Unsymmetrically substituted dibenzo[b,f][1,5]-diazocine-6,12(5H,11H)dione—A convenient scaffold for bioactive molecule design

Article abstract of DOI:10.3390/molecules25040906

A novel approach for the synthesis of unsymmetrically substituted dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones has been developed. This facile three-step method uses variously substituted 1H-benzo[d][1,3]oxazine-2,4-diones (isatoic anhydrides) and 2-aminobenzoic acids as a starting materials. The obtained products were further transformed into N-alkyl-, N-acetyl- and dithio analogues. Developed procedures allowed the synthesis of unsymmetrical dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones and three novel heterocyclic scaffolds: benzo[b]naphtho[2,3-f][1,5]diazocine-6,14(5H,13H)dione, pyrido[3,2-c][1,5]benzodiazocine-5,11(6H,12H)-dione and pyrazino[3,2-c][1,5]benzodiazocine-6,12(5H,11H)dione. For 11 of the compounds crystal structures were obtained. The preliminary cytotoxic effect against two cancer (HeLa, U87) and two normal lines (HEK293, EUFA30) as well as antibacterial activity were determined. The obtained dibenzo[b,f][1,5]diazocine(5H,11H)6,12-dione framework could serve as a privileged structure for the drug design and development.

Full text of DOI:10.3390/molecules25040906

molecules
Article
Unsymmetrically Substituted
Dibenzo[b,f][1,5]-diazocine-6,12(5H,11H)dione—A
Convenient Scaffold for Bioactive Molecule Design
Bartosz Bieszczad 1,
*
, Damian Garbicz 1, Damian Trzybin´ski 2, Damian Mielecki 1,
Krzysztof Woz´niak 2 , Elz˙bieta Grzesiuk 1 and Adam Mieczkowski 1,
*
1
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin´skiego 5a, 02-106 Warsaw,
Poland; dgarbicz@ibb.waw.pl (D.G.); damian@ibb.waw.pl (D.M.); elag@ibb.waw.pl (E.G.)
Biological and Chemical Research Centre, University of Warsaw, Zwirki i Wigury 101, 02-089 Warsaw,
2
˙
Poland; dtrzybinski@cnbc.uw.edu.pl (D.T.); kwozniak@chem.uw.edu.pl (K.W.)
Correspondence: b.bieszczad@ibb.waw.pl (B.B.); amiecz@ibb.waw.pl (A.M.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Andrea Penoni
Received: 3 February 2020; Accepted: 15 February 2020; Published: 18 February 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract:
A
novel approach for the synthesis of unsymmetrically substituted
This facile three-step
dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones has been developed.
method uses variously substituted 1H-benzo[d][1,3]oxazine-2,4-diones (isatoic anhydrides) and
2-aminobenzoic acids as a starting materials. The obtained products were further transformed
into N-alkyl-, N-acetyl- and dithio analogues. Developed procedures allowed the synthesis
of unsymmetrical dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones and three novel heterocyclic
scaffolds: benzo[b]naphtho[2,3-f][1,5]diazocine-6,14(5H,13H)dione, pyrido[3,2-c][1,5]benzodiazocine-
5,11(6H,12H)-dione and pyrazino[3,2-c][1,5]benzodiazocine-6,12(5H,11H)dione. For 11 of the
compounds crystal structures were obtained. The preliminary cytotoxic effect against two cancer
(HeLa, U87) and two normal lines (HEK293, EUFA30) as well as antibacterial activity were determined.
The obtained dibenzo[b,f][1,5]diazocine(5H,11H)6,12-dione framework could serve as a privileged
structure for the drug design and development.
Keywords: unsymmetrical dibenzo[b,f][1,5]diazocines; isatoic anhydrides; crystallographic analysis;
cytotoxicity; heterocycles
1. Introduction
Tricyclic dibenzodiazepines and their structural analogues (dibenzoazepines, dibenzothiazepines
and others) having two non-polar, aromatic benzene or heterocyclic rings separated by a
seven-membered ring containing heteroatoms such as sulfur, nitrogen or oxygen (1, Figure 1), are
very popular, privileged structures, useful for the development of drugs, as well as compounds with
various biological activities and applications (Figure 1). Modulation of biological properties is possible
by skillful chemical modifications, and variation of substituents and functional groups located in the
six-membered benzene rings and the seven-membered heterocyclic ring. This group of compounds
includes such important drugs as imipramine (
serotonin and norepinephrine reuptake inhibitor) [
and anticholinergic) [ ], quetiapine ( ; dibenzothiazepine; antipsychotic activity; dopamine, serotonin,
and adrenergic receptors antagonist) [ ], oxcarbazepine ( ; dibenzoazepine; anticonvulsant activity;
voltage-sensitive sodium channels blocker) [ ], and nevirapine ( ; dipyridodiazepine; anti-HIV;
non-nucleoside reverse transcriptase inhibitor) [ ]. Additionally, we recently reported [ ] the synthesis
2
; a dibenzoazepine with antidepressant activity and a
1
], clobenzepam ( ; dibenzodiazepine; antihistaminic
3
2
4
3
5
4,5
6
6
7
of structurally related, tricyclic pyrazinebenzodiazepines type
7. Surprisingly, these compounds
Molecules 2020, 25, 906; doi:10.3390/molecules25040906
www.mdpi.com/journal/molecules

Molecules 2020, 25, 906
2 of 20
exhibited a promising cytotoxic effect against two cancer cell lines: LoVo (human colon cancer) and
MV-4-11 (biphenotypic B myelomonocytic leukemia) [8].
Figure 1. Tricyclic 7-th membered dibenzoheterocycles and their analogues.
Tricyclic dibenzoheterocycles
rings are widely used in the design and search for new compounds of biological importance. On the
other hand, analogous tricyclic systems containing two non-polar, aromatic benzene rings separated by
a larger, eight-membered heterocyclic ring—tricyclic dibenzodiazocines and their structural analogs (
2–5, and their tricyclic analogues possessing pyridine 6 or piperidine
7
8
,
Figure 2) are not practically used in the design of biologically active compounds despite their high
potential. This situation is caused by the lack of appropriate synthetic routes leading to this type of
compounds. A rare example of the application of such heterocyclic scaffolds in the design of compounds
exhibiting biological activity involves the use of compound 9 (Figure 2) as a chemosensitizer, abolishing
the activity of a glycoprotein (GP 170) located in the cell membrane, and increasing the penetration of
the drug into the cell [9,10].
Figure 2. Tricyclic, 8-membered dibenzoheterocycles.
In our continuous research on the development of medicinally relevant compounds possessing
the dilactam structure [7,8], we recently focused on the dibenzo[b,f][1,5]diazocine scaffold 10 as a
possible framework for the design of biologically active substances. Our literature survey revealed that
symmetrical compounds type 10 (R1 = R2, R3 = R4) could be obtained in the dimerization reaction of
substituted 2-aminobenzoic acids (anthranilic acids) in the presence of phosphorus oxychloride [9] or
in the dimerization reaction of alkyl 2-aminobenzoates after treatment with sodium hydride [11]. These
methods, based on the dimerization of two moieties of 2-aminobenzoic acids or their esters, allow
for high-yield syntheses of symmetrical dibenzo[b,f][1,5]diazocines but are completely unsuitable for

Molecules 2020, 25, 906
3 of 20
synthesis of more complex and unsymmetrical compounds type 10, possessing various substituents
in the aromatic and dilactam rings (R1 R2, R3 R4). It is obvious that by limiting the ability
,
,
to modify the structure of a compound, one reduces its potential use in the design of biologically
active agents. This limits the modulation of biological activity, as well as physicochemical properties,
where the key to success is the skilful introduction and modification of substituents and side chains
attached to the heterocyclic scaffold. Compounds possessing the dibenzo[b,f][1,5]diazocine structure
10, could be also treated as small cyclic dipeptides, consisting of two aromatic
Consequently, synthetic efforts have been directed toward cyclisation of 2-(2-aminobenzamido)benzoic
acids 11 (Scheme 1) in the presence of classical peptide coupling reagents [11 12]. Unfortunately,
β-amino acid units.
,
as reported [12], and confirmed in our laboratory, this approach led exclusively to the formation of
yellow-coloured, bicyclic products possessing the 2-(2-aminophenyl)-4H-benzo[d][1,3]oxazin-4-one
structure 12. We also observed, and proved by the single-crystal X-ray diffraction analysis, that
crystallization of compound 12 (R1 = R2 = H) from water-methanol mixture, in the presence of
p-toluenesulphonic acid (1 equivalent), led to its hydrolysis to 2-(2-aminobenzamido)benzoic acid 11
(R1 = R2 = H), crystallized as a 4-toluenesulphonate salt 11*TsOH (for the crystallographic details see the
Supporting Information file). The application of some other cyclisation agents such as polyphosphate
ester (PPE) [12] or thionyl chloride [13] for cyclisation of primary amides also led to the product 12, while
treatment of the N-methyl amide with PPE resulted in the formation of the dibenzo[b,f][1,5]diazocine
scaffold [12]. These results suggest that the use of cyclisation agents such as carbodiimides, PPE or
thionyl chloride requires substitution or protection of amide group is necessary to avoid formation
of 12.
Scheme 1. DCC-mediated cyclisation of 2-(2-aminobenzamido)benzoic acid 11: Reagents and
Conditions: (a) DCC, DMF, rt, 18 h.
The only attempt toward the synthesis of unsymmetrical compounds type 10 (R1
, ,
R2, R3
R4) was reported in 2004 [13], where unprotected 2-aminobenzoic acids were initially treated with
thionyl chloride, and the obtained intermediates were coupled with a second molecule of unprotected,
differentially C-substituted 2-aminobenzoic acids. That short letter was only limited to the general
procedure of synthesis and physicochemical data for just one representative product containing the
dibenzo[b,f][1,5]diazocine structure. In contrast to the high yields of syntheses reported in the article,
in our hands this method led to the complex mixture of products, including benzo[d][1,3]oxazin-4-ones
12 and uncyclized 11. For this reason, we focused on the search for the alternative methods for
the synthesis of unsymmetrical dibenzo[b,f][1,5]diazocines 10 (R1
, ,
R2, R3 R4). In this paper, we
present a high-yielded, three-step procedure based on the synthesis of unsymmetrical and variously
substituted 2-(2-aminobenzamido)benzoic acids esters, followed by basic cyclisation in the presence of
sodium hydride.
2. Results and Discussion
2.1. Chemistry
In the present study, we developed two different synthetic approaches to the preparation of
asymmetrically substituted dibenzo[b,f][1,5]diazocine scaffold 10. The first method is based on
the application of variously N- and C- and N,C-disubstituted 1H-benzo[d][1,3]oxazine-2,4-diones

