3
(R1 = R2 = R3 = R4 = H) could also be synthesized from benzene-1,2-diamine and 1,2-phenylenebis
((1H-benzo[d][1,2,3]triazol-1-yl)methanone) obtained from phthaloyl dichloride and 1H-benzo
[d][1,2,3]triazole [18], while condensation of 5,6-diaminouracil with phtalic anhydride resulted in the
formation of fused pyrimido[4,5-b][1,4]diazocine ring [19].
Unfortunately, all the methods described in the literature led to the products
3 having no
substituents introduced to the dilactam ring (only secondary lactams R3 = R4 = H). This limitation
significantly decreased the possibilities of modification of this structure, introduction of side chains,
and thus the modulation of biological properties of compounds. The main goals of our research
were focused on the development of methods of synthesis of previously unknown, asymmetric,
N-substituted (R3 = H, R4
[b,f][1,4]diazocine-6,11-diones
,
H) and N,N’-disubstituted (R3
, , H) 5,12-dihydrodibenzo
R4
3
and the introduction of some other fused heterocyclic rings in
the place of the benzene ring to the tricyclic scaffold. We also focused our efforts on the development
of a synthetic method leading to the novel fused pentacyclic scaffold possessing two benzodiazocine
rings within its structure, as well as post-cyclisation modifications of obtained products. Additionally,
we confirmed the identity of the synthesized scaffolds by single-crystal X-ray diffraction analysis and
performed the detailed crystallographic analysis to gain deeper insight in their structural features
in the solid state. Finally, the cytotoxic effect of the synthesized compounds was determined using
one cell line derived from non-cancerous cells (EUFA30—human fibroblasts) and three tumor cells
(U87—human primary glioblastoma; HeLa—cervix adenocarcinoma, BICR18—laryngeal squamous
cell carcinoma).

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2. Results
2.1. Chemistry
For the preparation of N-substituted (R3 = H, R4 , H) and N,N’-disubstituted (R3
5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones , we decided to use two types of building blocks:
Appropriately modified benzene-1,2-diamines and properly prepared phthalic esters (Scheme 1). To our
, , H)
R4
3
4
5
knowledge, such derivatives containing modified and substituted eight-membered dilactam rings have
notyetbeendescribedintheliteraturesofar. Oursynthetic efforts leading to the fused diazocine-2,5-diones,
were started from the synthesis of 1,3,4,6-tetrahydrobenzo [b][1,4]diazocine-2,5-dione (
6), obtained
from benzene-1,2-diamine (4a) and dimethyl succinate ( ). In a similar way, 5,12-dihydrodibenzo[b,f]
7
[1,4]diazocine-6,11-dione (3a) was synthesized from benzene-1,2-diamine (4a) and dimethyl phthalate
(5a). These compounds 6 [20] and 3a [13,14,18] were synthesized previously, but for the first time the
crystal structures of 6 and 3a were reported by us. Surprisingly, we did not find any crystallographic
structure of compounds possessing neither 1,3,4,6-tetrahydrobenzo[b][1,4]diazocine-2,5-dione (
6) nor
5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione ( ) cores in the Cambridge Structural Database [21],
3
which made this group of chemical compounds an interesting and appropriate target for crystallographic
studies. To get some more information about the structural features of compounds possessing
the 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3a) core, we synthesized tricyclic derivatives
substituted with nitro (3b
with C-substituted benzene-1,2-diamines: 4-nitrobenzene-1,2-diamine (4b) and 4-benzoyl-1,2-diamine
4c), while dimethyl 4-nitrophthalate (5b) reacted with benzene-1,2-diamine (4a) which resulted in
the tricyclic 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones (3b 3d). Despite of the presence of
a nitro (3b 5d) or benzoyl group (3c), which, in our opinion should facilitate the crystallization
, 3d) or benzoyl group (3c). Dimethyl phthalate (5a) smoothly reacted
(
-
,
process, we were unable to obtain crystals appropriate for crystallographic analysis and consequently,
we focused our attention on the modification of the eight-membered diazocine-6,11-dione dilactam
ring. Such a modification could be desirable in the design and synthesis of novel bioactive compounds
and could be responsible for the modulation of biological effect and properties. This chosen direction
of research was supported by our previous results [7], where the alkylation of a seven-membered
dilactam ring in tricyclic heterocyclic derivative with benzyl bromide, potentiated the selectivity and
cytotoxic effect of the investigated compounds toward leukemic and colon cancer cell lines.
Scheme 1. The synthesis of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione scaffold.
For the preparation of previously unknown N-substituted (R3 = H, R4
[b,f][1,4]diazocine-6,11-diones
, we used commercially available N1-methylbenzene-1,2-diamine (4d
as well as 4,5-dichloro-N1-methylbenzene-1,2-diamine (4e) and N1-benzylbenzene-1,2-diamine (4f
, H) 5,12-dihydrodibenzo
3
)
)
obtained in facile two-step syntheses. N1-methylbenzene-1,2-diamine (4d) and 4,5-dichloro-N1-
methylbenzene-1,2-diamine (4e) reacted smoothly with dimethyl phthalate (5a), dimethyl 4,5-
dichlorophthalate (5c) or dimethyl 3,4,5,6-tetrachlorophthalate (5d), in the presence of sodium
hydride, giving requested products 3e–h with high yields (Table 1). We also succeeded in
the condensation of N1-methylbenzene-1,2-diamine (4d) with heterocyclic analogue of phtalic
ester, dimethyl thiophene-3,4-dicarboxylate (5e), which resulted in the formation of a novel
heterocyclic scaffold: 5-methyl-5,10-dihydrobenzo[b]thieno[3,4-f][1,4]diazocine-4,11-dione (3i). In a

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similar manner, N1-benzylbenzene-1,2-diamine (4f) reacted with dimethyl phthalate (5a), giving
5-benzyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3j) as a sole product.
Table 1. Compounds synthesized in the reaction shown in Scheme 1.
Amine
Ester
Product
4a
4a
4b
4c
4a
7
6 (27%)
3a (78%)
3b (53%)
3c (55%)
3d (50%)
5a
5a
5a
5b
4d
4e
5a
5a
3e (61%)
3f (55%)

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Table 1. Cont.
Amine
Ester
Product
4d
5c
3g (67%)
4d
4d
5d
5e
3h (15%)
3i (47%)
4f
5a
3j (74%)
We also investigated the reaction between benzene-1,2-diamine (4a) and tetraethyl benzene-1,2,4,5-
tetracarboxylate ( ) in a 2:1 ratio and observed the formation of a novel pentacyclic product 9a
8
,
possessing a unique scaffold consisting of two diazocine-6,11-dione rings separated by a benzene
ring (Scheme 2). Product 9a precipitated out of the reaction mixture after the reaction took
place and was separated by filtration under reduced pressure. In the obtained filtrate, we also
identified small amounts of the intermediate tricyclic diethyl 6,11-dioxo-5,6,11,12-tetrahydrodibenzo
[b,f][1,4]diazocine-8,9-dicarboxylate (3k), which was purified and isolated by column chromatography.
The treatment of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3a) with an excess of methyl iodide,
in the presence of sodium hydride, resulted in the formation of 5,12-dimethyl-5,12-dihydrodibenzo[b,f]
[1,4]diazocine-6,11-dione (3l) (Scheme 3). Surprisingly, in the absence of an alkylating reagent, we
observed a novel, unprecedented rearrangement of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione
(3a) to 2-(2-aminophenyl)isoindoline-1,3-dione (10), which structure was confirmed by X-ray
crystallography [22]. The structure of compound 10 resembles the structure of Thalidomide,
a drug used in anticancer therapy, and the described method of synthesis can be used to
obtain a variety of Thalidomide analogues [23,24]. Although, rearrangement of 3a under
acidic conditions, in the presence of phosphoryl chloride [25] or phosphorus pentachloride [13],
leading to 2-phenyl-1H-benzo[d]imidazole derivatives, has already been described in the literature,

Molecules 2020, 25, 2855
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the base-promoted contraction of eight-membered diazocine-6,11-dione ring in 3a to the five-membered
pyrrolidine-2,5-dione ring in 10 was observed and described by us for the first time.
Scheme 2. The synthesis of pentacyclic scaffold.
Scheme 3. Chemical modifications of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione scaffold.
For the synthesis of N,N’-disubstituted (R3
11-diones , we used 8,9-dichloro-5-methyl-5,12- dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3g
and 5-benzyl-5,12- dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3j) (Scheme 3).
, , H) 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,
R4
3
)
Compound 3j was treated with 2-chloro-N,N-dimethylethan-1-amine, in the presence of
sodium hydride, which resulted in the formation of 5-benzyl-12-(2-(dimethylamino)ethyl)-5,
12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3m). In a similar way, the treatment of 3g
with ethyl bromoacetate led to the formation of ethyl 2-(8,9-dichloro-12-methyl-6,11-dioxo-11,
12-dihydrodibenzo[b,f][1,4]diazocin-5(6H)-yl)acetate (3n) (Scheme 3). The heating of 3j in acetic

