New succinimides with potent anticancer activity: Synthesis, activation of stress signaling pathways and characterization of apoptosis in leukemia and cervical cancer cells

Article abstract of DOI:10.3390/ijms22094318

Based on previously identified dicarboximides with significant anticancer and immunomodulatory activities, a series of 26 new derivatives were designed and synthesized by the Diels–Alder reaction between appropriate diene and maleimide or hydroxymaleimide moieties. The resulting imides were functionalized with alkanolamine or alkylamine side chains and subsequently converted to their hydrochlorides. The structures of the obtained compounds were confirmed by 1H and 13C NMR and by ESI MS spectral analysis. Their cytotoxicity was evaluated in human leukemia (K562, MOLT4), cervical cancer (HeLa), and normal endothelial cells (HUVEC). The majority of derivatives exhibited high to moderate cytotoxicity and induced apoptosis in K562 cells. Microarray gene profiling demonstrated upregulation of proapoptotic genes involved in receptor‐mediated and mitochondrial cell death pathways as well as antiapoptotic genes involved in NF‐kB signaling. Selected dicarboximides activated JNK and p38 kinases in leukemia cells, suggesting that MAPKs may be involved in the regulation of apoptosis. The tested dicarboximides bind to DNA as assessed by a plasmid DNA cleavage protection assay. The selected dicarboximides offer new scaffolds for further development as anticancer drugs.

Full text of DOI:10.3390/ijms22094318

International Journal of
Molecular Sciences
Article
New Succinimides with Potent Anticancer Activity: Synthesis,
Activation of Stress Signaling Pathways and Characterization of
Apoptosis in Leukemia and Cervical Cancer Cells
Marcin Cies´lak 1,
* *
, Mariola Napiórkowska 2, , Julia Kaz´mierczak-Baran´ska 1 , Karolina Królewska-Golin´ska 1,
Anna Hawrył 3 , Iwona Wybran´ska 4 and Barbara Nawrot 1
1
Centre of Molecular and Macromolecular Studies, Department of Bioorganic Chemistry, Polish Academy
of Sciences, 112 Sienkiewicza, 90-363 Lodz, Poland; juliakazmierczak@tlen.pl (J.K.-B.);
kkrolews@cbmm.lodz.pl (K.K.-G.); bnawrot@cbmm.lodz.pl (B.N.)
Department of Biochemistry, Faculty of Medicine, Medical University of Warsaw, 1 Banacha,
02-097 Warsaw, Poland
Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Lublin, Chodz´ki 4a,
20-093 Lublin, Poland; anna.hawryl@umlub.pl
Clinical Biochemistry, Department of Genetics and Nutrigenomics, Faculty of Medicine,
Jagiellonian University Medical College, 15a Kopernika, 31-501 Kraków, Poland; mbwybran@cyf-kr.edu.pl
Correspondence: marcin@cbmm.lodz.pl (M.C.); mariola.krawiecka@wum.edu.pl (M.N.);
Tel.: +48-42-680-32-30 (M.C.); +48-22-572-06-39 (M.N.)
2
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Based on previously identified dicarboximides with significant anticancer and immunomod-
ulatory activities, a series of 26 new derivatives were designed and synthesized by the Diels–Alder
reaction between appropriate diene and maleimide or hydroxymaleimide moieties. The resulting
imides were functionalized with alkanolamine or alkylamine side chains and subsequently converted
to their hydrochlorides. The structures of the obtained compounds were confirmed by 1H and
13C NMR and by ESI MS spectral analysis. Their cytotoxicity was evaluated in human leukemia
(K562, MOLT4), cervical cancer (HeLa), and normal endothelial cells (HUVEC). The majority of
derivatives exhibited high to moderate cytotoxicity and induced apoptosis in K562 cells. Microarray
gene profiling demonstrated upregulation of proapoptotic genes involved in receptor-mediated
and mitochondrial cell death pathways as well as antiapoptotic genes involved in NF-kB signaling.
Selected dicarboximides activated JNK and p38 kinases in leukemia cells, suggesting that MAPKs
may be involved in the regulation of apoptosis. The tested dicarboximides bind to DNA as assessed
by a plasmid DNA cleavage protection assay. The selected dicarboximides offer new scaffolds for
further development as anticancer drugs.
Citation: Cies´lak, M.; Napiórkowska,
M.; Kaz´mierczak-Baran´ska, J.;
Królewska-Golin´ska, K.; Hawrył, A.;
Wybran´ska, I.; Nawrot, B. New
Succinimides with Potent Anticancer
Activity: Synthesis, Activation of
Stress Signaling Pathways and
Characterization of Apoptosis in
Leukemia and Cervical Cancer Cells.
Int. J. Mol. Sci. 2021, 22, 4318.
https://doi.org/10.3390/ijms22094318
Academic Editor: Angela Stefanachi
Received: 4 March 2021
Accepted: 19 April 2021
Published: 21 April 2021
Keywords: dicarboximides; chemical synthesis; cytotoxicity; apoptosis; kinases; anticancer; gene
profiling; SAR
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Cancer diseases are an important problem in the clinical medicine and pharmacology
of the 21st century. Annually, more than eight million people die from cancer worldwide. In
Europe alone, three million new cases are diagnosed and 1.7 million people die from cancer.
In Poland, cancer kills nearly 110,000 people, of which about 100,000 die from malignant
tumors. It is estimated that the ongoing increase in the incidence of cancer will make it the
Copyright:
© 2021 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 (https://
creativecommons.org/licenses/by/
4.0/).
main cause of death in Poland and worldwide in the coming decades [1,2]. Despite recent
progress in cancer treatment (for example development of immune checkpoint inhibitors),
chemotherapy (either alone or in combination with radiotherapy or biologic drugs) still
remains the first-line treatment in the majority of cancers. Therefore, new cytotoxic low
molecular weight compounds are actively sought and developed because they may offer
Int. J. Mol. Sci. 2021, 22, 4318. https://doi.org/10.3390/ijms22094318
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 4318
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better pharmacokinetic properties and/or less toxic profiles compared to already applied
chemotherapeutics (for example platinum compounds or taxanes).
Dicarboximides comprise a large group of compounds and can be divided into four
categories: succinimides, maleimides, glutarimides, and phthalimides. Succinimides and
phthalimides offer a wide range of chemical and biological applications. They are also
very important from a therapeutic perspective, as they are used as drugs (for example,
anticonvulsant–ethosuximide) or tested as drug candidates. These derivatives show anti-
fungal, antibacterial, antidepressant, or analgesic activities. Several reports describe the
antitumor activity of succinimides and phthalimides. Pyridinyl- and quinolinyl-derivatives
of succinimide demonstrated antitumor activity in leukemia cells by inhibition of DNA
synthesis [
3]. Trifluoromethylated mono- and bicyclic succinimides were found to in-
hibit the growth of myeloma, non-small cell lung cancer, and renal cancer cells [
4
]. N-
benzylcantharidinamide promoted the degradation of matrix metalloproteinase-9 (MMP-9)
mRNA in an antigen R-dependent manner in human hepatocellular carcinoma Hep3B
cells. This resulted in decreased expression of MMP-9 and reduced invasion of the highly
metastatic Hep3B cells [
totoxicity and induced apoptosis in human hepatoma (SKHep1), Burkitt’s B-cell lymphoma
(CA46), and chronic myelogenous leukemia (K562) cells [ ]. Dual-acting thiazolidinone
5]. Benzothiazole derivatives of phthalimide showed significant cy-
6
derivates of phthalimide with antiproliferative and immunosuppressive activity were also
identified. These compounds inhibited the proliferation of cancer cells and blocked the
secretion of pro-inflammatory cytokines, for example, TNF-
α
, IL-2, or Il-6 [7]. Probably
the best-known phthalimide-based drug is thalidomide (N-
α
-phthalimidoglutarimide).
This is a synthetic derivative of phthalimide N-substituted with glutarimide. Since 1957,
thalidomide has been used as an anti-emetic agent, but in 1961 it was withdrawn from
the market because of severe teratogenicity. However, further studies demonstrated the
potent anti-angiogenic and anti-inflammatory activity of thalidomide (and its derivatives—
lenalidomide and pomalidomide), which warranted its return as an essential medicine.
Currently, thalidomide is used as an anti-cancer drug (for multiple myeloma), for the treat-
ment of myelodysplastic syndrome and inflammatory disorders, like erythema nodosum
leprosum [8]. We have previously identified several lead dicarboximides (in particular Ic,
Ie, and II, Figure 1) structurally related to succinimides and phthalimides, which exhibited
high cytotoxicity and selectivity toward leukemia cells.
We also found that they targeted the ABC50 protein, showed immunomodulatory
activity, and induced apoptosis in leukemia cells [
hibited endonuclease-catalyzed digestion of DNA, suggesting its interaction with nucleic
acids [ ] and activated caspase 8 and 9, which are markers of receptor-mediated and mi-
9–11]. In particular, compound Ic in-
9
tochondrial apoptotic pathways, respectively [11]. However, the detailed mechanism of
apoptosis and the involvement of mitogen-activated protein kinases (MAPKs) were not in-
vestigated. These anticancer and immunomodulatory activities of dicarboximides indicate
their importance as potential drug candidates and significance for further development.
The main goal of this study was to obtain new derivatives of dicarboximides with
decreased lipophilicity and improved anticancer activity as compared to the lead com-
pounds (i.e., Ia–Ie and II). To achieve this aim, we designed, synthesized, and examined
the biological activity of new derivatives of dicarboximides. These new derivatives showed
potent cytotoxic and proapoptotic activity in cancer cells, and DNA intercalating proper-
ties. Microarray analysis of gene expression revealed that dicarboximides upregulated
genes involved in receptor-mediated and mitochondrial apoptotic pathways. Interestingly,
antiapoptotic genes associated with NF-kB signaling were also upregulated. They also
activated stress-induced MAPK signaling pathways in cancer cells.