Molecules 2020, 25, 906
4 of 20
(isatoic anhydrides) 13a–i as coupling partners with variously N- and C-substituted, unprotected
2-aminobenzoic acids 14a–d (Scheme 2).
O
R3
R3
HN
O
R1
N
R1
O
R4
R4
a
+
O
O
N
R2
O
NH
R2
OH
OH
13a-i
15a-k
14a-d
b
R3
O
R3
O
R1
N
R1
N
c
R4
R4
O
N
NH
R2
O
R2
O
10a-k
16a-k
Scheme 2. First synthetic strategy to the dibenzo[b,f][1,5]diazocine scaffold. Reagents and Conditions:
(a) NaOH, H2O, 80 ◦C, 30 min; (b) H2SO4, MeOH, reflux, 72 h; (c) THFanh., 60% NaH, reflux, 18 h.
The previous literature reported one example of the application of 1H-benzo[d]-
[1,3]oxazine-2,4-diones for the synthesis of the dibenzo[b,f][1,5]diazocine scaffold 10, which
was limited to symmetrically substituted products [14].
We observed that unsubstituted
1H-benzo[d][1,3]oxazine-2,4-dione (13c), C-substituted: 6-chloro (13a), 6-bromo- (13b) and
6-nitro-1H-benzo[d][1,3]oxazine-2,4-dione (13d), N-substituted: 1-methyl- (13e), 1-benzyl- (13g
and 1-(4-bromobenzyl)-1H-benzo[d][1,3]oxazine-2,4-dione (13h), as well as C,N-disubstituted:
6-chloro-1-methyl- (13f) and 6-bromo-1-(naphthalen-1-ylmethyl)-1H-benzo[d][1,3]oxazine-2,4-dione
)
(13i) could be used as coupling partners with unsubstituted 2-aminobenzoic acid (14a), C-substituted:
4,5-dimethoxy-2-aminobenzoic acid (14b) and 3-amino-2-naphthoic acids (14d) and N-substituted
2-(methylamino)benzoic acid (14c) (Table 1). The coupling step requires the presence of base, one
equivalent of sodium hydroxide, and is performed in an aqueous solution at 80 ◦C [11]. The obtained
non-symmetrically substituted 2-(2-aminobenzamido)benzoic acids 15a–k were not isolated but
directly transformed into the appropriate methyl esters 16a–k, by refluxing 15a–k in methanol,
in the presence of concentrated sulfuric acid [11]. The final cyclisation step of crude methyl
2-(2-aminobenzamido)benzoates 16a–k was performed with sodium hydride in refluxing anhydrous
THF, resulting in dibenzo[b,f][1,5]diazocines 10a–k. In the case of compounds 10a–e
the yield of this three-step synthesis was in the range of 30–35%, giving quite high efficiency for a
single step synthesis (average yield for one synthesis step). In the case of compounds 10f 10g and 10i
, 10h and 10j
,
,
we observed slightly lower efficiency, in the 25–29% range, while compound 10k was obtained with
18% yield.

Molecules 2020, 25, 906
5 of 20
Table 1. Synthesized compounds.
Substrate 1
Substrate 2
Product
Substrate 1
Substrate 2
Product
O
O
N
O
CH3
13a
14a
14a
13e
10a
10b
14b
14d
10g
10h
O
Br
O
N
O
H
13b
13f
O
Br
O
N
H
O
13g
13h
13i
14a
14a
13b
10i
10j
10c
14b
14c
10d
13c
10e
10f
14c
10k
13b
13d
14c
14c
Unfortunately, the unsubstituted 2-aminonicotinic acid (18a) and 3-amino-2-pyrazinecarboxylic
acid (18b) failed to react with 1H-benzo[d][1,3]oxazine-2,4-diones, which forced us to
investigate an alternative method for coupling of 18a-b with 2-aminobenzoic acids. In the
second method, 2-aminonicotinic acid (18a) and 3-amino-2-pyrazinecarboxylic acid (18b) were
transformed in the first synthesis step into 2-amino-N-sulfinylnicotinoyl chloride (19a) and
3-amino-N-sulfinylpyrazine-2-carbonyl chloride (19b) after treatment with SOCl2 in boiling toluene
(Scheme 3) [13].

Molecules 2020, 25, 906
6 of 20
Scheme 3. Second synthetic strategy to obtain the tricyclic diazocine-5,11(6H,12H)-dione scaffold.
Reagents and Conditions: (a) SOCl2, toluene, reflux, 3 h; (b) toluene, rt, 48 h; (c) THFanh., 60% NaH,
reflux, 18 h.
The resulting crude intermediates 19a,b underwent reaction with 2-amino-5-chlorobenzoate (20
)
to form methyl 2-(2-aminonicotinamido)-5-chlorobenzoate (21a) and methyl 2-(2-aminopyrazine-
3-carboxamido)-5-chlorobenzoate (21b). Intermediates 21a,b were not isolated, but rather
directly treated with sodium hydride in refluxing anhydrous THF, which led to the
formation of 8-chloropyrido[3,2-c][1,5]benzodiazocine-5,11(6H,12H)-dione (10l), in 17% yield, and
8-chloropyrazino[3,2-c][1,5]benzodiazocine-6,12(5H,11H)-dione (10m), in 19% yield (total yield for the
three-step synthesis).
We also performed post-cyclisation modifications of the reported dibenzo[b,f][1,5]diazocines,
including alkylation, acylation and thiolation of 8-membered dilactam diazocine-6,12-dione ring
(Scheme 4). The treatment of 5-methyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10d) with
ethyl bromoacetate, in the presence of 60% sodium hydride in mineral oil, led to the formation
of ethyl 2-(11-methyl-6,12-dioxo-11,12-dihydrodibenzo[b,f][1,5]diazocin-5(6H)-yl)acetate (10n), in
78% yield. Heating of 5-methyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10d) in boiling acetic
anhydride for 3 h, resulted in the N-acyl derivative, 5-acetyl-11-methyldibenzo[b,f][1,5]diazocine-
6,12(5H,11H)-dione
(10o), in 89% yield.
Finally, heating of 8-bromo-2,3-dimethoxy-
dibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10c) with p-tolyl Davy-reagent in boiling anhydrous
THF for 18 h, led to the formation of the corresponding dithiolactam derivative,
8-bromo-2,3-dimethoxydibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dithione (10p), in 58% yield.
O
CH3
N
N
O
O
a)
from 10d
b)
O
R4
S
H
O
R1
R3
R2
N
N
Br
O
O
c)
from 10c
10n
HN
HN
O
S
O
CH3
N
10c R1=Br, R2=R3=OCH3, R4=H
10d R1=R2=R3=H, R4=CH3
from 10d
10p
N
O
O
CH3
10o
Scheme 4. Reagents and Conditions: (a) ethyl bromoacetate, 60% NaH, DMSO, rt, 18 h; (b) Ac2O,
reflux, 3 h; (c) p-tolyl Davy-reagent, THFanh., reflux, 18 h.
2.2. Crystallographic Analysis
Attempts to obtain single-crystals suitable for X-ray diffraction measurements of all synthesized
dibenzo[b,f][1,5]diazocine-6,12(5H,11H)-diones 10a–o were undertaken. However, they were successful

Molecules 2020, 25, 906
7 of 20
in only eight cases (10b
,
10g
,
10h
,
10i
,
10j
,
10l
,
10m and 10o) (Figure 3). The crystal structures of three
intermediates (11*TsOH, 13i and 14c) were also determined (Figures 1–3 (ESI)). Crystals appropriate
for diffractometric analysis were grown by slow evaporation from ethanol (10h 10j 10m), acetone
10b 10l), ethyl acetate (10g 13i), hexane:ethyl acetate (14c), cyclohexane:ethyl acetate (10o), and
,
,
(
,
,
water:methanol (11*TsOH) at room temperature.
Figure 3. Asymmetric unit of the crystal lattice of final products 10b, 10g–j, 10l, 10m, 10o with
crystallographic atom numbering. Displacement ellipsoids are drawn at the 50% probability level. The
H-atoms are shown as small spheres of arbitrary radius.

Molecules 2020, 25, 906
8 of 20
The investigated compounds crystallized in the monoclinic P21/c (11*TsOH, 13i, 14c, 10b, 10g, 10l,
and 10m) or C2/c (10i), orthorhombic P212121 (10h) or Pbca (10o) and triclinic P-1 (10j) space groups,
with one molecule of a compound in the asymmetric unit of the crystal lattice (Figure 4, Figures S1 and
S2). The asymmetric unit of 10b contains one molecule of solvent (acetone). The conformation of 10a-o
resembles a butterfly. The refinement parameters and details of the crystallographic data are presented
in Table S1 (ESI). The values of valence and torsion angles, together with bond lengths are presented in
Tables S2−S4 (ESI).
Figure 4. Flow cytometry analysis of HEK293, HeLa and U87 cells stained with Annexin-FITC (A)
and propidium iodide (PI). Cells were treated with 200
µM compounds for 24 or 48 h. Camptothecin
(10 µM) and H2O2 (100 µM) were used as experimental controls.