Molecules 2020, 25, 2855
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anhydride at reflux temperature gave the N-acyl derivative, 5-acetyl-12-benzyl-5,12-dihydrodibenzo
[b,f][1,4]diazocine-6,11-dione (3o). We also attempted to alkylate the pentacyclic derivative 9a by
treating it with methyl iodide or benzyl bromide in the presence of sodium hydride (Scheme 4),
which resulted in the tetramethyl derivative 9b and tetrabenzyl derivative 9c. The 1H NMR spectrum
registered for 9a indicates the presence of two conformational isomers in the ratio of 1:0.25, which
can be deduced from two sets of signals originating from NH and Ar-H protons. These conformers
are the result of dihydrodiazocine-6,11-dione rings being able to adopt two possible orientations,
leading to a ‘U’-shaped or a ‘Z’-shaped molecule, as seen in the crystal structures of compounds
1
9c and 9a, respectively. Similarly, we noticed the presence of two sets of signals in the H NMR
spectrum of 9b (here in the ratio of 1:0.6), suggesting that in this case conformational equilibrium
1
of two possible orientations of the mentioned ring is also observed. The H NMR spectra in the
temperature range of 275–325 K (2–52 ◦C) registered for 9b dissolved in trifluoroacetic acid-d shows that
the temperature drift of 1H signals originating from both conformers is different, indicating somewhat
different dynamics in both forms (for temperature 1H NMR spectra showing this temperature drift see
Supporting Information, Figure S25b). Interestingly, in the 1H NMR spectrum of 9c only one set of 1H
signals is visible. This is probably the result of the presence of four bulky substituents at the nitrogen
atoms, which effectively prevents 9c from adopting a less energetically favorable conformation.
Scheme 4. Alkylation of pentacyclic scaffold.
2.2. Crystallographic Analysis
Attempts to obtain single-crystals suitable for X-ray diffraction measurements were undertaken
for all synthesized compounds. However, they were only successful in nine cases (3a
, 3g–3j, 6,
9a 9c and 10). Single crystals appropriate for this purpose were grown by slow evaporation from:
,
3a—DMSO:DMF (1:1), 3g—n-butyl acetate, 3h—ethanol, 3i—THF:water (9:1), 3j—ethanol,
9a—DMSO:NMP (1:1), 9c—dimethylacetamide (DMA), 10—ethanol.
6—DMF,
The investigated compounds crystallized in the monoclinic P21/n (3h
C2/c (3j) and C2/m (3a) or triclinic P-1 ( ) space groups. The asymmetric unit of the crystal lattice contain
half (3a and 9a), one (3g 3i 3j 9c and 10) or two molecules of given compound (3h) (Figure 2 and
Figures S1 S5 (ESI)). Three products crystallized in the form of solvates (3i 9a and 9c) (Figures S2 S4
,
3i
,
9a and 10), P21/c (3g
,
9c),
6
,
,
, 6,
−
,
−
(ESI)). The refinement parameters and details of the crystallographic data are presented in Table S1
(ESI). The values of bond lengths, valence and torsion angles are presented in Tables S2−S28 (ESI).
The conformation of the dibenzodiazocine skeleton in the case of all investigated tricyclic
derivatives (3a
above moiety can be emphasized using two parameters:
,
3g−3j) resembles a butterfly (Figure 3). Differences in the molecular geometry of the
α
, which is the angle between mean-planes
defined by the non-hydrogen atoms of the outer aromatic rings and D—a distance between geometrical
centers of gravity of these rings (Figure 3). The value of
α
for 3a
,
3g−j ranges from 72.3(3) to 87.74(4)◦
(Table 2); however, in most cases (3a 3g, 3i and 3j) the values are very close to each other. Detailed
,
analysis indicates that the angle is less obtuse in cases, where outer aromatic rings within the molecule
are differently substituted (3g and 3h) or composed (3j). This is particularly evident in 3h where one of
the phenyl moieties is bearing four chlorine atoms. In this case the mean-planes of the phenyl rings are
inclined to each other just by 72.3(3)/79.9(3)◦ (molecule A/molecule B). This can probably be explained
by the specific mutual orientation of the adjacent molecules of the compound in the crystal network.

Molecules 2020, 25, 2855
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The molecules of 3h are held together by the N
their neighboring phenyl rings are close enough to force compression of the dibenzodiazocine moieties.
−
H
···O hydrogen bond (Figure S1 (ESI)) in a way that
The respective orientation of the above aromatic rings manifests also in the presence of the π–π contact
between them (d(CgI···CgJ) = 3.644(5) Å, Figure S1 (ESI)). Interestingly this type of intermolecular
interaction was not identified in the crystals of the remaining tricyclic systems. As for the distance
between the geometric centers of the outer aromatic rings within the molecules of investigated tricyclic
compounds (D)—here the values are more scattered (4.231(5)
was observed in case of the 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3a), while the lowest
was for the 3h. Like in the case of , also here the smaller parameter values accompany compounds
bearing differently substituted or composed outer phenyl rings within the molecule (3g, 3h and 3j).
−
4.695(2) Å, Table 2). The highest value
α
Figure 2. Molecular structure of 3a, 3g-j and 6 with crystallographic atom numbering. Displacement
ellipsoids are drawn at the 50% probability level. The H-atoms are shown as small spheres of arbitrary
radius. Solvent molecule in case of 3i and the second crystallographically independent molecule of 3h
have been omitted for clarity. Symmetry code: (i) x, −y + 1, z.

Molecules 2020, 25, 2855
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Figure 3. The graphical representation of the conformation types of molecules of investigated tri- and
pentacyclic compounds with geometrical parameters used for their description.
Table 2. The values of geometric parameters describing the conformation of molecules of investigated
tri- and pentacyclic benzodiazocine derivatives.
Geometric Parameter
Compound
α [◦]
α’ [◦]
D [Å]
D’ [Å]
β
3a
3g
87.68(2)
86.68(6)
4.695(2)
4.680(2)
3h (molecule A/molecule B)
72.3(3)/79.9(3)
87.05(6)
4.231(5)/4.349(6)
4.372(2)
3i
3j
87.74(4)
4.567(7)
9a
9c
88.82(7)
64.78(6)
88.82(7)
69.52(6)
4.589(2)
4.057(2)
4.589(2)
4.170(2)
45.74(7)
Where:
benzodiazocine moiety; D and D’: distance between geometrical centers of gravity of outer aromatic rings of the
dibenzodiazocine moiety; : angle between mean-planes defined by the non-hydrogen atoms of the outer aromatic
α and α’: angle between mean-planes defined by the non-hydrogen atoms of the outer aromatic rings of the
β
rings of fused pentacyclic benzodiazocine moiety (see Figure 3).
The structure of both pentacyclic products composed of two fused dibenzodiazocine units (9a and
9c) is more complex. The molecular skeleton of 9a adopts the ‘Z’-shaped conformation (Figure 3).
The middle phenyl ring is situated directly at the inversion center, what makes both sides of the
molecule describable by the same values of geometric parameters. The values of
α in 9a are slightly
larger comparing to the previously analyzed tricyclic systems, while the value of D is comparable to 3j
(Table 2). In turn, the conformation of 9c, which is N-substituted by four bulky benzyl groups can be
described as the “U”-shaped (Figure 3). In the case of this derivative, the aromatic rings within the
respective dibenzodiazocine moieties are inclined to each other at more acute angles (64.78(6)◦ and
69.52(6)◦), and the distance between their geometric centers of gravity is the shortest among the all
investigated compounds (4.057(2) and 4.170(2) Å, Table 2). The outer phenyl rings of the pentacyclic
moiety are inclined to themselves at an angle of 45.74(7)◦. The mutual orientation of the benzyl
groups attached to the N12 and N25 nitrogen atoms relative to the main molecular unit results in the
appearance of weak intramolecular C
(Figure 4 and Figure S4, Table S30 (ESI)). In turn the substituent attached to the N19 atoms participating
in the formation of weak intramolecular C
−
H
···O hydrogen bonds involving the nearest carbonyl O-atoms
−
H
···π contact (Figure 4 and Figure S4, Table S31 (ESI)).
Besides the aforementioned differences in structural features, 9a and 9c differ significantly in a way in
which their molecules are organized in the crystals. Both systems crystallized with additional solvent
molecules incorporated in their crystal networks. What is interesting—in the crystal lattice of 9a two
different solvents (dimethyl sulfoxide and 1-methyl-pyrrolidin-2-one) are present. In the case of 9c,
the N,N-dimethylacetamide was found. In both of the investigated crystals, molecules are involved
in a network of intermolecular hydrogen bonds (Tables S29 and S30). In the crystal of 9c also the
intermolecular C
−
H
···π contacts were identified (Table S31 (ESI)). A close look at figures presenting the
supramolecular architecture of above solvates reveals that in the case of 9a, separate layers formed
by molecules of the compound and solvents can be distinguished (Figure S6 (ESI)), whereas in 9c
the molecules of the solvent are “interlocked” in pockets of the framework formed by the molecules of
the compound (Figure S7 (ESI)).
,

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Figure 4. Molecular structure of 9a and 9c with crystallographic atom numbering. Displacement
ellipsoids are drawn at the 50% probability level. The H-atoms are shown as small spheres of arbitrary
radius. The solvent molecules have been omitted for clarity. The intramolecular C
−
H
···O hydrogen
bonds are represented by dashed lines, while the C H
(i) −x + 2, −y + 1, −z + 1.
−
···π contact by dotted lines. Symmetry code:
2.3. Cytotoxic Effect of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones 3a-o
We evaluated the cytotoxic efficacy of 17 synthesized 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,
11-diones 3a-o on one normal (EUFA30) and three cancerous (HeLa, BICR18, U87) cell lines at
concentrations between 1 and 300
and 3o, showed cytotoxic effect at the same time maintaining the selectivity of action —- their IC50
ranged from several dozen (lowest: 93.7 M) to several hundred (highest: 322.8 M). Nevertheless,
µM (Figure 5). Among the tested compounds, five: 3f, 3g, 3h, 3n,
µ
µ
the IC50 for the normal cell line was higher than for the cancerous one. For the majority of the remaining
compounds, IC50 values were above the highest concentration used (Table 3).
To measure the induction of apoptosis, cells were treated with 200 µM of 3f, 3g, 3h, 3n, and 3o
compound, for 24 h (Figure 6). The experiment was performed on one normal (EUFA30) and one
cancer (HeLa) cell line. After 24 h of EUFA30 treatment we observed significant increase in dead cell
fractions: Early apoptosis, late apoptosis, and necrosis for almost all tested compounds: 6.9%, 1.6%
and 4.3% for 3f, 5.0%, 1.5% and 6.8% for 3g, 9.0%, 1.8% and 15.6% for 3h, 7.8%, 1.4% and 1.9% for 3n
,
9.5%, 6.4% and 0.8% for 3o, respectively, comparing to 0.3%, 0.4% and 5.7% in the non-treated control.
In the positive control (camptothecin—CPT) the proportion of dead cell fractions was 4.1%, 6.8% and
18.7%, respectively. The best active compound was 3h and the least 3n.