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Figure 1. Structures of lead compounds Ia–e and II.
2. Results
2.1. Synthesis
The starting imides 1–3 were obtained in Diels–Alder reactions between appropriate
dienes and maleimide, while the compounds and were obtained in the reaction with
4
5
N-(hydroxyethyl)-maleimide (Scheme 1). The epoxy-derivatives were obtained by con-
densation of starting imides with epichlorohydrin, and the products were condensed with
appropriate amines to give alkanolamine derivatives (Scheme 1). In the reaction between
imide 1 and 1,2-dichloroethane, the chloroalkyl derivative was obtained and condensed
with an appropriate amine (Scheme 1). All obtained amino-derivatives were converted
to their hydrochlorides by using methanol saturated with hydrochloride. The lead com-
pounds with the best activity (Ic, Ie, and II) described previously [9] were converted to their
acetates by using boiling acetic acid. The structures of new compounds were established
by spectroscopic methods (1H NMR, 13C NMR, and HRMS). In this case, ethyl groups in
positions 1 and 7 were replaced by methyl groups.
The main goal of the synthetic part was the synthesis of new derivatives of dicarbox-
imides with decreased lipophilicity in comparison to the lead compounds, i.e., Ia–Ie and II.
To reduce the lipophilicity of the new dicarboximides, a hydroxyalkyl chain connected with
an aliphatic amine was introduced to the imides (Scheme 1). At the same time, the imide
scaffold in derivatives from group
2
and
3
was the same as in lead compounds Ia
–Ie and II
,
respectively (Figure 1). The structure of the imide was changed in derivatives 1b
–
1g. The
lipophilicity of new derivatives was calculated using the Osiris Property Explorer program.
For some compounds, lipophilicity was determined experimentally by a reversed-phase
chromatography (detailed description is given in Materials and Methods and in the Sup-
plementary Data, Table S2). Table 1 summarizes the calculated (clogP) and experimental
(logkw) parameters of the lipophilicity of new derivatives.

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Scheme 1. Synthesis of starting imides, N–alkanolamine and N–alkylamino derivatives of dicarboximides
.
In some cases, the new derivatives had lower lipophilicity in comparison to the lead
compounds. For example, derivatives of imide had a median clogP and logkw of 3.19
and 4.85, respectively, while corresponding values for lead dicarboximides Ia Ie were
1
–
4.27 and 5.05. This decrease in lipophilicity can be attributed to the replacement of ethyl
groups by methyl groups in the imide scaffold and the presence of the hydroxyalkyl
chain. The introduction of a hydroxyalkyl chain in derivatives from the group of imide
2
resulted in a slight decrease in lipophilicity (median clogP and logkw of 4.1 and 5.03,
respectively) compared to parent compounds Ia Ie. However, pronounced exceptions
were alkanoldibutylamine derivatives 1e and 2e, which showed significantly increased
lipophilicity. The modifications introduced to imide derivatives did not significantly
–
3
affect their lipophilicity (median clogP and logkw of 5.69 and 6.28, respectively) compared
to the lead dicarboximide II (clogP 5.45 and logkw 6.34).
2.2. MTT Cytotoxicity Studies
The cytotoxicity of new derivatives of dicarboximides was tested in HeLa (cervix carci-
noma cell line), K562 (myelogenous leukemia cell line), MOLT-4 (Human T-Lymphoid Cell
Line), and HUVEC (normal endothelial) cells. First, the viability of cells was determined
after 48 h incubation with the screened compounds used at the concentration of 100 µM
(data not shown). For compounds that reduced the viability of cancer cells by more than
50%, the IC50 values were determined (Table 2). Cells exposed to 1% DMSO (as vehicle)
served as the control with 100% survival. Cells treated with 1 µM staurosporine served
as the internal control of the cytotoxicity experiments (the viability of cancer cells was
reduced to 44–49%, data not shown).

Int. J. Mol. Sci. 2021, 22, 4318
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Table 1. Calculated (clogP) and experimental (logkw) parameters of lipophilicity for synthesized
compounds. nd—not determined.
clogP
(Calculated)
Median
clogP
logkw
(Experimental)
Median
logkw
Imide Group Compound
Ia
Ib
Ic
Id
Ie
3.82
4.63
4.95
4.27
4.06
4.94
5.09
5.02
5.05
5.17
4.27
3.19
5.05
4.85
2
1
1b
1c
1d
1e
1f
1g
4
1h
1j
1i
2.48
3.22
3.15
4.97
3.47
2.31
2.69
3.84
2.88
4.04
4.78
4.91
4.87
nd
nd
4.83
nd
nd
nd
nd
2b
2c
2d
2e
2f
3.39
4.13
4.06
5.88
4.38
3.22
5.08
4.74
5.10
nd
nd
4.98
2
3
4.1
5.03
6.28
2g
II
5.45
6.34
3b
3c
3d
3e
3f
3g
5
5.02
5.76
5.69
7.51
6.01
4.85
5.23
6.31
6.06
6.28
nd
nd
6.28
nd
5.69
3
Test compounds showed significant cytotoxicity toward all tested cancer cell lines.
Derivatives of imide and (i.e., Ic*CH3COOH Ie*CH3COOH 2b 2g 3c 3g ), possess-
ing the same imide skeleton as the lead compounds, displayed the best activity with IC50
in the range of 1.9–26 M. In the group of derivatives of imide , the highest activity was
demonstrated by 1e against all tested cancer cell lines (IC50 of 3.2, 5.8, and 8 M, respec-
tively). It is noteworthy that derivatives 1b 1h, and 1i were the most toxic to MOLT-4 cells
(IC50 of 7, 20, and 15 M, respectively), whereas compound 1f was cytotoxic to K562 (IC50
of 18 M). Other derivatives of this class showed moderate activity (IC50 in the range of
28–105 M). Similar cytotoxicity as for cancer cells was observed in normal endothelial
2
3
,
,
–
,
–
, 5
µ
1
µ
,
µ
µ
µ
cells, with IC50 values in the range of 0.3–64 µM.
2.3. Activation of Apoptosis and Expression Profile of Apoptotic Genes
Six derivatives of dicarboximides, i.e., 3c
,
3d
,
3f
, 2c, 2d, and 2f, with the highest
cytotoxicity against K562 leukemia cells (IC50 < 6
µM) and with the same alkanolamine
fragment in the structure were further evaluated for the ability to induce apoptosis in
cancer cells. Caspases 3 and 7 (caspase 3/7) are markers of apoptosis, and their activity was
upregulated during this process. K562 cells were treated with 1% DMSO (negative control),
1 µM staurosporine (a strong inducer of apoptosis, a positive control), or a test compound at
the concentration of 5xIC50. After incubation for 18 h, K562 cells were lysed and the activity
of caspase 3/7 was measured using a profluorescent peptide substrate. Staurosporine
strongly activated caspase 3/7 in K562 cells, while DMSO had no effect (Figure 2). We