Molecules 2020, 25, 906
9 of 20
2.3. Cytotoxic and Antibacterial Effect of dibenzo[b,f][1,5]diazocine-6,12-diones 10a–p
We evaluated the cytotoxic efficacy of 16 synthesized dibenzo[b,f][1,5]diazocine-6,12-diones
10a–p on two normal (HEK293, EUFA30) and two cancerous (HeLa, U87) cell lines (concentrations
tested: 1–200
maintaining the selectivity of action—10b
(lowest—97.3 M) to several hundred (higher—205.7
µ
M; Table 2). Among the tested compounds, five showed a cytotoxic effect while
10f 10h 10j and 10p—their IC50 ranged from several dozen
M). Nevertheless, the IC50 for normal cell lines
,
,
,
µ
µ
was higher than for cancerous ones. For the majority of the remaining compounds, the IC50 values
were above 1 mM or could not be calculated.
Although the evaluated compounds showed a rather weak cytotoxic effect, we were
able to establish some relationships between the structure of the compounds and their
activity. 2-Bromodibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10b) showed a very weak cytotoxic
effect with some selectivity for the HeLa tumor cell line (IC50 = 97.3
derivative, 2-bromo-11-methyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10e), showed a weaker
cytotoxic effect than the parent compound (IC50 > 200 M for HeLa). The introduction of
µM). Its N-methyl
µ
an additional naphthalen-1-ylmethyl group resulted in the formation of 2-bromo-11-methyl-
5-(naphthalen-1-ylmethyl)dibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10k) showing noticeable
cytotoxicity on all cell lines tested (IC50 = 87.6
µM—EUFA, 119.0 µM—HEK293, 75.3 µM—HeLa,
75.4 M—U87). We observed that the presence of an additional, large and hydrophobic
µ
substituent attached to the dilactam ring was beneficial for enhancement of the cytoxic effect.
2-Chlorodibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10a) and 8-bromo-2,3-dimethoxydibenzo-
[b,f][1,5]diazocine-6,12(5H,11H)-dione (10c) showed no cytotoxic effect in the concentration
range tested. Structural modifications of 10a
:
introduction of another benzene ring into
the heterocyclic scaffold and a methyl group into the dilactam ring led to 2-chloro-5-methyl-
benzo[b]naphtho[2,3-f][1,5]diazocine-6,14(5H,13H)-dione (10h). These structural modifications
resulted in the enhancement of the cytotoxicity of 10h which exhibited cytotoxic effect
for all tested cell lines (IC50 = 170.5
148.6 M—U87). Finally, the conversion of 10c into its dithiolactam analogue: 8-bromo-2,3-dimethoxy-
dibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dithione (10p) caused the appearance of a cytotoxic effect
on cancer cell lines (IC50 > 200 M—EUFA, > 200 M—HEK293, 115.2 M—HeLa, 170.2 M—U87)
µM—EUFA, > 200 µM—HEK293, 107.4 µM—HeLa,
µ
µ
µ
µ
µ
suggesting that such structural modification is beneficial for increasing the cytotoxic effect of
these compounds.
To measure the induction of apoptosis, cells were treated with 200 µM of 10f, 10h, 10j, 10k and
10p compound, for 24 and 48 h (Figure 4). The experiment was performed on one normal (HEK293)
and two cancer (HeLa, U87) cell lines. After 24 h of treatment, the HEK293 cells showed significant
increase in necrosis and late apoptosis, as follows: 20% and 0.4% in control cells, 39% and 0.5% for 10f
,
32% and 3% for 10h, 22% and 10% for 10j. After 48 h of treatment, a slight increase in early and late
apoptosis was observed, namely, 6.6% and 3% for 10f, 3.8% and 3.2% for 10h, 4% and 4.4% for 10j, in
comparison to 1.3% and 0.5% in control cells.
The HeLa cells were the most sensitive to compound 10p, with a significant increase only in
necrotic phase. After 24 h, the percentage of necrotic cells was 34.5% in comparison to 21.1% in the
non-treated control, and 53.4% in comparison to 22.1% in control cells after 48 h treatment. The U87
cells were sensitive to three out of five compounds tested. In the control, after 24 h, there were 1.2%
of cells in early apoptosis, 1.6% in late apoptosis, and 8.6% in necrosis. For 10h treatment, cells were
at 4.1% in early apoptosis and 6.9% in late apoptosis; for 10j at 5.2% in late apoptosis and 17.8% in
necrosis; and for 10p at 14% in necrosis. After 48 h, we observed a significant increase in necrotic phase
only, as follows: in control to 17%, for 10h to 36.2%, for 10j to 28.1%, and for 10p to 46.6%.

Molecules 2020, 25, 906
10 of 20
Table 2. IC50
(µM) of dibenzo[b,f][1,5]diazocine-6,12-diones 10a–p based on the survival of non-cancerous (HEK293, EUFA30) and cancerous (U87, HeLa) cells after 24
and 48 h of treatment. “*”-Proliferation inhibition at 200 µM, the highest concentration used.
Cell Types
Compound
Eufa30
HEK293
HeLa
U87
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
[20.7]* ± 2.4
[27.0]* ± 15.7
[41.1]* ± 8.3
[17.0]* ± 27.7
[9.8]* ± 6.2
[7.7]* ± 6.4
[41.5]* ± 1.4
170.5 ± 21.1
[20.3]* ± 1.8
[36.4]* ± 11.4
87.6 ± 8.1
[17.7]* ± 11.2
[21.8]* ± 2.3
[0.0]*
[19.2]* ± 2.5
[18.5]* ± 7.2
[14.9]* ± 5.6
[12.9]* ± 8.2
[24.3]* ± 0.4
[4.7]* ± 3.1
[9.7]* ± 3.3
[37.0]* ± 18.9
[24.7]* ± 5.8
[30.8] ± 5.4
119.0 ± 7.1
[12.9]* ± 2.1
[7.9]* ± 4.0
[0.0]*
[9.7]* ± 5.2
[18.2]* ± 4.5
[9.3]* ± 5.5
[0.0]*
[12.2]* ± 0.1
97.3 ± 13.7
[0.7]* ± 0.1
[12.7]* ± 3.6
[0.8]* ± 2.5
[9.2]* ± 4.8
[0.9]* ± 2.4
[5.2]* ± 1.9
[6.1]* ± 2.0
107.6 ± 8.6
[5.3]* ± 2.3
202.6 ± 4.0
123.9 ± 5.4
[0.0]*
[18.9]* ± 9.1
[27.0]* ± 7.7
[20.8]* ± 4.2
[11.5]* ± 3.4
[24.4]* ± 3.1
[30.9]* ± 0.3
[22.6]* ± 3.1
148.6 ± 9.0
[27.3]* ± 6.4
[41.8]* ± 10.8
75.4 ± 8.7
[7.2]* ± 0.6
[15.9]* ± 4.5
[0.0]*
[2.5]* ± 1.4
[9.4]* ± 2.4
[3.6]* ± 2.9
[0.0]*
181.9 ± 5.6
[5.5]* ± 0.8
[42.4]* ± 5.7
197.4 ± 9.4
[4.9]* ± 2.2
[9.9]* ± 2.9
[0.0]*
[31.4]* ± 10.0
[34.3]* ± 8.3
[30.7]* ± 9.0
205.7 ± 12.8
[21.0]* ± 4.4
107.4 ± 10.0
[31.8]* ± 5.2
137.5 ± 14.1
75.3 ± 1.6
[16.5]* ± 3.0
[12.2]* ± 2.7
[13.2]* ± 4.1
[5.4]* ± 1.3
199.8 ± 13.9
[20.1]* ± 5.5
[38.2]* ± 2.7
74.4 ± 6.7
[11.2]* ± 1.7
[0.7]* ± 4.1
[7.2]* ± 4.2
[33.7]* ± 10.0
[25.2]* ± 2.2
192.0 ± 12.2
138.3 ± 12.0
[5.9]* ± 2.4
[5.6]* ± 2.0
[0.0]*
[12.8]* ± 3.5
[24.0]* ± 10.1
[8.2] ± 1.2
[9.9]* ± 2.5
[6.3]* ± 4.8
[0.0]*
[23.2]* ± 10.3
[39.1]* ± 5.2
[3.1]* ± 2.2
[32.9]* ± 3.3
115.2 ± 4.5
[24.2]* ± 2.8
[20.5]* ± 4.2
[9.9]* ± 0.8
[23.9]* ± 4.8
172.0 ± 10.0
[5.0]* ± 2.3
[0.0]*
[23.3]* ± 9.5
[33.4]* ± 2.1
[25.2]* ± 5.7
[18.3]* ± 7.3
[12.9]* ± 7.3
[5.9]* ± 1.5
203.9 ± 6.5
[0.0]*
[3.8]* ± 0.6
157.6 ± 9.6
202 ± 7.9
144.7 ± 8.5