Molecules 2020, 25, 2855
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Figure 5. The cytotoxic effect of indicated 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones on normal
EUFA30 and cancerous HeLa, BICR18, and U87 cells after 24 h based on AlamarBlue assay. Non-linear
fitting with logistic dose response model was employed.
Figure 6. Flow cytometry analysis of EUFA30 and HeLa cells stained with Annexin-FITC (A),
and propidium iodide (PI). Cells were treated with 200
µM of indicated compounds for 24 h.
Camptothecin (10 µM) was used as experimental control.

Molecules 2020, 25, 2855
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Table 3. IC50 (µM) of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones based on the survival of
non-cancerous (EUFA30) and cancerous (HeLa, BICR18, and U87) cells after 24 and 48 h of treatment.
Cell Types
EUFA30
HeLa
BICR18
U87
Compound
24 h
48 h
[9.0] * ± 5.2
24 h
48 h
24 h
48 h
24 h
48 h
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
6
[21.2] *
[31.0] *
[28.8] *
±
±
±
4.8
3.0 [18.6] *
5.1 N.A.
[20.7] *
1.8 [33.2] *
[14.5] *
±
±
±
±
±
2.4
6.2
4.4
4.6
3.3
[1.5] * ± 1.2
[4.7] * ± 1.5
N.A.
[7.1] * ± 2.7
[2.2] * ± 1.4
[10.0] * ± 3.3
[25.1] * ± 7.4
[22.7] * ± 8.6
[47.5] * ± 7.6
266.4 ± 27.6
93.7 ± 5.2
N.A.
N.A.
N.A.
[17.6] *
±
±
±
±
±
4.5
2.4
9.2
5.2
3.2
N.A.
N.A.
N.A.
N.A.
N.A.
±
[10.4] *
[15.0] *
[27.5] *
[34.1] *
[9.2] * ± 2.7
[4.2] * ± 2.9
[23.4] *
[3.9] * ± 2.9
[5.7] * ± 1.0
[7.8] * ± 2.1
[5.3] * ± 2.3
250.7 ± 5.1
[1.6] * ± 2.9
[3.3] * ± 2.3
N.A.
[2.5] * ± 5.3
[31.6] *
320.0 ± 36
[41.6] * 4.5 [29.5] *
±
8.9 [17.5] *
±
±
±
3.1 [19.8] *
N.A.
[19.1] *
8.5 298.4 ± 11.0
[7.6] * ± 4.2
[12.3] ± 1.8
177.7 ± 3.0
N.A.
[6.1] * ± 3.0
[5.6] * ± 3.2
N.A.
207.0 ± 23.6
322.8 ± 23.9
97.1 ± 13.2
[29.9] * ± 18.3
[2.4] * ± 2.4
293.6 ± 22.4
[2.8] * ± 1.9
N.A.
±
9.9 [43.1] *
±
4.7
144.5 ± 10.6
153.2 ± 4.1
132.1 ± 6.0
[29.4] *
[33.2] *
[36.7] *
[38.1] *
[42.2] *
[41.5] *
±
±
±
±
±
±
6.2
[1.5] * ± 1.4
[14.8] *
2.5 [34.3] *
[28.2] *
±
±
±
±
±
6.8
7.2
1.5
9.3
9.3
[24.0] * ± 5.7
[42.2] * ± 12.2
[16.8] * ± 5.9
[22.1] * ± 3.5
[30.1] * ± 9.7
[20.9] * ± 10.2
121.0 ± 11.4
[11.3] * ± 4.8
[11.4] * ± 3.7
[36.6] *
[33.0] *
[37.2] *
[39.0] *
[36.8] *
[30.7] *
±
±
±
±
±
±
4.8
2.8
5.5
4.7
2.8
8.7
8.0 [32.6] *
±
5.0
N.A.
[3.1] * ± 2.6
2.1 [19.5] *
±
5.8 [29.5] *
[2.8] * ± 1.6
N.A.
8.0
6.8
[9.9] * ± 5.7
[23.4] *
311.4 ± 14.1
N.A.
[4.7] * ± 2.8
[4.1] * ± 3.6
N.A.
N.A.
N.A.
N.A.
155.4 ± 14.6 [41.8] *
±
7.9 169.1 ± 13.4
[33.4] * ± 8.9
[4.3] * ± 1.4
N.A.
294.3 ± 3.2
N.A.
N.A.
240.2 ± 6.1
[33.8] * ± 4.6
[29.4] *
[25.6] *
±
5.9
6.6
[14.0] ± 6.1
[21.5] *
[4.9] * ± 3.1
±
4.3
[19.6] *
[22.9] *
±
5.8
4.3
N.A.
10
±
N.A.
±
N.A.
* proliferation inhibition expressed as a percentage at highest concentration used: 300 µM. N.A.: not active.
The HeLa cells were sensitive to all tested compounds with a significant increase in necrotic
phase only. After 24 h, the number of necrotic cells for 3f, 3g, 3h, 3n, and 3o were 15.3, 8.8, 15.3, 11.3,
and 27.7%, respectively, in comparison to 5.4% in the non-treated control. The best cytotoxic agent
appears to be 3o, the compound that killed cells similarly to CPT, a positive control with 31.2% of
necrotic cells.
In HeLa cells these compounds cause mainly necrosis in contrast to EUFA30 cells, where in the
most cases apoptosis, early and late, appeared in the dead cell fractions.
Although most compounds showed a weak cytotoxic effect, some relationships between structure
and activity could be observed. Compounds substituted with chlorine atoms in the benzene ring
3f, 3g, 3h, and 3n proved to be the most cytotoxic, and this effect was the strongest for compound
3h, substituted with four chlorine atoms (IC50 = 93.7
±
5.2
µ
M for BIRC18, IC50 = 97.1
±
13.2 µM for
U87). In the group of compounds substituted with chlorine atoms, the alkylation of 3g with ethyl
bromoacetate resulted in derivative 3n, did not significantly change the cytotoxicity for the cell lines
tested. The acylation of 3j resulting in the 3o derivative significantly enhanced the cytotoxic effect.
For compound 3j, the IC50 values was above 300 µM for all tested cell lines, while for 3o these values
ranged from 121.0 ± 11.4 µM (for BIRC18) to 240.2 ± 6.1 µM (for U87).
3. Materials and Methods
3.1. Chemistry
3.1.1. General Information
Commercially available chemicals were of reagent grade and used as received. The reactions
were monitored by thin layer chromatography (TLC), using silica gel plates (Kieselgel 60F254, E. Merck,
Darmstadt, Germany). Column chromatography was performed on silica gel 60 M (0.040–0.063 mm,
E. Merck, Darmstadt, Germany). Melting points are uncorrected and were measured on a Büchi
(New Castle, DE, USA) Melting Point B-540 apparatus. All 1H and 13C NMR spectra were registered
with a Bruker Avance III (Billerica, MA, USA) spectrometer operating at 500.13 and 125.77 MHz for 1H
and 13C, respectively, and equipped with a 5 mm probe head with Z-gradient coils. The experiments
were performed using pulse programs from the standard Bruker library for samples dissolved in
DMSO-d6, methanol-d4 or trifluoroacetic acid-d, depending on the sample. In each case spectra were
calibrated at residual solvent resonances. For the temperature 1H NMR spectra a BCU unit was used