Int. J. Mol. Sci. 2021, 22, 4318
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also observed a significant increase in the activity of caspase 3/7 in the presence of all
tested dicarboximides. Particularly, derivatives 2c
apoptosis, as they induced an over eight-fold increase in caspase activity compared to
control. Incubation of cells with compounds 3c 3d, and 3f activated caspase 3/7 to a
, 2d, and 2f were potent activators of
,
lesser extent. Altogether, these data suggest that the cytotoxicity of new derivatives of
dicarboximides is most likely due to the induction of apoptosis in the chronic myelogenous
leukemia (CML) K562 cells.
Table 2. The IC50 values (µM) for dicarboximides after 48 h incubation with a given cell line. Average
values and standard deviations (in parenthesis) from three experiments are shown. Compounds
were used as HCl salts except for Ic *, Ie *, and II * which were used as CH3COOH salts.
Compound
HeLa
K562
MOLT-4
HUVEC
1b
1c
1d
1e
1f
1g
4
30 (7.5)
26 (5.2)
59 (8.9)
3.2 (0.4)
55 (17.6)
>100
61 (14.8)
nd
>100
32 (8.3)
28 (6.7)
39 (9.4)
5.8 (1.0)
18 (3.8)
>100
105 (15.2)
nd
>100
7 (1.8)
28 (5)
37 (4.4)
8 (0.9)
40 (10.8)
>100
71 (14.8)
20 (4.7)
>100
20 (2.4)
23 (3.2)
22 (3.3)
30 (4.2)
21 (1.6)
nd
56 (4.8)
64 (7)
nd
1h
1j
1i
52 (19.2)
18 (3.1)
10 (2.6)
26 (5.6)
3.2 (0.8)
18 (4.9)
19 (4.5)
5.1 (0.7)
23 (3.6)
20 (2.4)
>100
30 (4.1)
20 (3.4)
8 (1.7)
8 (1.5)
4 (0.5)
5.8 (1.2)
4.1 (0.5)
3.9 (0.5)
20 (1.4)
16 (4.5)
>100
2 (0.6)
3.2 (0.4)
6 (1.4)
15 (1.2)
15 (3.2)
15 (2.3)
14 (0.9)
2.5 (0.1)
14 (2.6)
3.1 (0.2)
3.4 (0.3)
18 (2.5)
18 (5)
23 (1.8)
15 (6.3)
3.5 (0.2)
3.4 (0.3)
3.3 (0.2)
3.6 (0.2)
1.2 (0.06)
3.1 (0.3)
17 (1.6)
4 (0.5)
Ic *
Ie *
2b
2c
2d
2e
2f
2g
II *
3b
3c
3d
3e
3f
>100
nd
2 (0.4)
2.6 (0.3)
2.2 (0.3)
5.4 (0.5)
3 (1)
5.2 (0.9)
2.1 (0.1)
0.4 (0.03)
0.3 (0.02)
7 (0.6)
0.4 (0.02)
17 (1.8)
0.7 (0.08)
1.9 (0.6)
40 (8.4)
3 (0.9)
120 (16)
8 (1)
3 (0.6)
31 (3.6)
6.2 (1)
3g
5
* denotes compounds as acetate salts; nd—not determined.
Figure 2. Activity of caspase 3/7 in K562 cells treated with the test dicarboximides or staurosporine for 18 h. Abbreviations:
K562—untreated cells; DMSO—K562 cells treated with 1% DMSO; ST—staurosporine. The activity of caspase 3/7 in cells
exposed to 1% DMSO was normalized to 1.0. Mean values ± SD are shown.

Int. J. Mol. Sci. 2021, 22, 4318
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We also profiled the expression of 93 genes associated with apoptosis in leukemia
cells treated with the lead dicarboximide Ic. We showed previously that compound
Ic induced apoptosis in CML K562 cells and acute lymphoblastic leukemia (ALL) cells
MOLT-4, and that both receptor-mediated and mitochondrial apoptotic pathways may
be involved [9,11]. K562 leukemia cells were incubated for 18 h with 1% DMSO (control–
comparator), staurosporine (1
µ
M, positive control), or compound Ic (10 M). Subsequently,
µ
the total RNA was isolated and the expression of 93 mRNAs associated with apoptosis
was determined using DNA microarrays. The expression profile of apoptotic genes upon
treatment with Ic was similar to staurosporine, a known proapoptotic agent (Figure 3
and Supplementary Materials Table S1). The expression of 12 genes was significantly
upregulated in response to Ic (Figure 3).
Figure 3. Human apoptosis array analysis of gene expression in K562 exposed to compound Ic at 10
µM
(5 IC50) for 18 h. Staurosporine (1 M) served as a positive control. Only apoptosis-related genes with
×
µ
statistically significant changes in expression are shown (p < 0.05, t-Student). The fold of change was
expressed as log2 ratio of gene expression in a test sample vs control sample (cells + DMSO).
Eight of these genes encode proteins with proapoptotic activity. For example, TN-
FRSF10B (a membrane receptor from the TNF family) and RIPK1 (a Ser-Thr kinase, which
is a key regulator of TNF-mediated apoptosis) activate the receptor pathway of apoptosis.
PMAIP1 (Phorbol-12-Myristate-13-Acetate-Induced Protein 1), BAX (BCL2 Associated
X, a Bcl-2 family member), BBC3 (BCL2 Binding Component 3, a Bcl-2 family member),
and BNIP3 (BCL2 Interacting Protein 3) induce apoptosis via the mitochondrial pathway.
HTRA2 (HtrA Serine Peptidase 2) promotes apoptosis by binding and inhibiting IAPs
(inhibitor of apoptosis proteins), while CASP3 (caspase 3) is a major executioner protease
during cell apoptosis. Only one of the upregulated genes encodes a protein that inhibits
apoptosis, i.e., MCL1 (Myeloid Cell Leukemia Sequence 1), which belongs to the Bcl-2
family. Interestingly, compound Ic also increased the expression of genes that encode anti-
apoptotic proteins involved in the activation of the transcription factor NF-kB. Specifically,
IKBKB (Inhibitor Of Nuclear Factor Kappa B Kinase Subunit Beta) and IKBKG (Inhibitor Of
Nuclear Factor Kappa B Kinase Subunit Gamma) are protein kinases that activate NF-kB in
cells, and RelA is a subunit of NF-kB heterodimer.
2.4. Effect of Dicarboximides on MAP Kinase Signaling Pathways
In this study, we have demonstrated that new derivatives of dicarboximides are cyto-
toxic and induce apoptosis in leukemia cells. Similar effects were observed for previously
studied lead compounds Ic and II [9,11]; therefore, we investigated whether mitogen-
activated protein kinases (MAPKs) were involved in mediating dicarboximides-induced
cytotoxic effects and induction of apoptosis. MAPKs include extracellular signal-regulated
kinases (ERK1/2), p38 kinase, and c-Jun N-terminal kinase (JNK), which regulate cellular