Molecules 2020, 25, 906
11 of 20
Although compound 10k showed cytotoxicity to all tested human cell lines, we did not observe
any changes in the apoptosis/necrosis phases in relation to control. It is possible that the compound
shows not cytotoxic but cytostatic activity.
Many different natural products possessing a lactam ring (such as
or dilactam ring (diketopiperazines [16]) exhibit a strong antimicrobial effect. For this reason, we
evaluated compounds 10a for their antibacterial activity. Antibacterial studies were carried out
β-lactam antibiotics [15])
–
o
against two Gram-positive (Bacillus megaterium, Staphylococcus aureus RN4220) and six Gram-negative
bacterial strains (Escherichia coli AB1157, Pseudomonas putida KT2440, Shewanella oneidensis MR-1,
Salmonella typhimurium TA98, Salmonella typhimurium TA100, Pseudomonas aeruginosa PAO1). The results
showed none of the synthesized compounds exhibited antibacterial properties at the concentrations
of 12.5, 33.3, 50 and 100 µM. Even though the obtained products exhibited rather weak cytotoxic
and no antibacterial activity in the tested range of concentrations, further research with the use of
dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones as privileged structures useful in the design of bioactive
compounds is underway in our laboratory and will be published in due course.
3. Materials and Methods
3.1. Chemistry
3.1.1. General Information
Commercially available chemicals were of reagent grade and used as received. Purification of the
compounds was performed by column chromatography on silica gel 60 M (0.040–0.063 mm, E. Merck,
Darmstadt, Germany). Thin layer chromatography (TLC), using silica gel plates (Kieselgel 60F254, E.
Merck), was used to monitor reaction progress. A B-540 Melting Point apparatus (Büchi, New Castle,
DE, USA) was used to measure melting points. The 1H-NMR and 13C-NMR spectra, in DMSO-d6 and
CDCl3, were recorded at the Department of Chemistry, University of Warsaw, using an AVANCE III
HD 300 MHz spectrometer (Bruker, Billerica, MA, USA); shift values in parts per million are relative to
the SiMe4 internal reference. The resonance assignments were based on peak multiplicity and peak
integration of recorded spectra, Multiplets were assigned as bs (broad singlet), s (singlet), d (doublet),
t (triplet) dd (doublet of doublet), ddd (doublet of doublet of doublet) and m (multiplet). An LTQ
Orbitrap Velos instrument (Thermo Scientific, Waltham, MA, USA) located at the Mass Spectrometry
Laboratory of the Institute of Biochemistry and Biophysics PAS (Warsaw, Poland) was used to record
high resolution mass spectra. A 6200 FT/IR spectrometer (Jasco, Easton, MD, USA) at the Laboratory
of Optical Spectroscopy (Institute of Organic Chemistry PAS, Warsaw, Poland) was used to record
IR spectra.
3.1.2. General Procedure for the Synthesis of 2-(2-aminobenzamido)benzoic acids 15a–k
A suspension of 1H-benzo[d][1,3]oxazine-2,4-dione 13a–i (1 equiv.), 2-aminobenzoic acid 14a–d
(1 equiv.) and sodium hydroxide (1 equiv.) in water (10 mL/mmol) was heated at 80 ◦C for 30 min
until the evolution of carbon dioxide had ceased and a clear solution had formed. After cooling
the reaction mixture, the obtained solution was diluted with water, and the crude product was
precipitated by addition of glacial acetic acid, filtered and dried under vacuum. In cases of N-alkyl
1H-benzo[d][1,3]oxazine-2,4-diones where a gummy-like residue was formed, the crude product was
extracted with ethyl acetate, the organic phase was washed with brine and dried over anhydrous
magnesium sulfate. Evaporation of the solvent left a glass-like residue which was used in next step
without further purification.
3.1.3. General Procedure for the Synthesis of 2-(2-aminobenzamido)benzoic acids methyl esters 16a
2-(2-Aminobenzamido)benzoic acids 15a were dissolved in methanol (ca. 10 mL/mmol) then,
concentrated sulfuric acid was added (0.5 mL/mmol) (caution: exothermic). The resulted solution was
–k
–k

Molecules 2020, 25, 906
12 of 20
refluxed for 72 h. The excess of methanol was evaporated and the resulting residue was added to
water. The pH was adjusted to 8 by addition of NaOH and the crude products were extracted with
ethyl acetate. The combined organic layers were washed with 1 N sodium hydroxide, water and brine,
and then dried over anhydrous magnesium sulfate. Evaporation of solvent yielded a dark residue of
crude methyl esters 16a–k, which were used in the next step without further purification.
3.1.4. General Procedure for the Synthesis of dibenzo[b,f][1,5]diazocine-6,12(5H,11H)-diones 10a–k
from 2-(2-aminobenzamido)benzoic acids methyl esters 16a–k
2-(2-Aminobenzamido)benzoic acids methyl esters 16a–k were dissolved in the anhydrous THF
(20 mL/mmol) then 60% sodium hydride in mineral oil (2 equiv.) was added and the resulted solution
was refluxed for 18 h. The excess of THF was evaporated, the obtained residue was poured into 1 N
HCl and the crude product was extracted with ethyl acetate. The combined organic layers were washed
with 1 N HCl, water and brine, then dried over anhydrous magnesium sulfate. Crude products were
purified by column chromatography using hexane/EtOAc 1:1 then 2:8 v/v as eluent.
2-Chlorodibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10a): Yield 33%, colourless crystals, m.p.
◦
275–276 C, R.f. = 0.40 (hexane:ethyl acetate 2:8 v/v). 1H-NMR (DMSO-d6)
δ
10.33 (s, 1H, NH),
10.25 (s, 1H, NH), 7.45–7.21 (m, 5H, HAr), 7.11 (d, 1H, J = 2.4 Hz, HAr), 7.09 (d, 1H, J = 1.8 Hz, HAr);
13C-NMR (DMSO-d6)
δ 169.1, 167.6, 135.3, 134.4, 133.8, 133.3, 131.4, 130.7, 130.4, 128.2, 127.7, 127.6,
127.5, 125.9; IR (KBr): cm−1 3183, 3055, 2900, 1658, 1601, 1578, 1484, 1415, 1366, 1261, 1229, 1145, 1111;
HRMS (ESI): m/z [M+H]+ calcd for C14H10ClN2O2: 273.04253, 275.03958, found: 273.04202, 275.03904;
2-Bromod◦ibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10b): Yield 35%, colourless crystals, m.p.
283–284 C, R.f. = 0.43 (hexane:ethyl acetate 2:8 v/v). 1H-NMR (DMSO-d6)
δ 10.33 (s, 1H, NH),
10.24 (s, 1H, NH), 7.53 (dd, 1H, J = 2.4, 8.4 Hz, HAr), 7.49 (d, 1H, J = 2.4 Hz, HAr), 7.42–7.21 (m, 3H, HAr),
7.10 (d, 1H, J = 7.8 Hz, HAr), 7.03 (d, 1H, J = 8.4 Hz, HAr); 13C-NMR (DMSO-d6)
δ 169.0, 167.5, 135.5,
134.4, 134.2, 133.3, 133.2, 130.7, 130.6, 128.2, 127.8, 127.5, 125.9, 119.6; IR (KBr): cm−1 3182, 3056, 2900,
1657, 1602, 1480, 1411, 1362, 1260, 1229, 1142, 1100; HRMS (ESI): m/z [M+H]+ calcd for C14H10BrN2O2:
316.99202, 318.98997, found: 316.99140, 318.98930;
8-Bromo-2,3-dimethoxydibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10c): Yield 33%, colourless crystals,
1
m.p. 195–196 ◦C, R.f. = 0.28 (ethyl acetate). H-NMR (DMSO-d6)
δ
10.099 (s, 1H, NH), 10.057 (s, 1H,
NH), 7.54 (dd, 1H, J = 2.4, 8.4 Hz, HAr), 7.46 (d, 1H, J = 2.4 Hz, HAr), 7.01 (d, 1H, J = 8.4 Hz, HAr), 6.81
(s, 1H, HAr), 6.65 (s, 1H, HAr), 3.72 (s, 3H, OCH3), 3.70 (s, 3H, OCH3); 13C-NMR (DMSO-d6)
δ
169.0,
167.6, 150.0, 147.7, 135.6, 134.6, 133.2, 130.6, 127.85, 127.77, 124.9, 119.4, 110.4, 109.1, 55.70, 55.66; IR
(KBr): cm−1 3499, 3185, 3064, 2934, 2848, 1678, 1646, 1607, 1517, 1470, 1401, 1342, 1264, 1224, 1176, 1108,
1078, 1027; HRMS (ESI): m/z [M+H]+ calcd for C16H14BrN2O4: 377.01315, 379.01110, found: 377.01246,
379.01037;
5-Methyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10d): Yield 31%, colourless crystals, m.p.
◦
258–259 C, R.f. = 0.36 (hexane:ethyl acetate 2:8 v/v). 1H-NMR (DMSO-d6)
δ
10.25 (s, 1H, NH),
7.45–7.15 (m, 7H, HAr), 7.07–6.98 (m, 1H, HAr), 3.33 (s, 1H, CH3, partially overlapped with H2O signal);
13C-NMR (DMSO-d6)
δ 168.9, 167.4, 140.1, 134.7, 134.4, 133.3, 130.9, 130.2, 128.2, 127.9, 127.5, 127.1,
125.7, 125.4, 36.5; IR (KBr): cm−1 3257, 3067, 2979, 2934, 1991, 1938, 1836, 1673, 1623, 1599, 1575, 1470,
1415, 1386, 1351, 1302, 1260, 1226, 1185, 1161, 1141, 1082, 1034; HRMS (ESI): m/z [M+H]+ calcd for
C15H13N2O2: 253.09715, found: 253.09682;
2-Bromo-11-methyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10e): Yield 30%, colourless crystals, m.p.
1
247–248 ◦C, R.f. = 0.46 (hexane:ethyl acetate 2:8 v/v). H-NMR (DMSO-d6)
δ
10.30 (s, 1H, NH), 7.53–7.25
(m, 6H, HAr), 7.00 (d, 1H, J = 8.4 Hz, HAr), 3.32 (s, 1H, CH3, partially overlapped with H2O signal);
13C-NMR (DMSO-d6)
186.7, 165.8, 139.7, 136.3, 134.2, 133.1, 132.9, 131.2, 130.3, 128.5, 127.6. 127.5,
125.8, 119.4, 36.5; IR (KBr): cm−1 3172, 3087, 2881, 1681, 1635, 1597, 1473, 1427, 1399, 1370, 1345, 1300,
δ