Molecules 2020, 25, 2855
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for temperature control and stabilization. After reaching the required temperature measured samples
were equilibrated at this temperature for at least 10 min prior to measurement. Solvent signals in
NMR spectra were determined on the basis of literature data [26]. High resolution mass spectra were
performed by the Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics PAS, on a
LTQ Orbitrap Velos instrument, Thermo Scientific (Waltham, MA, USA). IR spectra were recorded
with the Jasco 6200 (Easton, MD, USA) FT/IR spectrometer in the Laboratory of Optical Spectroscopy,
Institute of Organic Chemistry PAS (Warsaw, Poland).
3.1.2. General Procedure for Synthesis 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones (3a-j)
To a solution of diamine 4a–f (1 equiv.) and diester 5a–e (1 equiv.) in anhydrous THF (5 mL/mmol),
60% sodium hydride in mineral oil (2 equiv.) was added and the resulting mixture was refluxed for 18 h.
The reaction mixture was poured into water, acidified with concentrated HCl to pH 2 and extracted
with ethyl acetate. The organic phase was washed with brine and dried over anhydrous magnesium
sulfate. The drying agent was filtered off and the resulting filtrate was evaporated under reduced
pressure. The crude product was purified either by crystallization or by column chromatography.
5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3a), crystallized from DMF, yield 78%, white crystals,
m.p. 286.0–287.0 ◦C. 1H NMR (500 MHz, DMSO-d6)
δ
10.18 (s, 2H, 2
×
NH), 7.65–6.85 (m, 8H, HAr);
13C NMR (125 MHz, DMSO-d6)
δ
170.4, 134.8, 132.3, 130.2, 127.5, 127.0, 126.6; IR (KBr): cm−1 3178,
3049, 2938, 2901, 2874, 1936, 1825, 1651, 1593, 1573, 1502, 1461, 1403, 1304, 1265, 1239, 1140, 1115, 1043;
HRMS (ESI): m/z [M + H]+ calcd for C14H11N2O2: 239.08150, found: 239.08174.
2-nitro-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3b), crystallized from ethyl acetate, yield 53%,
1
yellow crystals, m.p. 230.0 ◦C (decomposition), R.f. = 0.38 (hexane:ethyl acetate 2:8 v/v). H NMR (500
MHz, DMSO-d6)
δ
10.66 (s, 1H, NH), 10.44 (s, 1H, NH), 8.06 (dd, 1H, J = 2.0 Hz, J = 8.0 Hz, HAr), 7.99
170.2, 170.0, 145.5,
(d, 1H, J = 2.0 Hz, HAr), 7.51–7.30 (m, 5H, HAr); 13C NMR (125 MHz, DMSO-d6)
δ
140.9, 135.2, 131.6, 131.4, 130.72, 130.66, 127.6, 126.9, 126.8, 122.6, 122.1; IR (KBr): cm−1 3169, 3077, 3035,
2924, 2862, 1678, 1654, 1593, 1573, 1533, 1501, 1393, 1349, 1238, 1141, 1064; HRMS (ESI): m/z [M + H]+
calcd for C14H10N3O4: 284.06658, found: 284.06699.
2-benzoyl-5,12-dihydrodibenzo[b,f][1◦
,4]diazocine-6,11-dione (3c), crystallized from ethyl acetate, yield 55%,
beige crystals, m.p. 156.0–157.0 C, R.f. = 0.24 (hexane:ethyl acetate 2:8 v/v). 1H NMR (500 MHz,
DMSO-d6)
7.49–7.43 (m, 2H, HAr), 7.38–7.30 (m, 3H, HAr); 13C NMR (125 MHz, DMSO-d6)
138.6, 136.5, 135.5, 134.7, 132.9, 131.9, 131.8, 130.51,130.49, 129.5 (2 C), 128.9, 128.6 (2
δ
10.52 (s, 1H, NH), 10.32 (s, 1H, NH), 7.71–7.63 (m, 3H, HAr), 7.59–7.50 (m, 4H, HAr),
194.0, 170.3, 170.2,
C), 128.2, 126.9,
δ
×
×
126.80, 120.77; IR (KBr): cm−1 3171, 3060, 2931, 2873, 1734, 1685, 1659, 1601, 1574, 1492, 1446, 1392,
1314, 1278, 1263, 1239, 1179, 1141, 1125, 1078, 1043; HRMS (ESI): m/z [M + H]+ calcd for C21H15N2O3:
343.10772, found: 343.10731.
8-nitro-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3d), crystallized from ethyl acetate, yield 50%,
1
yellow crystals, m.p. 243.0–244.0 ◦C, R.f. = 0.40 (hexane:ethyl acetate 2:8 v/v). H NMR (500 MHz,
DMSO-d6)
δ 10.521 (s, 1H, NH), 10.492 (s, 1H, NH), 8.22 (dd, 1H, J = 2.5 Hz, J = 8.5 Hz, HAr), 8.11
(d, 1H, J = 2.5 Hz, HAr), 7.63 (d, 1H, J = 8.5 Hz, HAr), 7.26–7.15 (m, 4H, HAr); 13C NMR (125 MHz,
DMSO-d6)
δ 168.5, 168.2, 148.3, 137.7, 134.4, 134.3, 133.4, 128.7, 128.01, 127.98, 127.4, 127.2, 125.2, 121.9;
IR (KBr): cm−1 3245, 3078, 1674, 1645, 1612, 1582, 1527, 1503, 1487, 1446, 1396, 1346, 1301, 1236, 1149,
1065; HRMS (ESI): m/z [M + H]+ calcd for C14H10N3O4: 284.06658, found: 286.06693.
5-methyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3e) crystallized from ethyl acetate, yield 61%,
white crystals, m.p. 206.0–207.0 ◦C, R.f. = 0.36 (hexane:ethyl acetate 2:8 v/v). 1H NMR (500 MHz,
DMSO-d6)
7.19–7.12 (m, 1H, HAr), 3.30 (s, 3H, Me); 13C NMR (125 MHz, DMSO-d6)
133.3, 131.7, 130.2, 130.1, 128.3, 128.2, 127.2, 127.1, 126.4, 126.2, 36.6; IR (KBr): cm−1 3213, 3063, 2927,
δ
10.21 (s, 1H, NH), 7.47–7.41 (m, 1H, HAr), 7.40–7.34 (m, 2H, HAr), 7.31–7.19 (m, 4H, HAr),
δ
170.3, 168.6, 140.4, 135.1,

Molecules 2020, 25, 2855
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2883, 1965, 1935, 1828, 1675, 1626, 1594, 1571, 1502, 1488, 1420, 1392, 1346, 1309, 1256, 1236, 1191, 1162,
1140, 1080, 1036, 1007; HRMS (ESI): m/z [M + H]+ calcd for C15H13N2O2: 253.09715, found: 253.09709.
2,3-dichloro-5-methyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3f) crystallized from THF, yield
1
55%, white crystals, m.p. 198.0–199.0 ◦C, R.f. = 0.57 (hexane:ethyl acetate 2:8 v/v). H NMR (500 MHz,
DMSO-d6)
δ
10.35 (s, 1H, NH), 7.91 (s, 1H, HAr); 7.49 (s, 1H, HAr); 7.47–7.40 (m, 2H, HAr), 7.32–7.25
170.0, 168.3, 140.3, 135.4, 132.7, 131.1,
(m, 2H, HAr), 3.30 (s, 3H, Me); 13C NMR (125 MHz, DMSO-d6)
δ
130.6, 130.4, 130.3, 130.0, 129.1, 128.3, 126.6, 126.4, 36.6; IR (KBr): cm−1 3177, 3095, 3039, 2894, 1680,
1633, 1592, 1567, 1485, 1427, 1398, 1357, 1305, 1262, 1226, 1138, 1110, 1063, 1019; HRMS (ESI): m/z [M +
H]+ calcd for C15H11N2O2Cl2: 321.01921, 323.01626, found: 321.01957, 323.01658.
8,9-dichloro-5-methyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3g) crystallized from ethyl acetate,
yield 67%, white crystals, m.p. 207.0–208.0 ◦C, R.f. = 0.53 (hexane:ethyl acetate 2:8 v/v). 1H NMR
(500 MHz, DMSO-d6)
δ
10.43 (s, 1H, NH), 7.59 (s, 2H, HAr), 7.46 (d, 1H, J = 7.0 Hz, HAr), 7.35–7.24 (m,
167.8, 166.2,
2H, HAr), 7.19 (d, 1H, J = 7.0 Hz, HAr), 3.30 (s, 3H, Me); 13C NMR (125 MHz, DMSO-d6)
δ
139.9, 134.6, 133.5, 133.13, 133.05, 132.1, 128.8, 128.63, 128.59, 128.4, 127.43, 127.36, 36.7; IR (KBr): cm−1
3213, 3087, 2931, 1671, 1640, 1593, 1546, 1501, 1424, 1396, 1375, 1316, 1256, 1234, 1130, 1092, 1020; HRMS
(ESI): m/z [M + H]+ calcd for C15H11N2O2Cl2: 321.01921, 323.01626, found: 321.01957, 323.01664.
7,8,9,10-tetrachloro-5-methyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3h) column chromatography
methanol:chloroform 1:9 v/v, yield 15%, white crystals, m.p. 233.0 ◦C (decomposition), R.f. = 0.67
1
(hexane:ethyl acetate 2:8 v/v). H NMR (500 MHz, DMSO-d6)
δ
10.78 (s, 1H, NH), 7.50 (d, 1H, J = 7.0 Hz
,
HAr), 7.37–7.26 (m, 2H, HAr), 7.23 (d, 1H, J = 7.0 Hz, HAr), 3.31 (s, 3H, Me); 13C NMR (125 MHz,
DMSO-d6) δ 164.5, 163.3, 140.1, 134.7, 134.2, 134.1, 133.1, 131.7, 130.0, 129.9, 128.4, 128.2, 127.9, 127.8,
36.7; IR (KBr): cm−1 3347, 3209, 3107, 2936, 2889, 2857, 1678, 1652, 1596, 1537, 1497, 1433, 1401, 1353,
1299, 1264, 1232, 1149, 1098, 1038; HRMS (ESI): m/z [M + H]+ calcd for C15H9N2O2Cl4: 388.94126,
390.93831, 392.93536, found: 388.94181, 390.93890, 392.93563.
5-methyl-5,10-dihydrobenzo[b]thieno[3,4-f][1,4]diazocine-4,11-dione
(
3i
)
colum chromatography
hexane:ethyl acetate 2:8 v/v, yield 47%, white crystals, m.p. 241.0–242.0 ◦C, R.f. = 0.32 (hexane:ethyl
1
acetate 2:8 v/v). H NMR (500 MHz, DMSO-d6)
δ
10.78 (s, 1H, NH), 7.76–7.40 (m, 2H, HAr), 7.47–7.40
(m, 1H, HAr), 7.32–7.23 (m, 2H, HAr), 7.20–7.14 (m, 1H, HAr), 3.27 (s, 3H, Me); 13C NMR (125 MHz,
DMSO-d6)
δ 166.9, 165.2, 140.0, 135.1, 134.7, 134.2, 128.4, 128.3, 128.2, 127.8, 127.52, 127.48; IR (KBr):
cm−1 3176, 3108, 3055, 2904, 1735, 1645, 1587, 1503, 1469, 1366, 1301, 1283, 1243, 1160, 1105, 1044; HRMS
(ESI): m/z [M + H]+ calcd for C13H11N2O2S: 259.05357, found: 259.05396.
5-benzyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3j) crystallized from ethyl acetate, yield 74%,
white crystals, m.p. 205.0–206.0 ◦C, R.f. = 0.62 (hexane:ethyl acetate 2:8 v/v). 1H NMR (500 MHz,
DMSO-d6)
HAr), 7.09–7.02 (m, 1H, HAr), 5.30 (d, 1H, J = 15.0 Hz, CH2), 4.81 (d, 1H, J = 15.0 Hz, CH2); 13C NMR
(125 MHz, DMSO-d6) 170.2, 168.9, 138.9, 136.7, 135.7, 133.2, 131.6, 130.3, 130.1, 128.4 (2 C), 128.3,
127.8 (3
C), 127.3, 127.2, 127.0, 126.5, 126.2, 52.4; IR (KBr): cm−1 3160, 3038, 2900, 2869, 1655, 1596,
δ 10.09 (s, 1H, NH), 7.44–7.37 (m, 2H, HAr), 7.36–7.21 (m, 8H, HAr), 7.19–7.7.11 (m, 2H,
δ
×
×
1572, 1501, 1487, 1430, 1401, 1384, 1360, 1321, 1270, 1224, 1164, 1141, 1121, 1089, 1046, 1025; HRMS (ESI):
m/z [M + H]+ calcd for C21H17N2O2: 329.12845, found: 329.12877.
3.1.3. 5,12-dimethyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3l)
To a solution of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3a) (2.0 g, 8.4 mmol, 1.0 equiv.)
in anhydrous DMSO (20 mL), 60% sodium hydride in mineral oil (0.74 g, 18.4 mmol, 2.2 equiv.) was
added and resulting mixture was stirred for 30 min. After the addition of methyl iodide (1.2 mL,
18.4 mmol 2.2 equiv.), the reaction mixture was stirred for 18 h at room temperature, then was poured
into water and the crude product was extracted with ethyl acetate (3
washed with brine (1 50 mL) and dried over anhydrous magnesium sulfate. The crude product
was crystallized from ethyl acetate giving 3l as colorless crystals (1.9 g, yield 85%). M.p. 265.0–266.0
×
50 mL). The organic phase was
×