Int. J. Mol. Sci. 2021, 22, 4318
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responses to multiple stimuli like growth factors, cytokines, stress signals, or cytotoxic
drugs. In cells exposed to stress conditions (for example toxic compounds), p38 kinase
and JNK kinase are often activated, leading to cell cycle arrest and apoptosis. To inves-
tigate the effect of dicarboximides on MAPKs, leukemia cells were incubated with lead
compounds Ic and II, and subsequently, cell lysates were immunoblotted with specific
antibodies recognizing activated (i.e., phosphorylated) forms of MAP kinases. As positive
controls, we used cells treated with staurosporine (activates ERK1/2) or anisomycin (acti-
vator of JNK and p38 kinases). As expected, the incubation of K562 cells with compounds
Ic or II had no effect on the ERK pathway, which is usually activated by growth factors
and promotes cell proliferation (Figure 4a). However, dicarboximides Ic and II activated
JNK and p38 kinases. This is evidenced by increased phosphorylation of JNK and p38
kinase (Figure 4b,c, lanes 3 and 4) compared to control (lane 1). These data suggest that in
leukemia cells dicarboximides activate stress signaling pathways regulated by JNK and
p38 kinases, which subsequently leads to apoptotic death.
Figure 4. Effect of dicarboximides Ic and II on activation of ERK1/2 (
in K562 leukemia cells. Ctrl—control cells treated with 1% DMSO; Anisomycin 20
control, activator of JNK and p38 kinases); Ic (6 M); II (3 M); ST—Staurosporine 1
control, activator of ERK1/2); inh—JNK inhibitor 20 µM or p38 inhibitor 10 µM.
a
), JNK (
b
), and p38 (
M (positive
M (positive
c) kinases
µ
µ
µ
µ

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2.5. Interaction with DNA
A DNA-cleavage protection assay has been successfully used for the identification
of compounds that bind DNA either covalently or noncovalently. Such binding may
result in DNA being less susceptible to enzymatic cleavage [12]. Using this assay, we
showed previously that compound Ic may interact with DNA [
9
,10]. It was also reported
that naphtalenoimides or indolomaleimides intercalate into DNA [13
,
14]. With the aid
of circular dichroism (CD) spectroscopy, we demonstrated the direct binding of Ic to
DNA (Supplementary Materials Figure S1). In CD studies, a synthetic 23-nt duplex DNA
or bovine thymus DNA was incubated with compound Ic or daunorubicin (reference
compound). CD spectra indicated that compound Ic induced conformational changes
in DNA compared to the control sample (Supplementary Materials Figure S1). This ob-
servation suggests that the cytotoxicity of dicarboximides may result from binding to
and damaging the double-stranded DNA. Using a DNA-cleavage protection assay, we
investigated whether selected compounds 2c, 2d, and 2f and 3c, 3d, and 3f interact with a
plasmid DNA, resulting in increased resistance to endonucleolytic cleavage. Plasmid DNA
(pcDNA3.1HisC) containing a single BamHI cleavage site was incubated with test com-
pounds and subsequently digested with BamH1 restriction endonuclease. Daunorubicin (a
strong DNA-intercalating agent) was used as a reference compound. Non-digested plasmid
DNA existed predominantly in the circular form; however, the superhelical and linear
forms were also detected (Figure 5, lane 1). Digestion with the BamH1 enzyme converted
the plasmid into a linear form (Figure 5, lane 2; no circular DNA), while daunorubicin (at
100 µM) prevented linearization of DNA (Figure 5, lane 3; circular DNA present). In the
presence of test dicarboximides, the linear plasmid DNA was predominant; however, the
circular form was also detected (Figure 5 lane 4–9). This indicates that test dicarboximides
interact with DNA. Interestingly, this interaction seems to be stronger for 3c
(as more circular DNA is observed in lanes 4–6) than for the remaining test compounds
(lanes 7–9). This observation can be explained by the higher lipophilicity of 3c 3d, and 3f
compared to 2c, 2d, and 2f, which may facilitate their more efficient binding to DNA.
, 3d, and 3f
,
Figure 5. Effect of dicarboximides on digestion of pcDNA3.1HisC (total length 5.5 kbp) with BamH1 endonuclease observed
by agarose gel electrophoresis. M—marker DNA; Dar—daunorubicin.
3. Discussion
In our previous studies, we identified several derivatives of dicarboximides (Ia
– ,
Ie
11].
Ie, and II were particularly promising, as they showed specificity to cancer
II) that efficiently killed cancer cells and were not toxic to normal endothelial cells [
Compounds Ic
9–
,
cells only and induced apoptosis. However, they are lipophilic (Table 1) and have limited
water solubility. Based on their structures, we have designed and synthesized a library of

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26 new derivatives of dicarboximides with lower lipophilicity, which is a desired property
for potential new drug candidates. This was achieved mainly by the replacement of ethyl
groups in the imide scaffold by methyl groups at positions 1 and 7 as well as by the
introduction of a side chain with the hydroxyalkyl group substituted with alkylamino
groups (Scheme 1). This resulted in significantly lower lipophilicity of derivatives of imide
1
(median clogP 3.19), and imide
Ia–Ie (median clogP 4.27). However, chemical modifications introduced to derivatives of
imide rather increased their lipophilicity (median clogP 5.69) when compared to the parent
2 (median clogP of 4.1) compared to parent compounds
3
dicarboximide II (clogP 5.45) (Table 1). Lipophilicity is the most important property of new
drug candidates. According to Lipinski’s rule, clogP > 5 predicts poor absorption and/or
permeability of a drug [15]. In addition, high lipophilicity may increase the unspecific
cytotoxicity [16].
The cytotoxic properties of all investigated analogs were tested in human cells of
cancer and normal origin. Fourteen compounds (1b
significant cytotoxicity toward cancer cell lines (IC50 < 10
toxic than the lead compounds and exhibited cytotoxicity in normal endothelial cells
Table 2). Comparison of IC50 values indicates that compounds with lower lipophilicity are
,
1e,
2b
–2f, 3c–3g, 5, Ie*) displayed
µM); however, they were less
(
less cytotoxic. This is particularly evident for derivatives of dicarboximid
the lowest cytotoxicity.
1, which show
Preliminary studies on derivatives 3c, 3d, 3f, 2c, 2d, and 2f showed that these com-
pounds activated caspase 3 and 7 in K562 cells (Figure 2). This suggests that the cytotoxicity
of the new derivatives is associated with the induction of apoptosis in CML cells. Interest-
ingly, proapoptotic activity of dicarboximides seems to depend on lipophilicity. Derivatives
of imide 2 (median clogP 4.1) were significantly more potent than derivatives of imide 3 (me-
dian clogP 5.69). Apoptosis may proceed through two major pathways: receptor-mediated
(extrinsic) and mitochondrial (intrinsic) [17]. The effect of dicarboximides on apoptotic
pathways was investigated by microarray-based gene expression profiling. We found
that several pro-apoptotic genes involved in the receptor and mitochondrial apoptotic
pathways were upregulated in K562 cells treated with Ic. The expression profile induced
by Ic was similar to staurosporine, a natural compound that activates mainly the mito-
chondrial pathway of apoptosis [18]. Specifically, compound Ic upregulated the expression
of TNFRSF10B and RIPK1, which promote apoptosis via the receptor (extrinsic) pathway.
TNFRSF10B belongs to a TNF family of transmembrane receptors. Upon binding the
TRAIL ligand (TNF-related apoptosis-inducing ligand), TNFRSF10B is activated leading
to caspase 8-mediated apoptosis [19]. Interestingly, in response to cytotoxic agents cancer
cells overexpress TNFRSF10B, and this seems to be sufficient to trigger ligand-independent
apoptosis [20]. RIPK1 (Receptor Interacting Serine/Threonine Kinase 1) is involved in
signaling events downstream of TNFR1 that (depending on the cellular context) may lead
to apoptosis through recruitment of FADD and subsequent activation of caspase 8 [21] or
promote necroptosis through phosphorylation of RIPK3 and MLKL [22]. Compound Ic also
increased the expression of protein genes involved in the mitochondrial (intrinsic) pathway
of apoptosis: PMAIP1 (Phorbol-12-Myristate-13-Acetate-Induced Protein 1), BAX (BCL2
Associated X, a Bcl-2 family member), BBC3 (BCL2 Binding Component 3, a Bcl-2 family
member), and BNIP3 (BCL2 Interacting Protein 3). BAX is a member of the Bcl-2 family of
proteins and a key activator of the intrinsic apoptotic pathway. In response to cellular stress,
BAX is activated and oligomerizes, forming pores in the mitochondrial outer membrane.
This enables cytochrome c and/or SMAC/Diablo proteins to leak into the cytoplasm and
activate caspase 3 [23]. BBC3 is a pro-apoptotic protein that belongs to a class of BH3-only
proteins. BBC3 binds other anti-apoptotic Bcl2 proteins (for example antiapoptotic MCL1)
using its BH3 domains and thus promotes apoptosis. Interestingly, the expression of BBC3
is induced by DNA damaging agents and regulated by p53 [24]. This could explain the
increased expression of BBC3 in K562 cells treated with Ic, as we demonstrated previously
that this compound interacts with DNA and thus may be considered a DNA-damaging
agent [9]. Altogether, these results suggest that dicarboximides activate both receptor-