Molecules 2020, 25, 906
13 of 20
1274, 1240, 1180, 1135, 1079, 1038, 1014; HRMS (ESI): m/z [M+H]+ calcd for C15H12BrN2O2: 331.00767,
333.00562, found: 331.00718, 333.00503;
11-Methyl-2-nitrodibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10f): Yield 25%, colourless crystals, m.p.
1
225–226 ◦C, R.f. = 0.46 (hexane:ethyl acetate 2:8 v/v). H-NMR (DMSO-d6)
δ
10.74 (s, 1H, NH), 8.18-8.09
(m, 2H, HAr), 7.50–7.41 (m, 2H, HAr), 7.36–7.26 (m, 3H, HAr), 3.37 (s, 1H, CH3, overlapped with H2O
signal); 13C-NMR (DMSO-d6)
δ 168.6, 165.3, 145.5, 140.5, 139.6, 134.9, 132.4, 131.4, 128.7, 127.8, 126.4,
125.9, 125.3, 123.6, 36.6; IR (KBr): cm−1 3177, 3056, 2907, 1946, 1712, 1676, 1628, 1528, 1485, 1335, 1260,
1239, 1178, 1137, 1089; HRMS (ESI): m/z [M+H]+ calcd for C15H12N3O4: 298.08223, found: 298.08160;
2,3-Dimethoxy-11-methyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10g): Yield 29%, colourless crystals,
1
m.p. 271–272 ◦C, R.f. = 0.23 (ethyl acetate). H-NMR (, DMSO-d6)
δ 9.97 (s, 1H, NH), 7.46–7.37 (m,
2H, HAr), 7.37–7.27 (m, 3H, HAr), 6.77 (s, 1H, HAr), 6.59 (s, 1H, HAr), 3.68 (s, 3H, OCH3), 3.67 (s, 3H,
OCH3), 3.30 (s, 1H, CH3); 13C-NMR (DMSO-d6)
δ 168.9, 167.3, 149.6, 147.4, 140.5, 133.3, 130.9, 128.1,
129.0, 127.6, 126.1, 125.6, 110.2, 108.7, 55.65, 55.62, 36.6; IR (KBr): cm−1 3193, 3000, 2936, 2841, 1674,
1626, 1513, 1478, 1454, 1431, 1398, 1364, 1302, 1258, 1223, 1156, 1133, 1105, 1082, 1036, 1021; HRMS (ESI):
m/z [M+H]+ calcd for C17H17N2O4: 313.11828, found: 313.11760;
2-Chloro-5-methylbenzo[b]naphtho[2,3-f][1,5]diazocine-6,14(5H,13H)-dione (10h): Yield 34%, beige crystals,
1
m.p. 270–271 ◦C, R.f. = 0.56 (hexane:ethyl acetate 2:8 v/v). H-NMR (DMSO-d6)
δ
10.59 (s, 1H, NH),
7.98–7.82 (m, 3H, HAr), 7.62 (s, 1H, HAr), 7.58–7.36 (m, 5H, HAr), 3.37 (s, 1H, CH3); 13C-NMR (DMSO-d6)
δ
167.6, 167.3, 138.6, 135.6, 133.4, 133.0, 132.6, 131.8, 131.1, 131.0, 128.1, 128.0, 127.78, 127.76, 127.5,
127.1, 126.9, 123.6, 36.5; IR (KBr): cm−1 3157, 3117, 3052, 2965, 2924, 2887, 1939, 1913, 1839, 1768, 1670,
1630, 1594, 1475, 1420, 1351, 1302, 1274, 1228, 1164, 1129, 1106; HRMS (ESI): m/z [M+H]+ calcd for
C19H14ClN2O2: 337.07383, 339.07088, found: 337.07313, 339.07024;
5-Benzyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10i): Yield 29%, beige crystals, m.p. 212–213 ◦C,
1
R.f. = 0.60 (hexane:ethyl acetate). H-NMR (DMSO-d6)
δ
10.26 (s, 1H, NH), 7.37–7.18 (m, 12H, HAr),
7.05–7.00 (m, 1H, HAr), 5.32 (d, 1H, J = 15.0 Hz, CH2), 4.81 (d, 1H, J = 15.0 Hz, CH2); 13C-NMR
(DMSO-d6)
δ 168.9, 167.8, 138.9, 136.7, 134.8, 134.3, 133.9, 130.8, 130.3, 128.3, 128.2, 127.9, 127.84, 127.76,
127.3, 127.1, 125.9, 125.4, 52.4; IR (KBr): cm−1 3166, 3037, 2952, 2898, 1969, 1829, 1735, 1664, 1638,
1598, 1486, 1454, 1402, 1379, 1301, 1240, 1153, 1104, 1076, 1042; HRMS (ESI): m/z [M+H]+ calcd for
C21H17N2O2: 329.12845, found: 329.12792;
5-(4-Bromobenzyl)dibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10j): Yield 30%, colourless crystals, m.p.
1
222–223 ◦C, R.f. = 0.66 (hexane:ethyl acetate). H-NMR (DMSO-d6)
δ 10.24 (s, 1H, NH), 7.51–7.15 (m,
12H, HAr), 7.06–6.98 (m, 1H, HAr), 5.35 (d, 1H, J = 15.0 Hz, CH2), 4.72 (d, 1H, J = 15.0 Hz, CH2); 13C-
NMR (DMSO-d6)
δ 168.6, 167.7, 138.6, 136.1, 134.8, 134.2, 133.9, 131.2, 130.9, 130.35, 130.32, 128.4, 127.8,
127.7, 127.1, 126.0, 125.4, 120.5, 51.7; IR (KBr): cm−1 3219, 3059, 2939, 1901, 1672, 1633, 1598, 1488, 1467,
1395, 1353, 1307, 1285, 1258, 1217, 1158, 1103, 1068, 1024, 1009; HRMS (ESI): m/z [M+H]+ calcd for
C21H16BrN2O2: 407.03897, 409.03692, found: 407.03841, 409.03626;
2-Bromo-11-methyl-5-(naphthalen-1-ylmethyl)dibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10k): Yield
1
18%, colourless crystals, m.p. 255–256 ◦C, R.f. = 0.40 (hexane:ethyl acetate 7:3 v/v). H-NMR (DMSO-d6)
δ
8.18–8.06 (m, 1H, HAr), 7.98–7.88 (m, 1H, HAr), 7.82 (d, 1H, J = 8.1 Hz, HAr), 7.60–7.22 (m, 9H, HAr),
7.16–7.07 (m, 2H, HAr), 6.16 (d, 1H, J = 15.0 Hz, CH2), 4.98 (d, 1H, J = 15.0 Hz, CH2), 3.05 (s, 1H,
CH3); 13C-NMR (DMSO-d6)
166.8, 164.5, 139.8, 137.3, 136.6, 133.7, 133.4, 133.3, 131.4, 131.3, 131.0,
δ
130.0, 128.6, 128.5, 128.4, 128.3, 127.6, 127.4, 126.3, 125.8, 125.7, 125.0, 123.3, 120.5, 48.8, 35.8; IR (KBr):
cm−1 3039, 3037, 3006, 2978, 2930, 2904, 2875, 1731, 1663, 1641, 1597, 1510, 1469, 1452, 1422, 1409, 1355,
1285, 1259, 1209, 1175, 1155, 1123, 1082, 1042, 1016; HRMS (ESI): m/z [M+H]+ calcd for C26H20BrN2O2:
471.07027, 473.06819, found: 471.07027, 473.06822;