Molecules 2020, 25, 2855
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1
◦C, R.f. = 0.42 (hexane:ethyl acetate 2:8 v/v). H NMR (500 MHz, DMSO-d6) δ 7.48–7.41 (m, 1H, HAr),
7.38–7.32 (m, 1H, HAr), 7.31–7.25 (m, 1H, HAr), 7.24–7.18 (m, 1H, HAr), 3.35 (s, 6H, 2 Me, overlapped
×
with H2O); 13C NMR (125 MHz, DMSO-d6)
δ 168.4, 140.4, 132.8, 130.0, 128.9, 127.2, 126.1, 36.2; IR (KBr):
cm−1 3278, 3065, 3045, 2937, 1651, 1593, 1571, 1502, 1486, 1454, 1420, 1388, 1310, 1277, 1162, 1119, 1034,
1003; HRMS (ESI): m/z [M + H]+ calcd for C16H15N2O2: 267.11280, found: 267.11304.
3.1.4. 5-benzyl-12-(2-(dimethylamino)ethyl)-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3m)
To a solution of 5-benzyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3j) (1.5 g, 4.6 mmol,
1.0 equiv.) in anhydrous DMSO (30 mL), 60% sodium hydride in mineral oil (0.45 g, 11.4 mmol,
2.5 equiv.) was added, and the resulting mixture was stirred for 30 min. After the addition of
2-chloro-N,N-dimethylethan-1-amine hydrochloride (0.82 g, 5.7 mmol, 1.2 equiv.) the reaction mixture
was stirred for 18 h at room temperature, then was poured into water and the crude product was
extracted with ethyl acetate (3
dried over anhydrous magnesium sulfate. The crude product was purified by column chromatography
(triethylamine:ethyl acetate 5:95 v/v) giving 3m as white crystals (1.29 g, yield 71%). M.p. 154.0–155.0 ◦C,
×
50 mL). The organic phase was washed with brine (1
×
50 mL) and
1
R.f. = 0.38 (triethylamine:ethyl acetate 5:95 v/v). H NMR (500 MHz, DMSO-d6)
δ
7.40–7.14 (m, 13H,
HAr), 5.30 (d, 1H, J = 15.0 Hz, CH2),4.80 (d, 1H, J = 15.0 Hz, CH2), 3.47 (dt, 1H, J = 7.0 Hz, J = 14.0 Hz
CH2), 2.71 (dt, 1H, J = 7.0 Hz, J = 14.0 Hz, CH2), 2.34 (t, 2H, J = 7.0 Hz, CH2), 2.01 (s, 6H, 2
Me); 13
NMR (125 MHz, DMSO-d6) 168.5, 168.3, 140.2, 139.3, 137.2, 133.0, 132.4, 130.1, 130.0, 129.0, 128.8,
128.7 (2 C), 128.6 (2 C), 127.7, 127.5 (2
C), 126.0, 125.9, 56.5, 51.7, 46.4, 45.1; IR (KBr): cm−1 3276,
,
×
C
δ
×
×
×
3061, 2982, 2952, 2858, 2823, 2798, 2775, 1981, 1653, 1583, 1572, 1498, 1486, 1458, 1440, 1401, 1380, 1364,
1330, 1319, 1239, 1218, 1195, 1154, 1120, 1080, 1060, 1042, 1025; HRMS (ESI): m/z [M + H]+ calcd for
C25H26N3O2: 400.20195, found: 400.20245.
3.1.5. Ethyl 2-(8,9-dichloro-12-methyl-6,11-dioxo-11,12-dihydrodibenzo[b,f]
[1,4]diazocin-5(6H)-yl)acetate (3n)
To a solution of 8,9-dichloro-5-methyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3g
)
(2.0 g, 6.2 mmol, 1.0 equiv.) in anhydrous DMSO (20 mL), 60% sodium hydride in mineral oil (0.37 g,
9.4 mmol, 1.5 equiv.) was added and the resulting mixture was stirred for 30 min. After the addition
of ethyl bromoacetate (1.0 mL, 9.4 mmol, 1.5 equiv.) the reaction mixture was stirred for 18 h at
room temperature, then was poured into water and the crude product was extracted with ethyl
acetate (3 × 50 mL). The organic phase was washed with brine (1
×
50 mL) and dried over anhydrous
magnesium sulfate. The crude product was purified by column chromatography (hexane:ethyl acetate
7:3 v/v) giving 3n as white crystals (1.93 g, yield 76%). M.p. 114.0–115.0 ◦C, R.f. = 0.32 (hexane:ethyl
1
acetate 6:4 v/v). H NMR (500 MHz, DMSO-d6)
δ
7.58 (s, 1H, HAr), 7.52–7.42 (m, 3H, HAr), 7.37–7.29 (m,
2H, HAr), 4.78 (d, 1H, J = 17.0 Hz, CH2), 4.49 (d, 1H, J = 17.0 Hz, CH2), 4.15 (q, 2H, J = 7.0 Hz, CH2),
3.36 (s, 3H, Me, partially overlapped with H2O signal), 1.20 (t, 3H, J = 7.0 Hz, Me); 13C NMR (125 MHz,
DMSO-d6) δ 168.1, 166.5, 165.7, 140.2, 138.9, 133.3, 133.0, 132.1, 129.4, 129.3, 128.5, 128.3, 127.6, 127.1,
61.0, 51.0, 36.6, 14.0; IR (KBr): cm−1 3291, 3087, 2986, 2927, 2849, 1757, 1660, 1587, 1548, 1501, 1475, 1449,
1416, 1390, 1353, 1319, 1254, 1204, 1156, 1135, 1096, 1061, 1025, 1011; HRMS (ESI): m/z [M + H]+ calcd
for C19H17N2O4Cl2: 407.05599, 409.05304, found: 407.05674, 409.05381.
3.1.6. Acylation of 5-benzyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3j)
A suspension of 5-benzyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3j) (1.0 g, 3.0 mmol)
in acetic anhydride (15 mL) was refluxed for 1 h, then the excess of anhydride was evaporated
and crude 5-acetyl-12-benzyl-5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3o) was crystallized
from mixture of cyclohexane:ethyl acetate 9:1 giving 3o as colorless crystals (1.06 g, 94% yield).
1
M.p. 174.0–175.0 ◦C, R.f. = 0.54 (hexane:ethyl acetate 6:4 v/v). H NMR (500 MHz, DMSO-d6)
δ
7.49–7.17
(m, 13H, HAr), 5.21 (d, 1H, J = 14.5 Hz, CH2), 4.78 (d, 1H, J = 14.5 Hz, CH2), 2.31 (s, 3H, Me); 13C NMR
(125 MHz, DMSO-d6) 171.0, 169.5, 168.3, 139.7, 136.8, 136.3, 132.7, 132.0, 131.7, 130.6, 130.2, 130.1,
δ