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mediated and mitochondrial pathways of apoptosis by the upregulation of several genes
involved in both pathways.
Interestingly, compound Ic also increased the expression of anti-apoptotic genes encod-
ing IKBKB/IKK
(Inhibitor Of Nuclear Factor Kappa B Kinase Subunit Gamma), and RelA, which serves
as a subunit of NF- B heterodimer. These proteins are involved in the activation and
function of transcription factor NF- B, which regulates the expression of several genes
involved in inflammation, immune responses, cell survival, and tumorigenesis and inhibits
apoptosis. Serine kinase IKK and regulatory protein Ikk are key components of the
IKK complex, which is essential for the activation of NF- B transcription factors. The
anti-apoptotic activity of IKK and IKK has been confirmed in studies using knockout
mice. IKK -deficient mice died during embryonic development due to the uncontrolled
β (Inhibitor Of Nuclear Factor Kappa B Kinase Subunit Beta), IKBKG/IKKγ
κ
κ
β
γ
κ
β
γ
β
apoptosis of liver cells [25]. A similar phenotype, i.e., liver apoptosis was observed in the
case of Ikkγ-deficient male embryos, leading to their death on day (E)12 [26].
The cytotoxic effects of dicarboximides may also be mediated by MAPKs, a family of
Ser-Thr kinases that consists of ERK1/2, p38, and JNK kinase. We demonstrated that in
cells exposed to lead dicarboximides Ic and II, p38 and JNK kinases are activated while
there was no effect on ERK1/2 (Figure 4). ERK1/2 are activated mainly by growth factors
(for example PDGF or EGF) and positively control cell proliferation by promoting G1- to
S-phase transition [27]. On the other hand, p38 and JNK kinases are activated by stress
conditions, for example, oxidative stress, ionizing radiation, cytotoxic/genotoxic agents,
DNA damage, inflammatory cytokines, or deprivation of growth factors. In response
to stress conditions, JNK transactivates the c-Jun transcription factor or phosphorylates
proteins associated with apoptosis, leading to activation of both mitochondrial and receptor-
mediated pathways of apoptosis [28]. Similarly, activation of p38 leads to inhibition of cell
cycle progression and induction of apoptosis [29,30]. Our results suggest that p38 and JNK
can contribute to the activation of apoptosis induced by Ic and II.
We also examined whether DNA may be targeted by test derivatives. The obtained
results showed that dicarboximides inhibited the digestion of plasmid DNA with BamHI
endonuclease, albeit to a lower extent than daunorubicin (Figure 5). These data suggest
that the test dicarboximides may intercalate to DNA and possibly cause DNA damage.
This property of dicarboximides (including compound Ic) could explain the increased
expression of the BBC3 gene in K562 cells exposed to Ic, as discussed above.
The SAR analysis indicated that the volume of substituents at positions 1 and 7 of
the imide system is an important factor in determining the cytotoxicity of dicarboximides
(Figure 1). This conclusion is supported by comparison of the cytotoxic and apoptotic
activities in three sets of compounds: 1c, 2c, and 3c; 1d, 2d, and 3d; or 1f, 2f, and 3f. The
derivatives containing ethyl or phenyl groups at positions 1 and 7 showed lower IC50
towards HeLa, K456, and MOLT-1 cells than compounds with methyl groups. The type
of alkylamine substituent in the side chain is also important for inducing cytotoxicity
and apoptosis-signaling pathways. Dicarboximides with alkylamine substituent bearing
a protonated nitrogen atom at physiological pH (pKa of amines is > 9.0, [31]) were the
most active. For example, tert-butyl derivatives (2c and 3c), piperidynyl derivatives (2f
and 3f), dibutyl derivatives (1e, 2e, and 3e), and diethylamine derivative (3d) showed the
highest cytotoxicity against cancer cells tested. It is worth noting that all of the above-
mentioned compounds have values of clogP > 4. On the other hand, compounds containing
the morpholine system i.e., 1g
derivatives within the corresponding group of imides
,
2g, and 3g (with the lowest clogP values among other
, and , respectively) exhibited
1
,
2
3
the lowest cytotoxicity towards tested cell lines. This result suggests that the presence
of an acceptor oxygen atom at the morpholine ring, but not the cyclic structure of the
substituent, is responsible for undesirable interactions that are not offered by the piperidine
ring of 1f
group is further supported by comparison of compounds of scaffold
not cytotoxic when the imide function is substituted with hydroxyalkyl (4) or chloroethyl
,
2f, and 3f. The favorable role of the protonated nitrogen atom of the alkylamino
1, which are less or