Molecules 2020, 25, 906
14 of 20
3.1.5. General Procedure for the Synthesis of 8-chloropyrido[3,2-c][1,5] benzodiazocine-5,11-
(6H,12H)-dione (10l) and 8-chloropyrazino[3,2-c][1,5]benzodiazocine- 6,12(5H,11H)-dione (10m)
Synthesis of 2-amino-N-sulfinylnicotinoyl chloride (19a) and 3-amino-N-sulfinyl-pyrazine-2-carbonyl
chloride (19b).
2-Aminonicotinic acid (18a) or 3-amino-2-pyrazinecarboxylic acid (18b) was suspended in dry
toluene (10 mL/mmol), then thionyl chloride (5 equiv.) was added dropwise. The obtained slurry was
refluxed for 3 h, until clear solution was formed. The excess of solvent was evaporated, and the residue
was co-evaporated with toluene to remove traces of thionyl chloride. Crude products 19a,b were used
in the next step without further purification.
Synthesis of methyl 2-(2-aminonicotinamido)-5-chlorobenzoate (21a) and methyl 2-(2-aminopyrazine-
3-carboxamido)-5-chlorobenzoate (21b).
2-Amino-N-sulfinylnicotinoyl chloride (19a) or 3-amino-N-sulfinylpyrazine-2-carbonyl chloride
(19b) (1 equiv.) was dissolved in dry toluene (10 mL/mmol), then the solution of methyl
2-amino-5-chlorobenzoate (20) (1 equiv.) in toluene (10 mL/mmol) was added dropwise. The
mixture was stirred at ambient temperature for 48 h, then the solvent was evaporated and the obtained
residue was dissolved in ethyl acetate. The organic phase was washed two times with 1 N NaOH,
water and brine, then dried over anhydrous magnesium sulfate. Evaporation of the solvent resulted in
crude methyl esters 21a-b which were used in next step without further purification.
Cyclisation of methyl 2-(2-aminonicotinamido)-5-chlorobenzoate (21a) and methyl 2-(2-aminopyrazine-
3-carboxamido)-5-chlorobenzoate (21b).
Methyl 2-(2-aminonicotinamido)-5-chlorobenzoate (21a, 1 equiv.) and methyl 2-(2-aminopyrazine-
3-carboxamido)-5-chlorobenzoate (21b, 1 equiv.) were dissolved in anhydrous THF (20 mL/mmol)
then 60% sodium hydride in mineral oil (2 equiv.) was added. The resulting solution was refluxed for
18 h. The excess of THF was evaporated, residue was poured into 1 N HCl and the crude product
was extracted with ethyl acetate. The combined organic layers were washed with 1 N HCl, water and
brine, then dried with anhydrous magnesium sulfate. Crude products 10l,m were purified by column
chromatography using hexane/EtOAc 1:1 then 2:8 v/v as eluent.
8-Chloropyrido[3,2-c][1,5]benzodiazocine-5,11(6H,12H)-dione (10l): Yield 17%, colourless crystals, m.p.
1
308-309 ◦C, R.f. = 0.26 (hexane:ethyl acetate). H-NMR (DMSO-d6)
δ
10.84 (s, 1H, NH), 10.46 (s, 1H,
NH), 8.46 (dd, 1H, J = 1.8, 4.8 Hz, HAr), 7.84 (dd, 1H, J = 1.8, 7.5 Hz, HAr), 7.49–7.40 (m, 2H, HAr), 7.48
(dd, 1H, J = 4.8, 7.8 Hz, HAr), 7.18–7.11 (m, 1H, HAr); 13C-NMR (DMSO-d6)
δ 167.7, 167.3, 150.6, 147.0,
138.1, 134.6, 133.6, 131.7, 130.8, 128.0, 127.90, 127.64, 123.1; IR (KBr): cm−1 3182, 3066, 2937, 2902, 1924,
1675, 1596, 1486, 1459, 1433, 1410, 1328, 1281, 1254, 1226, 1150, 1110; HRMS (ESI): m/z [M+H]+ calcd for
C13H9ClN3O2: 274.03778, 276.03483, found: 274.0372, 276.03436;
8-Chloropyrazino[3,2-c][1,5]benzodiazocine-6,12(5H,11H)-dione (10m): Yield 19%, colourless crystals, m.p.
1
346–347 ◦C, R.f. = 0.35 (hexane:ethyl acetate). H-NMR (DMSO-d6)
δ
11.24 (s, 1H, NH), 10.77 (s, 1H,
NH), 8.65–8.50 (m, 2H, HAr), 7.55–7.40 (m, 2H, HAr), 7.30–7.18 (m, 2H, HAr); 13C-NMR (DMSO-d6)
δ
167.1, 165.5, 144.9, 144.5, 144.1, 143.7, 134.1, 132.8, 132.2, 131.1, 128.2, 127.8; IR (KBr): cm−1 3249, 3164,
3057, 2961, 2891, 1928, 1685, 1571, 1537, 1484, 1458, 1406, 1343, 1276, 1236, 1211, 1150, 1133, 1108, 1064;
HRMS (ESI): m/z [M+H]+ calcd for C12H8ClN4O2: 275.03303, 277.03008, found: 275.03247, 277.02957;
3.1.6. Synthesis of ethyl 2-(11-methyl-6,12-dioxo-11,12-dihydrodibenzo[b,f][1,5]diazocin-
5(6H)-yl)acetate (10n)
5-Methyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10d, 252 mg, 1 mmol, 1 equiv.) was
dissolved in anhydrous DMSO (5 mL), then 60% NaH dispersed in mineral oil (48 mg, 1.2 mmol,
1.2 equiv.) was added. The resulted suspension was stirred at room temperature for 30 min. until

Molecules 2020, 25, 906
15 of 20
evolution of gas ceased. Then, ethyl bromoacetate (133
µL, 1.2 mmol, 1.2 equiv.) was added dropwise,
and the resulting solution was stirred for 18 h at room temperature. The next day, the reaction mixture
was poured into water (20 mL) and extracted with ethyl acetate (3 20 mL). Combined organic layers
were washed with brine (1 20 mL) and dried over anhydrous magnesium sulfate, followed by
×
×
evaporation under reduced pressure. Crude product was purified by column chromatography using
hexane/EtOAc 1:1 then 2:8 v/v as eluent. Yield 78% (264 mg), colourless crystals, m.p. 125–126 ◦C,
1
R.f. = 0.66 (hexane:ethyl acetate 2:8 v/v). H-NMR (DMSO-d6)
δ 7.45-7.15 (m, 8H, HAr), 4.62 (d, 1H,
J = 17.1 Hz, CH2), 4.47 (d, 1H, J = 17.1 Hz, CH2), 4.23–4.10 (m, 2H, CH2), 1.22 (t, 3H, J = 7.2 Hz, CH3);
13C-NMR (DMSO-d6)
δ 168.3, 167.6, 166.9, 140.1, 139.0, 134.3, 133.2, 131.0, 130.8, 128.2, 128.1, 127.6,
127.2, 125.6, 125.2, 60.9, 51.1, 36.1, 14.0; IR (KBr): cm−1 3458, 3300, 3058, 2981, 2936, 1953, 1840, 1739,
1656, 1599, 1456, 1411, 1411, 1375, 1324, 1308, 1281, 1260, 1217, 1120, 1088, 1056, 1023; HRMS (ESI): m/z
[M+H]+ calcd for C19H19N2O4: 339.13393, found: 339.13393;
3.1.7. Synthesis of 5-acetyl-11-methyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10o)
The dispersion of 5-methyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10d) (504 mg, 2 mmol,
1 equiv.) in acetic anhydride (10 mL) was refluxed for 3 h. The excess anhydride was evaporated
under reduced pressure and the residue was crystallized from a mixture of cyclohexane and ethyl
acetate (9:1) to give pure product. Yield 89% (524 mg), colourless crystals, m.p. 193–194 ◦C, R.f. = 0.74
1
(hexane:ethyl acetate 2:8 v/v). H-NMR (DMSO-d6)
δ
7.46-7.19 (m, 8H, HAr), 3.34 (s, 1H, CH3), 2.61 (s,
1H, CH3); 13C-NMR (DMSO-d6)
δ 171.5, 168.6, 166.7, 139.7, 135.4, 134.3, 133.8, 132.1, 130.3, 129.4, 129.0,
128.3, 127.8, 127.0, 126.2, 36.0, 26.7; IR (KBr): cm−1 3412, 3379, 3296, 3061, 2938, 1975, 1941, 1718, 1703,
1658, 1599, 1485, 1453, 1420, 1371, 1304, 1251, 1206, 1142, 1084, 1038, 1013; HRMS (ESI): m/z [M+H]+
calcd for C17H15N2O3: 295.10772, found: 295.10735;
3.1.8. Synthesis of 8-bromo-2,3-dimethoxydibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dithione (10p)
Bromo-2,3-dimethoxydibenzo[b,f][1,5]diazocine-6,12(5H,11H)-dione (10c, 376 mg, 1 mmol,
1 equiv.) was dissolved in dry THF and then p-tolyl Davy-reagent (873 mg, 2 mmol, 2 equiv.)
was added. The resulting suspension was refluxed for 18 h, then the reaction mixture was cooled
to room temperature. The obtained clear, yellow solution was evaporated with small amount of
chromatographic silica gel. The crude product was purified by column chromatography usi◦ng
hexane/EtOAc 9:1 then 7:3 v/v as eluent. Yield 58% (237 mg), yellow solid, m.p. 186–187 C
1
(decomposition), R.f. = 0.27 (hexane:ethyl acetate 7:3 v/v). H-NMR (DMSO-d6)
δ
12.30 (s, 1H, NH),
12.28 (s, 1H, NH), 7.56–7.49 (m, 2H, HAr), 6.99 (d, 1H, J = 5.1 Hz, HAr), 6.88 (s, 1H, HAr), 6.64 (s, 1H,
HAr), 3.73 (s, 3H, OMe), 3.70 (s, 3H, OMe); 13C-NMR (DMSO-d6)
δ 200.2, 197.5, 149.8, 148.0, 141.2, 133.1,
132.9, 131.2, 131.1, 126.9, 126.6, 120.4, 111.4, 107.7, 55.9, 55.7; IR (KBr): cm−1 3439, 3248, 2925, 2644, 1602,
1506, 1403, 1345, 1265, 1227,1200, 1141, 1079, 1030; HRMS (ESI): m/z [M+H]+ calcd for C16H14BrN2O2S2:
408.96746, 410.96541, found: 408.96725, 410.96512;
3.1.9. General Procedure for the Synthesis of 1-Substituted 1H-benzo[d][1,3]oxazine-2,4-diones 13f–i
To a stirred solution of 6-chloro-1H-benzo[d][1,3]oxazine-2,4-dione (13a), 6-bromo-1H-
benzo[d][1,3]oxazine-2,4-dione (13b) or 1H-benzo[d][1,3]oxazine-2,4-dione (13c) (1 equiv.) in DMSO
(10 mL/mmol), 60% sodium hydride (1.5 equiv.) was added and the resulting suspension was stirred for
15 min. at room temperature, until the evolution of gas ceased. Then, the appropriate halide (1.5 equiv.):
methyl iodide for 13a, 1-(bromomethyl)naphthalene for 13b or benzyl bromide and 4-bromobenzyl
bromide for 13c, was added and the resulting mixture was stirred at room temperature for 18 h. Next
day, the reaction mixture was poured into water (100 mL/10 mL DMSO) and extracted with ethyl
acetate (3
×
100 mL/100 mL H2O). Combined organic layers were washed with brine (100 mL/300 mL
EtOAc) and dried over anhydrous magnesium sulfate. Crude products were crystallized from mixture
of ethyl acetate and hexane.