Molecules 2020, 25, 2855
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128.7 (2
×
C), 128.4, 128.3 (2
×
C), 127.6, 127.0, 126.52, 126.47, 51.7, 26.4; IR (KBr): cm−1 3064, 3004, 2934,
1949, 1831, 1709, 1649, 1595, 1572, 1496, 1455, 1433, 1393, 1368, 1303, 1278, 1253, 1203, 1156, 1139, 1078,
1045, 1019; HRMS (ESI): m/z [M + H]+ calcd for C23H19N2O3: 371.13902, found: 371.13952.
3.1.7. 1,3,4,6-tetrahydrobenzo[b][1,4]diazocine-2,5-dione (6)
Following the procedure of Elgureo et al. [20] purified by column chromatography methanol:chloroform
1:9 v/v, yield 27%, white crystals, m.p. 244.0–245.0 ◦C, R.f. = 0.22 (methanol:chloroform 1:9 v/v).1H NMR
(500 MHz, DMSO-d6)
δ
9.55 (2, 2H, 2
×
NH); 7.34–7.27 (m, 2H, HAr), 7.22–7.15 (m, 2H, HAr), 2.33 (bs,
4H, 2
×
CH2); 13C NMR (125 MHz, DMSO-d6)
δ
172.2, 134.8, 127.4, 126.9, 30.5; IR (KBr): cm−1 3179,
3051, 2920, 1992, 1947, 1681, 1644, 1587, 1504, 1455, 1409, 1332, 1308, 1232, 1176, 1124, 1050, 1010; HRMS
(ESI): m/z [M + H]+ calcd for C23H19N2O3: 191.08150, found: 191.08141.
3.1.8. Synthesis of 9a
To a solution of tetraethyl benzene-1,2,4,5-tetracarboxylate (8.0 g, 21.9 mmol, 1.0 equiv.) and
phenylenediamine (4.7 g, 43.7 mmol, 2.0 equiv.) in anhydrous THF (100 mL), 60% sodium hydride
(3.7 g, 91.8 mmol, 4.2 equiv.) was added and the resulting mixture was refluxed overnight. Then the
mixture was poured into water (500 mL), acidified with concentrated HCl to pH 2. The obtained
precipitate, containing crude 9a, was filtered through a Schott funnel, washed several times with
ethyl acetate and crystallized from the mixture of DMSO:NMP 1:1. The filtrate was extracted with
ethyl acetate (3
×
100 mL), the combined organic phases containing tricyclic product 3k were washed
with brine (1 × 50 mL) and dried over magnesium sulfate. Evaporation of the solvent gave the crude
product which was purified by column chromatography (100% ethyl acetate), giving the final product
3k as a white crystalline solid.
9a: yield 59% (5.13 g), white crystals, m.p. 375.0–376.0 ◦C. 1H NMR (500 MHz, DMSO-d6)
(2 s, 4H, 4
NH), 7.30–7.16 (m, 8H, HAr), 7.12–7.00 (m, 2H, HAr); 13C NMR (125 MHz, DMSO-d6)
δ 10.16. 10.10
×
×
δ
, main conformer only: 168.5, 134.3, 133.7, 127.5, 126.9, 124.6; IR (KBr): cm−1 3465, 3189, 3059, 2886,
1669, 1592, 1552, 1503, 1415, 1371, 1302, 1229, 1154, 1113, 1038; HRMS (ESI): m/z [M + H]+ calcd for
C22H15N4O4: 399.10878, found: 399.10842.
diethyl 6,11-dioxo-5,6,11,12-tetrahydrodibenzo[b,f][1,4]diazocine-8,9-dicarboxylate (3k
white crystals, m.p. 242.0–243.0 ◦C, R.f. = 0.28 (hexane:ethyl acetate 2:8 v/v). 1H NMR (500 MHz,
DMSO-d6) 10.48 (s, 2H, 2 NH), 7.67 (s, 2H, HAr), 7.26–7.18 (m, 4H, HAr), 4.25 (q, 4H, J = 7.0 Hz,
Me); 13C NMR (125 MHz, DMSO-d6)
168.5, 165.5, 134.6, 134.4,
): yield 6% (0.5 g),
δ
×
2 × CH2), 1.24 (t, 6H, J = 7.0 Hz, 2
×
δ
133.1, 128.0, 127.31, 127.25, 61.9, 13.8; IR (KBr): cm−1 3195, 3062, 2984, 2937, 2901, 1736, 1718, 1665,
1592, 1561, 1501, 1444, 1391, 1363, 1291, 1168, 1144, 1110, 1015; HRMS (ESI): m/z [M + H]+ calcd for
C20H19N2O6: 383.12376, found: 383.12371.
3.1.9. General Procedure for Alkylation of 9a
To a solution of 9a (1 equiv.) in anhydrous DMSO (2 mL/mmol), 60% sodium hydride in mineral
oil (5 equiv.) was added. The resulting mixture was stirred for 30 min, then appropriate halide (6 eq.),
methyl iodide or benzyl bromide, was added. The reaction mixture was stirred for 18 h at room
temperature, then was poured into water and the precipitated product was filtered through a Schott
funnel. The obtained solids were washed with ethyl acetate, then with acetone and dried in air to give
pure products.
1
(
9b) yield 82%, white crystals, m.p. 400.0 ◦C, R.f. = 0.56 (methanol:chloroform 1:9 v/v). H NMR
(500 MHz, CF3COOD)
NMR (125 MHz, CF3COOD)
δ
7.42 (s, 2H, HAr), 7.37–7.05 (m, 8H, HAr), 3.46, 3.41 (2
×
s, 12H, 4
×
Me); 13
C
δ
, both conformers: 172.2, 141.0, 140.4, 136.3, 136.2, 133.1, 132.4, 128.8,
128.7, 127.8, 127.2; IR (KBr): cm−1 3464, 3040, 2936, 1649, 1594, 1553, 1503, 1448, 1418, 1383, 1310, 1272,
1177, 1122, 1045; HRMS (ESI): m/z [M + H]+ calcd for C26H23N4O4: 455.17138, found: 455.17135;

Molecules 2020, 25, 2855
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◦
(
9c) yield 90%, white crystals, m.p. 385.0–386.0 C, R.f. = 0.67 (hexane:ethyl acetate 2:8 v/v).
1H NMR (500 MHz, DMSO-d6)
7.45–7.30 (m, 12H, HAr), 7.28 (s, 2H, HAr), 7.27–7.20 (m, 4H, HAr),
7.17–7.01 (m, 12H, HAr), 4.62 (d, 4H, J = 15.0 Hz, 2 CH2), 3.96 (d, 4H, J = 15.0 Hz, 2
CH2); 13C NMR
(125 MHz, DMSO-d6) 166.7, 138.9, 136.8, 134.0, 129.5, 128.9, 128.4, 127.9, 127.7, 123.9, 51.2; IR (KBr):
δ
×
×
δ
cm−1 3290, 3084, 3062, 3029, 2935, 1955, 1665, 1651, 1598, 1584, 1495, 1455, 1431, 1399, 1362, 1324, 1298,
1261, 1224, 1201, 1157, 1148, 1119, 1079, 1053, 1030; HRMS (ESI): m/z [M + H]+ calcd for C50H38N4O4:
759.29658, found: 759.29776;
3.1.10. Rearrangement of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3a) to
2-(2-aminophenyl)isoindoline-1,3-dione (10)
To a suspension of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione (3a) (1.0 g, 4.2 mmol, 1 equiv.)
in DMSO, 60% (0.18 g, 4.6 mmol, 1.1 equiv.) sodium hydride in mineral oil was added and the resulting
mixture was stirred overnight at room temperature. The red clear solution which was formed was
poured into water and extracted with ethyl acetate (3
×
50 mL), the organic phase was washed with
brine (1 50 mL) and dried over anhydrous magnesium sulfate. The crude product was purified
×
by column chromatography (hexane:ethyl acetate 7:3 v/v), giving 10 as a yellow solid (0.58 g, 58%
yield). m.p. 195.0–196.0 ◦C, R.f. = 0.35 (hexane:ethyl acetate 6:4 v/v). H NMR (500 MHz, DMSO-d6)
δ
1
7.95–7.83 (m, 4H, HAr), 7.12 (t, 1H, J = 7.5 Hz, HAr), 7.01 (d, 1H, J = 7.5 Hz, HAr), 6.75 (d, 1H, J = 7.5 Hz,
HAr), 6.56 (t, 1H, J = 7.5 Hz, HAr), 6.36 (s, 2H, NH2); 13C NMR (125 MHz, DMSO-d6)
δ 167.6, 146.5,
134.1, 132.5, 130.0, 129.7, 123.1, 115.9, 115.32, 115.28; IR (KBr): cm−1 3458, 3380, 3030, 1783, 1708, 1623,
1578, 1502, 1461, 1377, 1314, 1271, 1216, 1159, 1143, 1100, 1083, 1049, 1026; HRMS (ESI): m/z [M + H]+
calcd for C14H11N2O2: 239.08150, found: 239.08171;
3.1.11. Synthesis of 4,5-dichloro-N1-methylbenzene-1,2-diamine (4e)
To a solution of 4,5-dichloro-2-nitroaniline (30.0 g, 0.14 mol, 1.0 equiv.) in DMF (150 mL),
60% sodium hydride in mineral oil (6.4 g, 0.16 mol, 1.1 equiv.) was added. After 30 min of stirring in
room temperature, methyl iodide (13.5 mL, 0.22 mol, 1.5 equiv.) was added to the reaction mixture
and the solution warmed up spontaneously. After 1h, the reaction mixture was poured into water
(500 mL) and the product was extracted with ethyl acetate (3
×
100 mL). The organic phase was
washed with brine (1 50 mL) and dried over anhydrous magnesium sulfate. The drying agent
×
was filtered off, the resulting filtrate was concentrated under reduced pressure and left in the fridge
for crystallization. The orange crystals of 4,5-dichloro-N-methyl-2-nitroaniline were formed, which
were washed with a small amount of ethyl acetate and dried. Yield: 25.0 g (78%). A slurry of
4,5-dichloro-N-methyl-2-nitroaniline (15.0 g, 0.07 mol, 1.0 equiv.) and iron powder (19.0 g, 0.34 mol,
5.0 equiv.) in a mixture of 95% ethanol (200 mL) and glacial acetic acid (100 mL) was stirred at 60 ◦C for
2h. The progress of reaction was monitored by TLC, when the reaction was completed; the excess of
volatiles was evaporated under reduced pressure. The obtained residue was treated with water and the
pH was adjusted to ca. 8 using sodium hydroxide. The crude product was extracted with ethyl acetate
(
3 × 150 mL), organic phase was washed with brine (1
×
50 mL) and dried over anhydrous magnesium
sulfate. Evaporation of the solvent resulted in the 4,5-dichloro-N1-methylbenzene-1,2-diamine (4e),
obtained as a brown solid (12.6 g, 97% yield) which was used in the next step without further
1
purification. M.p. 100.0–101.0 ◦C, R.f. = 0.35 (hexane:ethyl acetate 9:1 v/v). H NMR (500 MHz, CDCl3)
δ
6.72 (s, 1H, HAr), 6.62 (s, 1H, HAr), 3.30 (bs, 3H, NH2+NH), 2.81 (s, 3H, Me); 13C NMR (125 MHz,
CDCl3) δ
138.8, 132.8, 123.2, 120.3, 117.1, 112.0, 31.0; HRMS (ESI): m/z [M + H]+ calcd for C7H9Cl2N2:
191.01373, 193.01078, 175.00785, found: 191.01403, 193.01110, 175.00788;
3.1.12. Synthesis of N1-benzylbenzene-1,2-diamine (4f)
A solution of 1-fluoro-2-nitrobenzene (15.0 mL, 0.14 mol, 1.0 equiv.), benzylamine (17.0 mL,
0.15 mol, 1.1 equiv.) and TEA (25.0 mL, 0.18 mol, 1.3 equiv.) in DMSO (10 mL) was stirred at 100 ◦C for
18 h. After cooling down, the obtained orange solution was poured into water and the reaction product