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substituent (1h), respectively, but exhibit higher cytotoxicity when 1h is substituted with
a tert-butyl amine residue (1i). However, the presence of the protonated alkylamino
group is not an obligatory feature to provide high cytotoxicity, since, e.g., compound 5,
containing a hydroxyalkyl side chain, exhibited significant cytotoxicity. The activity of the
lead compounds is also influenced by the type of salts, i.e., acetate vs. hydrochloride. The
acetate salts showed lower cytotoxicity and lacked selectivity against the studied cell lines.
It can be hypothesized that the change of polarization of the molecule by the acetate anion
and the decrease in the hydrophilicity could affect the activity of these derivatives.
4. Materials and Methods
4.1. Chemistry
All chemicals used in the reaction were supplied from Sigma-Aldrich (Saint Louis,
MO, USA) or POCH (Polskie Odczynniki Chemiczne, Gliwice, Poland). Melting points
were determined by the capillary method using the Electrothermal 9100 apparatus and
were uncorrected. The nuclear magnetic resonance spectra were recorded in DMSO-d6
or CDCl3 on VMNRS300 spectrometer (Bruker, Billerica MA, USA) operating at 300 MHz
(1HNMR) and 75 MHz (13CNMR). Chemical shifts (
δ) are expressed in parts per million
relative to tetramethylsilane (TMS) used as the internal reference. Coupling constants (J)
values are given in hertz (Hz), and spin multiples are given as s (singlet), d (doublet),
t (triplet), and m (multiplet). Mass spectral ESI (electrospray ionization) measurements
were carried out on a MicrOTOF II Bruker instrument with a TOF detector. The spectra
were obtained in the positive ion mode. Flash chromatography was performed on Merck
Kieselgel (Darmstadt, Germany) 0.05–0.2 mm reinst (70–325 mesh ASTM) silica gel using
chloroform or chloroform–methanol as an eluent system at the v/v ratio of 50:0.2 or 50:0.5.
The progress of the reactions described in the experimental section was monitored by TLC
on silica gel (plates with fluorescent indicator 254 nm, layer thickness 0.2 mm, Kieselgel G.
Merck), using chloroform–methanol as an eluent system at the v/v ratio of 9.8:0.2 or 9.5:05.
4.1.1. Synthesis of Imides 1–5 and 1h
The starting imides 1–3 were prepared by the methods described earlier [10,32].
4.1.2. Synthesis of 1,7-Dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-
trione (1)
A mixture of 2,5-dimethyl-3,4-diphenylcyclopenta-2,4-dien-1-one (0.02 mol) and maleimide
(0.022 mol) was heated for 6 h in 20 mL of benzene. During the synthesis, the solution
became red, and a white solid precipitated from it. When the reaction was completed, the
solvent was evaporated under reduced pressure, and the solid residue was crystallized
from benzene.
◦
M.W. = 357.4018; C23H19NO3; Yield: 77%, white powder, m.p. 220.6–221.1 C (from
1
benzen); HNMR (300 MHz, DMSO-d6+ TMS,
δ
/ppm): 1.39 (6H, s, -CH3); 3.38 (2H, s,
C2-H, C6-H); 6.98 (4H, m, Ar-H); 7.19 (6H, m, Ar-H); 11.66 (1H, s, NH); Anal. Calc for
C23H19N3O3: 77.28% C; 5.32% H, 3.92% N; found 77.40% C; 5.30% H; 3.99% N; 13CNMR:
δ
11.7, 48.5, 55.5, 127.4, 127.9, 129.4, 133.2, 141.3, 177.5, 199.5; HRMS (m/z): calculated value
for [M + Na+] 380.1257; found 380.1259.
4.1.3. Synthesis of 1,7-Diethyl-8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-
trione (2)
A mixture of 2,5-diethyl-3,4-diphenylcyclopenta-2,4-dien-1-one (0.01 mol) and maleimide
(0.012 mol) in benzene (15 mL) was refluxed for 14 h. When the reaction was completed,
the solvent was evaporated under reduced pressure, and the solid residue was crystallized
from benzene.
M.W. = 385.455; C25H23NO3; Yield: 92%; white powder, m.p. 206.8–209 ◦C (from benzen);
1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 0.87 (6H, t, J = 7.5 Hz, -CH3); 2.05 (m, 4H,
-CH2-), 3.58 (2H, s, C2-H, C6-H); 6.97 (4H, m, Ar-H); 7.18 (6H, m, Ar-H); 11.70 (1H, s, NH);

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Anal. Calc for C25H23NO3: 77.93% C; 5.98% H, 3.63% N; found 77.94% C; 5.97% H; 3.62%
N; 13CNMR:
δ 9.1, 19.2, 43.9, 55.3, 60.4, 128.0, 129.4, 133.1, 142.4, 171.6, 197.8; HRMS (m/z):
calculated value for [M + Na+] 408.1570; found 408.1570.
4.1.4. Synthesis of 1,7,8,9-Tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (3)
A mixture of 1,2,3,4-tetraphenylcyclopenta-1,3-diene (0.02 mol) and maleimide (0.022 mol)
was heated for 6 h in 20 mL of benzene. When the reaction was completed, the solvent was
evaporated under reduced pressure, and the solid residue was crystallized from benzene.
◦
M.W. = 467.5571; C33H25NO2; Yield: 81%, white powder, m.p. 246.5–246.9 C (from
benzen); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ
/ppm): 2.23 (1H, d, J = 8.4, C10-H), 3.23
(1H, d, J = 8.7, C10-H), 4.20 (2H, s, C2-H, C6-H); 6.60 (4H, m, Ar-H); 6.88 (6H, m, Ar-H);
7.24 (6H, m, Ar-H); 7.74 (4H, d, J = 7.2, Ar-H); 11.44 (1H, s, NH); Anal. Calc for C33H25NO2:
84.79% C; 5.35% H, 3.00% N; found 84.85% C; 5.39% H; 3.08% N; 13CNMR:
δ 52.9, 62.9, 65.1,
126.4, 126.6, 127.1, 127.7, 129.1, 129.6, 134.5, 139.8, 145.0, 178.0; HRMS (m/z): calculated
value for [M + Na+] 490.1778; found 490.1777.
4.1.5. Synthesis of N-Hydroxyethylimides 4 and 5
A mixture of an appropriate diene:
- 2,5-dimethyl-3,4-diphenylcyclopenta-2,4-dien-1-one (0.02 mol) for compound 4
- 1,2,3,4-tetraphenylcyclopenta-1,3-diene (0.02 mol) for compound 5 and N-hydroxymaleimide
(0.022 mol) was heated for 6 h in 20 mL of benzene. When the reaction was completed, the
solvent was evaporated under reduced pressure, and the solid residue was crystallized
from benzene.
4-(2-hydroxyethyl)-1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-
trione (4)
M.W. = 401.4544; C25H23NO4; Yield: 84%; white powder, m.p. 186–187 ◦C (from MeOH/
(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ
/ppm): 1.57 (6H, s, -CH3), 2.16 (1H, s, -OH),
3.25 (2H, m, C2-H, C6-H), 3.73 (4H, m, -CH2-), 6.93 (4H, m, Ar-H), 7.26 (6H, m, Ar-H);
13CNMR:
δ 12.2, 47.4, 47.9, 56.5, 60.1, 127.7, 128.1, 129.4, 132.9, 141.5, 175.8, 176.1, 199.3;
HRMS (m/z): calculated value for [M + Na+] 424.1519; found 424.1519.
4-(2-hydroxyethyl)-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (5)
M.W. = 511.6096; C35H29NO; Yield: 61%, white powder, m.p. 197–198 ◦C (from benzen);
1HNMR (300 MHz, DMSO-d6+ TMS,
δ
/ppm): 2.12 (1H, br.s, -OH), 2.32 (1H, d, J = 4.5,
C6-H), 3.17 (1H, d, J = 4.5, C2-H), 3.74 (2H, m, C10-H, C20-H); 4.17 (2H, s, C10-H), 6.55 (4H,
m, Ar-H); 6.89 (6H, m, Ar-H); 7.32 (6H, m, Ar-H); 7.70 (4H, m, Ar-H); 13CNMR:
42.2, 52.2,
δ
60.5, 63.6, 65.9, 126.8, 127.1, 127.4, 128.1, 129.0, 129.8, 133.8, 139.2, 145.0, 177.1; HRMS (m/z):
calculated value for [M + Na+] 543.5988; found 534.2045.
4.1.6. Synthesis of 4-(2-Chloroethyl)-1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6
dec-8-ene-3,5,10-trione (1h)
]
To the solution of imide
a (0.01 mol) in acetonitrile (30 mL), 1,2-dichloroethane
(0.02 mol) was added. The reaction was heated for 72 h. When the reaction was com-
pleted, the solvent was evaporated under reduced pressure, and the residue was purified
by column chromatography on silica gel (eluent: chloroform).
4-(2-chloroethyl)-1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-
trione (1h)
◦
M.W. = 419.9000; C25H22ClNO3; Yield: 74%; white powder, m.p. 190–191 C (from hexane);
1HNMR (300 MHz, CDCl3,
δ
/ppm): 1.58 (6H, -CH3), 3.28 (2H, m, C2-H, C6-H), 3.64 (2H,
t, J = 6.4 Hz, C10-H,), 3.86 (2H, t, J = 6 Hz, C20-H), 6.91 (4H, m, Ar-H), 7.25 (6H, m, Ar-H);
13CNMR:
12.2, 39.5, 40.4, 47.9, 56.5, 127.7, 128,1, 129.4, 132.9, 141.5, 175.1, 199.2; HRMS
(m/z): calculated value for [M + Na+] 442.8893; found 442.1175.
δ