Molecules 2020, 25, 906
16 of 20
6-Chloro-1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione (13f): Yield 62%, yellow crystals, m.p. 201–202 ◦C,
1
R.f. = 0.33 (hexane:ethyl acetate 7:3). H-NMR (DMSO-d6)
δ 7.94 (d, 1H, J = 2.4 Hz, HAr), 7.89 (dd, 1H,
J = 2.4, 9.0 Hz, HAr), 7.48 (d, 1H, J = 9.0 Hz, HAr), 3.46 (s, 1H, Me); 13C-NMR (DMSO-d6)
δ 158.0, 147.4,
141.1, 136.6, 127.9, 127.6, 117.1, 113.3, 31.9; HRMS (ESI): m/z [M+H]+ calcd for C9H7ClNO3: 212.01090,
214.00795, found: 212.01049, 214.00754;
1-Benzyl-1H-benzo[d][1,3]oxazine-2,4-dione (13g): Yield 57%, beige crystals, m.p. 141–142 ◦C, R.f. = 0.54
1
(hexane:ethyl acetate 7:3). H-NMR (DMSO-d6)
δ 8.04 (dd, 1H, J = 1.5, 7.8 Hz, HAr), 7.73 (ddd, 1H,
J = 1.5, 7.2, 8.7 Hz, HAr), 7.46–7.20 (m, 7H, HAr), 5.30 (s, 2H, CH2); 13C-NMR (DMSO-d6)
δ 158.9, 148.3,
141.3, 137.0, 135.3, 129.5, 128.6, 127.4, 126.6, 123.7, 115.1, 112.1, 47.6; HRMS (ESI): m/z [M+H]+ calcd for
C15H12NO3: 254.08116, found: 254.08104;
1-(4-Bromobenzyl)-1H-benzo[d][1,3]oxazine-2,4-dione (13h): Yield 62%, beige crystals, m.p. 182–183 ◦C, R.f.
1
= 0.50 (hexane:ethyl acetate 7:3). H-NMR (DMSO-d6)
δ
8.04 (dd, 1H, J = 1.5, 7.8 Hz, HAr), 7.74 (ddd,
1H, J = 1.5, 7.2, 8.7 Hz, HAr), 7.85–7.49 (m, 2H, HAr), 7.44–7.36 (m, 2H, HAr), 7.35–7.26 (m, 1H, HAr),
7.22 (dd, 1H, J = 8.4 Hz, HAr), 5.27 (s, 2H, CH2); 13C-NMR (DMSO-d6)
δ 158.8, 148.3, 141.2, 137.0, 134.8,
131.5, 129.5, 129.0, 123.8, 120.5, 115.0, 112.2, 47.0; HRMS (ESI): m/z [M+H]+ calcd for C15H11BrNO3:
331.99168, 333.98964, found: 331.99138, 333.98930;
6-Bromo-1-(naphthalen-1-ylmethyl)-1H-benzo[d][1,3]oxazine-2,4-dione (13i): Yield 51%, white solid, m.p.
1
230–231 ◦C, R.f. = 0.74 (hexane:ethyl acetate 7:3). H-NMR (DMSO-d6)
δ
8.26–8.16 (m, 1H, HAr), 8.15 (d,
1H, J = 2.4 Hz, HAr), 8.04–7.97 (m, 1H, HAr), 7.92–7.78 (m, 2H, HAr), 7.72–7.56 (m, 2H, HAr), 7.42–7.33
(m, 2H, HAr), 7.02 (d, 1H, J = 9.0 Hz, HAr), 5.73 (s, 2H, CH2); 13C-NMR (DMSO-d6)
δ
157.9, 147.8, 140.8,
139.2, 133.3, 131.0, 129.9, 129.5, 128.7, 127.7, 126.5, 126.2, 125.4, 123.0, 122.2, 117.7, 115.3, 114.52, 114.48,
46.4; HRMS (ESI): m/z [M+H]+ calcd for C19H13BrNO3: 382.0073, 384.00529 found: 382.0645, 384.00443;
3.1.10. DCC-mediated Synthesis of 2-(2-aminophenyl)-4H-benzo[d][1,3]oxazin-4-one (12, R1 = R2 = H)
A suspension of 1H-benzo[d][1,3]oxazine-2,4-dione (13c, 489 mg, 3 mmol, 1 equiv.), 2-aminobenzoic
acid (14a, 411 mg,◦3 mmol, 1 equiv.), sodium hydroxide (120 mg, 3 mmol, 1 equiv.) in water (30 mL)
was heated at 80 C for 30 min. until the evolution of carbon dioxide ceased and clear solution
was formed. After cooling, the obtained solution was diluted with water, crude product was
precipitated by addition of glacial acetic acid and the resulting precipitate was dried under reduced
pressure. The crude 2-(2-aminobenzamido)benzoic acid (11) was dissolved in 50 mL of DMF, then
N,N’-dicyclohexylcarbodiimide (DCC) (681 mg, 3.3 mmol, 1.1 equiv.) was added and the reaction
mixture was stirred at room temperature for 18 h. The next day, the obtained solution was poured into
water (100 mL) and the product was extracted with ethyl acetate (3
×
100 mL). Combined organic layers
were washed with brine (1 50 mL), dried over anhydrous magnesium sulfate followed by evaporation
×
of volatiles under reduced pressure. Crude product was purified by column chromatography using
hexane: ethyl acetate 9:1 v/v as eluent. yield 84% (600 mg), yellow solid, m.p. 171–172 ◦C, R.f. = 0.69
1
(hexane:ethyl acetate 7:3). H-NMR (CDCl3)
δ 8.19 (d, 1H, J = 4.8 Hz, HAr), 8.08 (d, 1H, J = 4.8 Hz,
HAr), 7.76 (t, 1H, J = 4.7 Hz, HAr), 7.56 (d, 1H, J = 5.1 Hz, HAr), 7.44 (t, 1H, J = 4.5 Hz, HAr), 7.26 (t,
1H, J = 4.7 Hz, HAr), 6.78–6.68 (m, 2H, HAr), 6.47 (bs, 2H, NH2); 13C-NMR (CDCl3)
δ 159.4, 158.0,
149.8, 146.7, 136.5, 133.7, 129.7, 128.7, 127.8, 126.4, 116.9, 116.8, 116.6, 110.1; IR (KBr): cm−1 3448, 3311,
1743, 1625, 1592, 1550, 1489, 1471, 1447, 1356, 1331, 1310, 1235, 1220, 1167, 1054, 1013; HRMS (ESI): m/z
[M+H]+ calcd for C14H11N2O2: 239.08150, found: 239.08128;
3.1.11. The Synthesis of methyl 2-amino-5-chlorobenzoate (20)
To stirred slurry of 6-chloro-1H-benzo[d][1,3]oxazine-2,4-dione (13a, 591 mg, 3 mmol, 1 equiv.)
in methanol (30 mL), sodium methoxide (486 mg, 9 mmol, 3 equiv.) was added, and the reaction
mixture was refluxed for 3 h. After cooling down, the excess of solvent was evaporated under reduced
pressure; the obtained residue was treated with water (100 mL) and extracted with ethyl acetate (3
50 mL). Combined organic layers were washed with water (1 × 100 mL), brine (1 × 50 mL) and dried
×

Molecules 2020, 25, 906
17 of 20
over anhydrous magnesium sulfate. Evaporation of solvent gave pure product as slight yellow oil
which solidified upon storage. Yield 97% (538 mg), m.p. 70–71 ◦C, R.f. = 0.80 (hexane:ethyl acetate
1
7:3). H-NMR (CDCl3)
δ
7.81 (d, 1H, J = 2.4 Hz, HAr), 7.19 (dd, 1H, J = 2.4, 9.0 Hz, HAr), 6.59 (d, 1H,
J = 9.0 Hz, HAr), 5.73 (bs, 2H, NH2), 3.86 (s, 3H, Me); 13C-NMR (CDCl3)
120.7, 118.1, 111.6, 51.9; HRMS (ESI): m/z [M+H]+ calcd for C8H9ClNO2: 186.03163, 188.02868 found:
δ 167.7, 149.1, 134.2, 130.5,
186.03171, 188.02870.
3.2. X-ray Data Collection and Data Refinement
Good quality single-crystals of 10b, 10g, 10h, 10i, 10j, 10l, 10m, 10o 11*TsOH, 13i and 14c, were
selected for the X-ray diffraction experiments at T = 100(2) K. Diffraction data were collected on
a SuperNova Dual Source diffractometer (Agilent Technologies, Yarnton, Oxfordshire, UK) with
CuK
Technologies SuperNova Single Source diffractometer with MoK
using CrysAlis RED software [17]. The analytical numeric absorption correction using a multifaceted
crystal model based on expressions derived by Clark and Reid [18] (10b 10l 10j 10h, 11*TsOH and
13i) and multi-scan empirical absorption correction using spherical harmonics (10i 10g 10m 10o
α
radiation (
λ
= 1.54184 Å) (10g
,
10h
,
10i
,
10j
,
10l
,
10m
,
10o 11*TsOH, 13i and 14c) or an Agilent
α
radiation ( = 0.71073 Å) (10b),
λ
,
,
,
,
,
and 14c), implemented in SCALE3 ABSPACK scaling algorithm, were applied [17]. The structural
determination procedure was carried out using the SHELX package [19]. 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 programme [19]. All H-atoms linked to the N and
O-atoms were located on a Fourier difference map and refined as riding 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 14c, length of the O H bond was restrained
−
to 0.82 Å. Other H-atoms were positioned geometrically, with C–H equal to 0.93, 0.97 and 0.98 Å for the
aromatic, methylene and methine H-atoms, respectively, and constrained to ride on their parent atoms
with Uiso(H) = xUeq(C), where x = 1.2 for the aromatic, methylene and methine H-atoms. In the case of
10i, a few distinct peaks on the difference Fourier map indicated the presence of a disordered solvent
molecule. However, all of the attempts to model disordered solvents used for crystallization failed.
Therefore, the solvent contribution was removed by applying the appropriate MASK procedure in the
Olex2 programme [20]. Calculated total solvent accessible volume was 982.1 Å3 occupied by 286.6
electrons per unit cell. The figures for this publication were prepared using Olex2 programme [20].
3.3. Cell Culturing
Following cell lines: HeLa, U87, HEK293, EUFA30 were cultured in DMEM medium (Life
Technology, Carlsbad, CA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA,
USA) and 0.1% antibiotics (penicillin, streptomycin, Life Technology, Carlsbad, CA, USA). Cells were
grown in atmosphere of 5 and 95% CO2 and air, respectively, at 37 ◦C.
3.4. Cytotoxicity Assay
Exponentially growing cells at the density of 2
cultured for 18 h, and treated with newly synthesized compounds at concentrations 1–200
×
103 cells/well were seeded onto a 96-well plate,
M, or
µ
with DMSO as a control, for 24 or 48 h. Alamar Blue (Thermo Fisher Scientific, Waltham, MA, USA)
was added accordingly to manufacturer 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,
Brea, CA, USA). The experiments were repeated at least three times with three replicates for each
inhibitor concentration. After background subtraction, inhibition rates, IC50, were calculated as the
component concentration inhibiting cell growth by 50%. All the calculations were performed using
Origin 9.0 software.