Molecules 2020, 25, 2855
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was extracted with ethyl acetate (3
×
100 mL). The organic phase was washed with brine (1
×
50 mL)
and dried over anhydrous magnesium sulfate. Evaporation of the solvent gave N-benzyl-2-nitroaniline
as a red solid (29.0 g, 89.5% yield), which was used in the next step without further purification.
A slurry of N-benzyl-2-nitroaniline (10.0 g, 44.0 mmol, 1.0 equiv.) and iron powder (12.3 g, 220.0 mmol,
5.0 equiv.) in a mixture of 95% ethanol (100 mL) and glacial acetic acid (50 mL) was stirred at 60 ◦C for
2 h. The progress of reaction was monitored by TLC, when the reaction was completed, the excess of
volatiles was evaporated under reduced pressure. The obtained residue was treated with water, the pH
was adjusted to ca. 8 using sodium hydroxide and the reaction product was extracted with ethyl
acetate (3
×
150 mL). The organic phase was washed with brine (1
×
50 mL) and dried over anhydrous
magnesium sulfate. The drying agent was filtered off and the resulting filtrate was evaporated under
reduced pressure, giving N1-benzylbenzene-1,2-diamine (4f) as a brown solid (8.3 g, 96% yield) used
in the next step without further purification. M.p. 49.0–50.0 ◦C, R.f. = 0.33 (hexane:ethyl acetate 9:1
1
v/v). H NMR (500 MHz, CDCl3)
δ
7.48–7.43 (m, 2H, HAr), 7.43–7.37 (m, 2H, HAr), 7.36–7.31 (m, 1H,
HAr), 6.89-6.83 (m, 1H, HAr), 6.80-6.69 (m, 3H, HAr), 4.35 (s, 2H, CH2), 3.46 (bs, 3H, NH2 + NH); 13
C
NMR (125 MHz, CDCl3)
δ 139.4, 137.7, 134.2, 128.7, 127.9, 127.3, 120.8, 118.9, 116.6, 112.1, 48.7; HRMS
(ESI): m/z [M + H]+ calcd for C13H15N2: 199.12298, found: 199.12313;
3.1.13. General Procedure for Synthesis of Phthalic Acids Methyl Esters 5b–d
To a stirred slurry of appropriate phthalic acid (0.1 mol) in methanol (200 mL), concentrated
sulphuric acid (20 mL) was added dropwise (caution: exothermic!). The reaction mixture was stirred
for ten minutes in room temperature which led to the dissolution of phthalic acid. The obtained
solution was refluxed for 18 h, then the excess of methanol was evaporated under reduced pressure
and the remaining residue was poured into water. The pH was adjusted to ca. 8 by the addition of solid
sodium hydroxide and the reaction product was extracted with ethyl acetate (3
×
100 mL) The organic
phase was washed with brine (1 50 mL) and dried over anhydrous magnesium sulfate. The drying
×
agent was filtered off and the resulting filtrate was evaporated under reduced pressure. Evaporation of
the solvent gave the crude product which was used in next step without further purification.
dimethyl 4-nitrophthalate (5b): yield 76%, beige solid, m.p. 66.0–67.0 ◦C, R.f. = 0.20 (hexane:ethyl acetate
1
9:1 v/v). H NMR (500 MHz, CDCl3)
δ 8.58 (d, 1H, J = 2.5 Hz, HAr), 8.36 (dd, 1H, J = 2.5 Hz, J = 8.5 Hz,
HAr), 7.81 (d, 1H, J = 8.5 Hz, HAr), 3.94 (s, 3H, Me); 3.93 (s, 3H, Me); 13C NMR (125 MHz, CDCl3)
166.8, 165.5, 148.9, 138.2, 132.7, 130.1, 126.2, 124.5, 53.4; HRMS (ESI): m/z [M + H]+ calcd for C10H10NO6:
δ
240.05026, found: 240.05031;
dimethyl 4,5-dichlorophthalate (5c): yield 91%, pale oil, R.f. = 0.49 (hexane:ethyl acetate 9:1 v/v). 1H
NMR (500 MHz, CDCl3)
δ
7.77 (s, 2H, HAr), 3.88 (s, 6H, 2
×
Me); 13C NMR (125 MHz, CDCl3)
δ 166.0,
135.8, 131.4, 131.0, 53.1; HRMS (ESI): m/z [M + H]+ calcd for C10H9O4Cl2: 262.98724, 264.98429, found:
262.98744, 264.98445;
◦
dimethyl 3,4,5,6-tetrachlorophthalate (5d): yield 35%, white solid, m.p. 92.0–93.0 C, R.f. = 0.49
1
(hexane:ethyl acetate 9:1 v/v). H NMR (500 MHz, CDCl3)
δ
3.92 (s, 6H, 2
×
Me); 13C NMR (125 MHz,
CDCl3) δ
164.3, 136.2, 132.1, 130.7, 53.6; HRMS (ESI): m/z [M + H]+ calcd for C10H7O4Cl4: 330.90930,
332.90635, 334.90340, found: 330.90909, 332.90613, 334.90316.
3.1.14. Synthesis of dimethyl thiophene-3,4-dicarboxylate (5e)
To a slurry of tiophene-3,4-dicarboxylic acid (4.0 g, 23.3 mmol, 1.0 equiv.) and potassium carbonate
(8.1 g, 58.3 mmol, 2.5 equiv.) in DMF (20 mL), methyl iodide (3.2 mL, 51.1 mmol, 2.2 equiv.) was added
dropwise. The resulting mixture was stirred for 18 h at 40 ◦C, then poured into water (100 mL) and
the pH was adjusted to ca. 8 using sodium hydroxide. The reaction product was extracted with ethyl
acetate (3
×
50 mL), the organic phase was washed with brine (1
×
50 mL) and dried over anhydrous
magnesium sulfate. The drying agent was filtered off, the resulting filtrate was concentrated under
reduced pressure and left in the fridge for crystallization. Big, colorless crystals of 5e were formed