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4.1.7. General Conditions of Synthesis of New N-Alkylamino Derivatives of 1,7-Dimethyl-
8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione (1i–1j)
The 4-(2-chloroethyl)-1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-
trione was dissolved in acetonitrile (30 mL), and then anhydrous K2CO3 (0.01 mol) and
an appropriate amine (0.01 mol) were added. The reaction was carried out in reflux,
respectively, for 24–48 h in the boiling temperature of the solvent. The reaction was
monitored by TLC. When the reaction was completed, the solvent was evaporated, and
the residue was purified by column chromatography on silica gel (eluent: chloroform or
chloroform/methanol 50:0.2 v/v).
4-[2-(morpholin-4-yl)ethy]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-
3,5,10-trione (1j)
M.W. = 470.5595 (free amine); C29H30N2O4*HCl; Yield: 70%; white powder, m.p. 227–
228 ◦C (from EtOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 1.41 (6H, s,
-CH3), 3.09 (2H, m, -CH2-morph), 3.30 (2H, m, -CH2-morph), 3.39 (3H, m, C2-H, C6-H),
3.86 (4H, m, C10-H, -CH2-morph), 3.91 (2H, m, C20-H), 6.87 (4H, m, Ar-H), 7.18 (6H, m,
Ar-H), 11.57 (1H, br.s, NH+); 13CNMR:
δ 11.8, 32.5, 48.1, 50.9, 52.3, 55.6, 62.8, 127.5, 128,1,
129.2, 133.1, 141.4, 176.3, 199.2; HRMS (m/z): calculated value for [M + H+] 471.2278;
found 471.2276.
4-[2-(piperidin-1-yl)ethy]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-
3,5,10-trione (1i)
M.W. = 468.5866 (free amine); C30H32N2O3*HCl; Yield: 64%; white powder, m.p. 214–
◦
216 C (from EtOH/(Et)2O); 1HNMR (300 MHz, CDCl3,
δ/ppm): 1.42 (6H, -CH3), 1.76 (4H,
m, -CH2-piper), 2.85 (2H, m, C10-H), 3.16 (2H, m, C20-H), 3.36 (1H, m, C6-H), 3.49 (1H, m,
C2-H), 3.54 (4H, m, -CH2-piper), 3.82 (2H, m, -CH2-piper), 6.86 (4H, m, Ar-H), 7.19 (6H, m,
Ar-H), 10.37 (1H, br.s, NH+); 13CNMR:
δ 11.8, 15.1, 21.2, 22.0, 32.9, 48.0, 51.9, 55.6, 127.5,
128,1, 129.2, 133.0, 141.4, 176.1, 199.1; HRMS (m/z): calculated value for [M + H+] 469.2486;
found 469.2485.
4.1.8. General Conditions of the Synthesis of New Alkanoloamine Derivatives of
Dicarboximides (1b–1g, 2b–2g, 3b–3g)
The new alkanolamine derivatives were obtained in two-step reactions according to
the method described previously [10]. Step I: the mixture of an appropriate imide:
1: 1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione
2: 1,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione
3: 1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione
(0.01 mol) with 1-chloro-2,3-epoxypropane (50–60 mL), in the presence of an anhy-
drous K2CO3 (0.01 mol) was stirred on a magnetic stirrer under reflux with a tube with
CaCl2. The reaction was carried out. at room temperature for 15 h. When the reaction
was completed (TLC control), the inorganic precipitate was filtered off and the filtrate
was concentrated. The obtained oily product was purified by column chromatography on
silica gel (eluent: chloroform and chloroform/methanol 50:0.2). Step II: to the solution
of an appropriate 4-(oxirane-2-ylmethyl)-4-imide (1a, 2a, 3a) (0.001 mol) in a mixture of
methanol/water (10:1 v/v), the appropriate amine (0.001 mol) was added. Reactions were
carried out at room temperature on a magnetic stirrer under reflux for 15–20 h. When the
reaction was completed (TLC control), the excess of the solvent was evaporated and the
crude product was purified by column chromatography on silica gel (eluent: chloroform or
chloroform/methanol (50:0.2, 50:0.5)).
4-[3-(ethylamino)-2-hydroxypropyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo [5.2.1.02,6
]
dec-8-ene-3,5,10-trione (1b)
M.W. = 458.5488 (free amine); C28H30N2O4*HCl; Yield: 55%; white powder, m.p. 198–
201 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
/ppm): 1.18 (3H, t,
δ