Molecules 2020, 25, 906
18 of 20
3.5. Flow Cytometry
The apoptosis detection kit (Annexin V-FITC BD Biosciences, Franklin Lakes, NJ, USA) was used
to detect apoptosis by flow cytometry. Cells were seeded in 6-well plates at concentration of 5
105 cells/well, cultured for 18 h, and tested compound was applied for indicated time. Afterwards,
cells were washed with PBS, resuspended in binding buffer at concentration of 2 106 cells/mL. The
anti-Annexin V FITC-conjugated antibody and propidium iodide were added to 100 l aliquots. Then,
mixtures were incubated for 15 min at room temperature, supplemented with binding buffer to 500
×
×
µ
µl
and processed by BD FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed in
Flowing Software version 2.5.1 (Flowing Software, http://www.uskonaskel.fi/flowingsoftware).
3.6. Disk Diffusion Test
Bacterial inoculum was spread on LB plates solidified with 1.5% agar. Standard 6 mm paper discs
were placed on the surface of the plates and 4
plotted on the discs.
µL of tested compound at the desired concentration
4. Conclusions
In this article we have presented that asymmetrically substituted dibenzo[b,f][1,5]-
diazocine-6,12(5H,11H)diones, which may be treated as convenient privileged structures useful
in the design of biologically active compounds, can be obtained according to two, general methods
based on reactions of: (a) 1H-benzo[d][1,3]oxazine-2,4-diones and unprotected 2-aminobenzoic
acids, and (b) unprotected 2-aminobenzoic acid analogues, activated with thionyl chloride, and
2-aminobenzoic acid esters. The procedures described allow a wide range of modifications of all
structural elements of the dibenzo[b,f][1,5]diazocine-6,12(5H,11H)dione framework: the introduction
of various substituents and functional groups into benzene rings as well as diverse substituents and
side chains attached to the eight-membered diazocine ring. We have shown that these modifications
can be introduced both, through the selection of appropriately modified building blocks as well
as further chemical modifications of the obtained dibenzo[b,f][1,5]diazocine-6,12(5H,11H)dione
scaffold. The wide range of synthetic methods has also allowed to obtain three new heterocyclic
frameworks: benzo[b]naphtho[2,3-f][1,5]diazocine-6,14(5H,13H)-dione 10h, pyrido[3,2-c][1,5]benzo-
diazocine-5,11(6H,12H)-dione 10l, and pyrazino[3,2-c][1,5]benzodiazocine-6,12(5H,11H)-dione 10m
.
The usefulness of the synthetic methods has been confirmed by obtaining a representative library of 16
compounds, evaluated as possible cytotoxic and antimicrobial agents.
Supplementary Materials: 1H-NMR, 13C-NMR, IR, HRMS (ESI) and X-Ray analysis data are available online.
CCDC 1956772 19567781 and 1976509 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 A.M., B.B., D.T., D.G. Biological data
analysis was performed by D.G., E.G., D.M. Synthesis and structure elucidation were performed by B.B., A.M.
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 MNiSW grant (‘Diamentowy Grant’ DI2015 007245, B.B.). This study
was also supported by the National Science Centre Poland MAESTRO grant-DEC-2012/04/A/ST5/00609 (D.T.
and K.W.). 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 was sponsored in part by the Centre for Preclinical Research and Technology (CePT), a project
co-sponsored by European Regional Development Fund and Innovative Economy, The National Cohesion Strategy
of Poland.

Molecules 2020, 25, 906
19 of 20
Acknowledgments: We are greatly indebted to Małgorzata Łobodzka for bacterial strains. 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.
References
1.
Gilman, P.K. Tricyclic antidepressant pharmacology and therapeutic drug interactions updated. Br. J.
Pharmacol. 2007, 151, 737–748. [CrossRef] [PubMed]
2.
3.
Saxena, A.K.; Saxena, M. Developments in antihistamines (H1). Prog. Drug Res. 1992, 39, 35–125. [PubMed]
Kasper, S.; Muller-Spahn, F. Review of quetiapine and its clinical applications in schizophrenia. Expert Opin.
Pharmacother. 2000, 1, 783–801. [CrossRef] [PubMed]
4.
Wellington, K.; Goa, K.L. Oxcarbazepine an update of its efficacy in the management of epilepsy. CNS Drugs
2001, 15, 137–163. [CrossRef] [PubMed]
5.
6.
Kallis, M.M.; Huff, N.A. Oxcarbazepine, an antiepileptic agent. Clin. Ther. 2001, 23, 680–700. [CrossRef]
Murphy, R.L.; Montaner, J. Drug Evaluations anti-infectives: Nevirapine: A review of its development,
pharmacological profile and potential for clinical use. Expert Opin. Investig. Drugs 1996, 5, 1183–1199.
[CrossRef]
7.
8.
Mieczkowski, A.; Trzybin´ski, D.; Wilczek, M.; Psurski, M.; Bagin´ski, M.; Bieszczad, B.; Mroczkowska, M.;
Woz´niak,
K.
(S)-2-(4-Chlorobenzoyl)-1,2,3,4-tetrahydrobenzo[e]pyrazino[1,2-a][1,4]diazepine-6,12
(11H,12aH)-dione—Synthesis and crystallographic studies. Molbank 2017, 2017, M964. [CrossRef]
Mieczkowski, A.; Psurski, M.; Bagin´ski, M.; Bieszczad, B.; Mroczkowska, M.; Wilczek, M.;
Czajkowska, J.; Trzybin´ski, D.; Woz´niak, K.; Wietrzyk, J. Novel (S)-1,3,4,12a-tetrahydropyrazino[2,1-c]
[1,4]benzodiazepine-6,12(2H,11H)-dione derivatives: Selective inhibition of MV-4-11 biphenotypic B
myelomonocytic leukemia cells’ growth is accompanied by reactive oxygen species overproduction and
apoptosis. Bioorg. Med. Chem. Lett. 2018, 28, 618–625. [CrossRef] [PubMed]
9.
Nonnenmacher, E.; Hevertt, A.; Mahamoudt, A.; Aubert, C.; Molnartt, J.; Barbe, J. A novel route to new
dibenzo[b,f][1,5]diazocine derivatives as chemosensitizers. Org. Prep. Proced. Int. 1997, 29, 711–715.
[CrossRef]
10. Nonnenmacher, E.; Brouant, P.; Mrozek, A.; Karolak-Wojciechowska, J.; Barbe, J. Structure and conformational
analysis of 5,11-dibenzyldibenzo[b,f][1,5]diazocine-6,12-dione. A novel approach for new chemosensitizers.
J. Mol. Struct. 2000, 522, 263–269. [CrossRef]
11. Hoorfar, A.; Ollis, W.D.; Price, J.A.; Stephanidou Stephanatou, J.; Stoddart, J.F. Conformational behaviour
of medium-sized rings. Part 11. Dianthranilides and trianthranilides. J. Chem. Soc. Perkin Trans. 1982
,
1649–1699. [CrossRef]
12. Nadkarni, S.S.; Hosangadi, B.D. Studies in large ring compounds: Part IX—Observations on cyclodehydration
of anthraniloylanthranilic acids. Ind. J. Chem. 1988, 27B, 225–228.
13. Hassner, A.; Sun, B.; Gellermann, G.; Meir, S. Dilactams. Synthesis of nonsymmetrical dibenzodiazocinediones.
Tetrahedron Lett. 2004, 45, 1377–1379. [CrossRef]
14. Yokoyama, A.; Karasawa, M.; Taniguchi, M.; Yokozawa, T. Successive formation of two amide linkages
between two benzene rings. Chem. Lett. 2013, 42, 641–642. [CrossRef]
15. Aoki, H.; Okuhara, M. Natural
[PubMed]
β-lactam antibiotics. Ann. Rev. Microbial. 1980, 34, 159–181. [CrossRef]
16. Borthwick, A.D. 2,5-Diketopiperazines: Synthesis, reactions, medicinal chemistry, and bioactive natural
products. Chem. Rev. 2012, 112, 3641–3716. [CrossRef] [PubMed]
17. CrysAlis CCD and CrysAlis RED; Oxford Diffraction Ltd.: Yarnton, UK, 2008.
18. Clarck, R.C.; Reid, J.S. The analytical calculation of absorption in multifaceted crystals. Acta Crystallogr. A
1995, A51, 887–897. [CrossRef]
19. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. A 2008, 64, 112–122. [CrossRef] [PubMed]

Molecules 2020, 25, 906
20 of 20
20. Dolomanov, O.V.; Bourhis, L.J.; Gildea, R.J.; Howard, J.A.K.; Puschmann, H. OLEX2: A complete structure
solution, refinement and analysis program. J. Appl. Crystallogr. 2009, 42, 339–341. [CrossRef]
Sample Availability: Samples of the compounds 10a–p are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).