Molecules 2020, 25, 2855
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1
(1.83 g, 40% yield). M.p. 59.0–60.0 ◦C, R.f. = 0.25 (hexane:ethyl acetate 9:1 v/v). H NMR (500 MHz,
CDCl3)
δ
7.84 (s, 2H, HAr), 3.85 (s, 6H, 2
×
Me); 13C NMR (125 MHz, CDCl3)
δ 163.5, 133.2, 131.9, 52.42;
HRMS (ESI): m/z [M + H]+ calcd for C8H9O4S: 201.02161, found: 201.02177;
3.1.15. Synthesis of tetraethyl benzene-1,2,4,5-tetracarboxylate (8)
Following the procedure of Berthold et.al. [27] the slurry of pyromellitic dianhydride (25.0 g,
0.11 mol) in absolute ethanol (250 mL) was refluxed for 2 h, then sulfuric acid (15 mL) was added
dropwise. The reaction mixture was refluxed for an additional 18 h, then the excess ethanol was
evaporated. The residue was poured into water and the pH was adjusted to ca. 8 using sodium
hydroxide. The reaction product was extracted with ethyl acetate (3
×
100 mL), the organic phase was
washed with brine (1
×
50 mL) and dried over anhydrous magnesium sulfate. Evaporation of the
solvent gave
8
as an oily residue, which solidified upon storage (27.16 g, 64% yield). M.p. 54.0–55.0 ◦C,
1
R.f. = 0.28 (hexane:ethyl acetate 9:1 v/v). H NMR (500 MHz, CDCl3)
δ
8.02 (s, 2H, HAr), 4.36 (q, 8H,
J = 7.0 Hz, 4
×
CH2), 1.35 (t, 12H, J = 7.0 Hz, 4
×
Me); 13C NMR (125 MHz, CDCl3)
δ 166.1, 134.4, 129.6,
62.3, 14.1; HRMS (ESI): m/z [M + H]+ calcd for C18H23O8: 367.13674, found: 367.13834;
3.2. X-ray Data Collection and Data Refinement
Good quality single-crystals of 3a
experiments at T = 100(2) K. Diffraction data were collected on the Agilent Technologies (Santa Clara,
CA, USA) SuperNova Dual Source diffractometer with CuK radiation ( = 1.54184 Å) (3a 3g–j 9a 9c
and 10) and the Agilent Technologies SuperNova Single Source diffractometer with MoK radiation
λ = 0.71073 Å) ( ), using CrysAlis RED software [28]. The multi-scan empirical absorption correction
using spherical harmonics (3a 3i 3j 9c, and 10), the analytical numeric absorption correction
, 3g–j, 9a, 9c, and 10, were selected for the X-ray diffraction
α
λ
,
,
,
,
α
(
6
,
,
, 6,
using a multifaceted crystal model based on expressions derived by Clark and Reid [29] (3g and
9a) and numerical absorption correction based on gaussian integration over a multifaceted crystal
model (3h), implemented in SCALE3 ABSPACK scaling algorithm were applied [28]. The structural
determination procedure was carried out using the SHELX package [30]. The structures were solved
with direct methods and then successive least-square refinement was carried out based on the full-matrix
least-squares method on F2 using the SHELXL program [30]. All H-atoms linked to the N and O-atoms
were located on Fourier differential maps and refined with Uiso(H) = xUeq(N,O), where x = 1.2 for the
amine and 1.5 for the hydroxyl H-atoms, respectively. In all the cases, the N
−
H bonds were subject to
the DFIX 0.87 restraint. In the case of 3i, the length of the O H bond was restrained to 0.85 Å, and
−
the distance between the H-atoms of water was restrained to 1.39 Å. Other H-atoms were positioned
geometrically, with C–H equal to 0.93, 0.96 and 0.97 Å for the aromatic, methyl and methylene H-atoms,
respectively, and constrained to ride on their parent atoms with Uiso(H) = xUeq(C), where x = 1.2
for the aromatic and methylene H-atoms, and 1.5 for the methyl H-atoms, respectively. The 10 was
refined as two-component twin with a 0.502(2):0.498(2) domain ratio (second component was rotated
by the –179.98◦ around [100] (direct)). The molecular interactions in crystals have been identified using
the PLATON program [31]. The figures for this publication were prepared using Olex2 and Mercury
programs [32,33].
3.3. Cell Culturing
HeLa, U87, BICR18, EUFA30celllineswereculturedinDMEMmedium(LifeTechnology)supplemented
with 10% fetal bovine serum (Life Technology) and 0.1% antibiotics, penicillin and streptomycin
(Life Technology). Cells were grown in a humidified atmosphere of CO2/air (5/95%) at 37 ◦C.
3.4. Cytotoxicity Assay
Exponentially growing cells were seeded onto a 96-well plate at the density of 2
cultured for 18 h, and treated with newly synthesized derivatives at concentrations 1–300
DMSO as a control, for 24 or 48 h. Alamar Blue (Invitrogen) was added accordingly to manufacturer
×
103 cells/well,
M, or with
µ

Molecules 2020, 25, 2855
20 of 22
protocol. After 4 h, light emission at 590 nm was measured with excitation at 560 nm using a scanning
multiwell spectrophotometer (DTX 880, Beckman Coulter). The experiments were carried out at
least three times with three replicates for each inhibitor concentration. After background subtraction,
inhibition rate, IC50 was calculated as the concentration of the component inhibiting cell growth by
50%. All the calculations were performed using Origin 9.0 software.
3.5. Flow Cytometry
The Annexin V-FITC apoptosis detection kit (BD Biosciences) was used to detect apoptosis by
flow cytometry. Cells were seeded at Petri dishes, cultured for 18 h with tested agents applied for
24 h. DMSO was added as the non-treated control and camptothecin (CPT) as the positive control.
Subsequently, cells were washed with PBS, resuspended in binding buffer at a concentration of
2 × 106 cells/mL, and anti-Annexin V FITC-conjugated antibody and propidium iodide were added to
100
µL aliquots. The mixtures were incubated for 15 min at room temperature, supplemented with
binding buffer to 500
µL and processed by BD FACSCalibur (BD Biosciences). Data were analyzed in
Flowing Software version 2.5.1 (Flowing Software, http://www.uskonaskel.fi/flowingsoftware).
4. Conclusions
We have shown, that previously unknown, unsymmetrically N-substituted and N,N-disubstituted
5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones, bearing various substituents on the dilactam ring,
could be obtained in our novel, convenient protocol for the synthesis. The developed procedure
allows for the introduction of diverse modifications and side chains into the dilactam ring as well
as various functional groups attached to the benzene rings. The investigated synthesis was proved
useful by obtaining five dilactam-unsubstituted (3a–d, 3k) and twelve dilactam-substituted (3e–j,
3l–o) tricyclic dibenzodiazocine derivatives and one benzothienodiazocine (3i). We have shown that
the desired modifications of the dibenzodiazocine scaffold can be introduced both at the stage of
appropriate selection of substrates for synthesis and subsequent post-cyclisation modifications, using
the alkylation or acylation reactions of the resulting tricyclic system. Such compounds, due to their
structural similarities to tricyclic dibenzodiazepine drugs, could be treated as a privileged structures,
useful scaffolds for design and discovery of novel bioactive substances. The performed single-crystal
X-ray structural analysis revealed the structural features of newly synthesized compounds, which
supports design and development of new chemical materials or therapeutic agents with given or
assumed chemical or biological properties. The investigated method, also allowing the synthesis
of novel pentacyclic scaffold, consists of three benzene and two diazocine rings (9a–c), and led to
the observation of a new type of rearrangement, contraction of the diazocine ring to the isoindoline
ring (10). Although the synthesized compounds, tested on selected cancerous and non-cancerous cell
lines, exhibited a rather weak cytotoxic effect, our synthetic methods are useful to design compounds
with improved biological activity. Such studies on the biological applications of dibenzodiazocine
derivatives are ongoing in our laboratory and will be published in due course.
Supplementary Materials: 1H-NMR (3a–o
9a–c 10), dept135 (3a–o 4e 4f 5b–e
9a–c 10) and X-ray analysis data (3a
,
4e
9a–c
3j
,
4f
,
5b–e
10) IR (3a–o
9a 9c
,
6
,
8
,
9a–c
,
10), 13C-NMR (3a–o
9a–c 10), HRMS (3a–o
,
4e
,
4f
,
5b–e
,
6
,
8
6
,
,
,
,
,
,
,
6
,
8
,
,
,
6,
,
,
4e
,
4f 5b–e
,
,
8
,
,
,
3g
–
,
6
,
,
,
10) are available online. CCDC 1998020–1998028
contain the supplementary crystallographic data for this paper. These data can be obtained freely via http:
//www.ccdc.cam.ac.uk/data_request/cif, by e-mailing data_request@ccdc.cam.ac.uk or by contacting directly the
Cambridge Crystallographic Data Centre (12 Union Road, Cambridge CB2 1EZ, UK. Fax: +44 1223 336033).
Author Contributions: Design, conception and writing were performed by B.B., A.M., D.G., D.T.; biological
data analysis were collected and analyzed by D.G., E.G.; synthesis, purification and structure elucidation were
performed by B.B., A.M.; recording and analysis of NMR spectra was performed by M.K.D.; crystallographic
analysis was performed by D.T., K.W. All authors reviewed and approved the final version. All authors have read
and agreed to the published version of the manuscript.
Funding: This work was supported by the Polish Ministry of Science and Higher Education (MNiSW)
“DIAMENTOWY GRANT V” (decision number DI2015 007245; B.B.).

Molecules 2020, 25, 2855
21 of 22
Acknowledgments: This study was carried out at the Biological and Chemical Research Centre, University of
Warsaw, established within the project co-financed by European Union from the European Regional Development
Fund under the Operational Programme Innovative Economy, 2007 – 2013. The X-ray diffraction data collection
was accomplished at the Core Facility for Crystallographic and Biophysical research to support the development of
medicinal products. The “Core facility for crystallographic and biophysical research to support the development
of medicinal products” project is carried out within the TEAM-TECH Core Facility programme of the Foundation
for Polish Science co-financed by the European Union under the European Regional Development Fund. The
equipment used (A.M., B.B.) was sponsored in part by the Centre for Preclinical Research and Technology (CePT),
a project cosponsored by the European Regional Development Fund and Innovative Economy, The National
Cohesion Strategy of Poland. We thank Jacek Ole˛dzki for recording the ES-MS spectra.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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