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J = 7.2 Hz, -CH3), 1.42 (6H, s, -CH3), 2.78 (1H, m, C6-H), 2.80 (2H, m, -CH2-) 3.05 (1H, m,
C2-H), 3.37(2H, m, C30-H), 3.51 (2H, m, C10-H), 4.10 (1H, s, C20-H), 5.88 (1H, s, -OH), 6.87
(4H, m, Ar-H), 7.18 (6H, m, Ar-H), 8.65 (1H, br.s, -NH2+), 9.09 (1H, br.s, -NH2+); 13CNMR:
δ
10.7, 11.8, 42.3, 42.7, 47.4, 47.6, 49.3, 55.7, 63.4, 127.5, 128,1, 129.3, 133.2, 141.3, 175.8, 176.2,
199.4; HRMS (m/z): calculated value for [M + H+] 459.2284; found 459.2284.
4-[3-(tert-butylamino)-2-hydroxypropyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo [5.2.1.02,6
]
dec-8-ene-3,5,10-trione (1c)
M.W. = 486.6019 (free amine); C30H34N2O4*HCl; Yield: 57%; white powder, m.p. 248–
250 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 1.26 (9H, s,
-C(CH3)3), 1.43 (6H, d, J = 5.1 Hz, -CH3), 2.50 (1H, m, C6-H), 3.16 (1H, m, C2-H), 3.52 (4H,
m, C30-H, C10-H), 4.14 (1H, m, C20-H), 5.83 (1H, s, -OH), 6.88 (4H, m, Ar-H), 7.18 (6H, m,
Ar-H), 8.39 (1H, br.s, -NH2+), 9.04 (1H, br.s, -NH2+); 13CNMR:
δ 11.8, 24.9, 42.8, 44.5, 47.3,
47.6, 48.5, 55.6, 56.2, 63.7, 127.4, 128.0, 129.3, 133.2, 141.3, 175.7, 176.3, 199.4; HRMS (m/z):
calculated value for [M + H+] 487.2598; found 487.2597.
4-[3-(diethylamino)-2-hydroxypropyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6
]
dec-8-ene-3,5,10-trione (1d)
M.W. = 486.6019 (free amine); C30H34N2O4*HCl; Yield: 72%; white powder, m.p. 177–
179 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 1.18 (6H, t,
J = 7.2 Hz, -CH3), 1.42 (6H, d, J = 4.2 Hz, -CH3), 3.16 (6H, m, C6-H, C2-H, C30-H, C10-H),
3.52 (2H, m, -CH2-), 3.73 (2H, m, -CH2-), 4.19 (1H, s, -OH), 6.87 (4H, m, Ar-H), 7.17 (6H, m,
Ar-H), 9.87 (1H, br.s, -NH+); 13CNMR:
δ 8.3, 11.8, 42.8, 46.7, 47.4, 47.6, 54.0, 55.7, 62.5, 127.5,
128.0, 129.3, 133.1, 141.3, 175.8, 176.2, 199.3; HRMS (m/z): calculated value for [M + H+]
487.2598; found 487.2597.
4-[3-(dibutylamino)-2-hydroxypropyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo [5.2.1.02,6
]
dec-8-ene-3,5,10-trione (1e)
M.W. = 542.7082 (free amine); C34H42N2O4*HCl; Yield: 74%; white powder, m.p. 192–
196 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 0.89 (6H, t,
J = 7.35 Hz, -CH3), 1.27 (4H, m, -CH2-), 1.42 (6H, d, J = 4.2 Hz, -CH3), 1.60 (4H, m, -CH2-),
3.05 (4H, m, -CH2-), 3.20 (1H, m, C6-H), 3.31 (2H, m, C30-H), 3.51 (3H, m, C10-H, C20-H),
4.19 (1H, m, C20-H) 5.94 (1H, s, -OH), 6.88 (4H, m, Ar-H), 7.17 (6H, m, Ar-H), 9.81 (1H, br.s,
-NH+); 13CNMR:
δ 11.8, 13.4, 19.3, 24.5, 47.4, 47.6, 52.2, 52.8, 55.1, 55.7, 62.6, 127.5, 128.0,
129.3, 133.1, 141.3, 175.8, 176.2, 199.2; HRMS (m/z): calculated value for [M + H+] 543.3224;
found 543.3223.
4-[2-hydroxy-3-(piperidin-1-yl)propyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6
]
dec-8-ene-3,5,10-trione (1f)
M.W. = 498.6126 (free amine); C31H34N2O4*HCl; Yield: 45%; white powder, m.p. 170–
171 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 1.42 (6H, m,
-CH3), 1.74 (4H, m, -CH2-pip), 2.50 (3H, m, C6-H, C30-H), 2.88 (2H, m, -C10-H), 3.32 (7H,
m, C2-H, -CH2-pip), 4.42 (1H, m, C20-H) 5.97 (1H, s, -OH), 6.88 (4H, m, Ar-H), 7.18 (6H, m,
Ar-H), 9.97 (1H, br.s, -NH+); 13CNMR:
δ 11.8, 21.2, 22.1, 42.7, 47.6, 52.1, 53.4, 55.6, 58.7, 62.3,
127.5, 128.0, 129.3, 133.1, 141.4, 175.8, 176.1, 199.3; HRMS (m/z): calculated value for [M +
H+] 499.2598; found 499.2597.
4-[-2-hydroxy-3-(morpholin-4-yl)propyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclo [5.2.1.02,6
]
dec-8-ene-3,5,10-trione (1g)
M.W. = 500.5854 (free amine); C30H32N2O5*HCl; Yield: 54%; white powder, m.p. 210–
◦
1
212 C (from MeOH/(Et)2O); HNMR (300 MHz, DMSO-d6+ TMS, δ/ppm): 1.42 (6H,
m, -CH3), 3.07 (2H, m, C2-H, C6-H), 3.38 (6H, m, -CH2-morpholin), 3.52 (2H, m, -CH2-
morpholin), 3.88 (4H, m, C30-H, C10-H), 4.30 (1H, m, C20-H), 6.01 (1H, s, -OH), 6.87 (4H, m,
Ar-H), 7.18 (6H, m, Ar-H), 10.07 (1H, br.s, -NH+); 13CNMR:
δ 11.8, 42.6, 47.5, 51.0, 52.5, 55.6,

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58.7, 62.2, 62.9, 127.5, 128.1, 129.3, 133.1, 141.4, 175.8, 176.1, 199.3; HRMS (m/z): calculated
value for [M + H+] 501.2384; found 501.2379.
4-[3-(ethylamino)-2-hydroxypropyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo [5.2.1.02,6]dec-
8-ene-3,5,10-trione (2b)
M.W. = 486.6019 (free amine); C30H34N2O4*HCl; Yield: 55%; white powder, m.p. 197–
200 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 0.89 (6H, m,
-CH3), 1.18 (3H, t, J = 7.2 Hz, -CH3), 1.87 (2H, m, -CH2-), 2.11 (2H, m, -CH2-), 2.80 (1H, m,
0
0
C6-H), 2.88 (2H, m, -CH2-) 3.06 (1H, m, C2-H), 3.41(2H, m, C3 -H), 3.54 (2H, m, C1 -H), 4.13
(1H, s, C20-H), 5.89 (1H, br.s, -OH), 6.87 (4H, m, Ar-H), 7.16 (6H, m, Ar-H), 8.67 (1H, br.s,
-NH2+), 9.10 (1H, br.s, -NH2+); 13CNMR:
δ 9.0, 10.7, 18.9, 39.5, 42.2, 42.7, 43.6, 43.7, 49.2,
59.2, 63.3, 127.4, 128.0, 129.2, 133.5, 141.7, 141.8, 176.0, 176.4, 199.0; HRMS (m/z): calculated
value for [M + H+] 487.6093; found 487.2591.
4-[3-(tert-butylamino)-2-hydroxypropyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo [5.2.1.02,6
]
dec-8-ene-3,5,10-trione (2c)
M.W. = 514.6551 (free amine); C32H38N2O4*HCl; Yield: 67%; white powder, m.p. 231–
235 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 0.88 (6H, d, J
= 5.1 Hz, -CH3), 1.26 (9H, m, -C(CH3)3), 1.87 0(2H, m, -CH2-), 2.09 (2H, m, -CH2-), 2.78 0(1H,
0
m, C6-H), 3.08 (1H, m, C2-H), 3.40 (2H, m, C3 -H0, 3.69 (2H, m, C1 -H), 4.14 (1H, m, C2 -H),
5.80 (1H, s, -OH), 6.88 (4H, m, Ar-H), 7.16 (6H, m, Ar-H), 8.36 (1H, br.s, -NH2+), 8.86 (1H,
br.s, -NH2+); 13CNMR:
δ 9.0, 18.8, 24.9, 42.9, 43.6, 44.3, 56.2, 59.2, 59.3, 63.6, 127.4, 128.0,
129.1, 133.5, 133.5, 141.7, 141.8, 175.9, 176.5, 198.9; HRMS (m/z): calculated value for [M +
H+] 515.6625; found 515.2904.
4-[3-(diethylamino)-2-hydroxypropyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo [5.2.1.02,6
]
dec-8-ene-3,5,10-trione (2d)
M.W. = 514.6551 (free amine); C30H34N2O4*HCl; Yield: 62%; white powder, m.p. 177–
180 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
/ppm): 0.88 (6H, m,
-CH3), 1.19 (6H, t, J = 7.1 Hz, -CH3), 2.06 (2H, m, -CH2-), 2.08 (2H, m, -CH2-),3.16 (6H, m,
C6-H, C2-H, C30-H, C10-H), 3.51 (4H, m, -CH2-), 4.20 (1H, m, C20-H), 5.98 (1H, br. s, -OH),
6.88 (4H, m, Ar-H), 7.16 (6H, m, Ar-H), 9.87 (1H, br.s, -NH+); 13CNMR:
9.3, 9.0, 18.8, 42.8,
δ
·
δ
43.7, 46.7, 47.4, 48.5, 54.0, 59.2, 62.5, 127.4, 128.0, 129.1, 133.5, 141.7, 176.0, 176.4, 199.0;
HRMS (m/z): calculated value for [M + H+] 515.6625; found 515.2904.
4-[3-(dibutylamino)-2-hydroxypropyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6
]
dec-8-ene-3,5,10-trione (2e)
◦
M.W. = 570.7614 (free amine); C36H46N2O4*HCl; Yield: 64%; white powder, m.p. 92–110 C
(from MeOH/(Et)2O); 1H NMR (300 MHz, DMSO-d6+ TMS,
δ
/ppm): 0.88 (12H, m, -CH3),
1.29 (4H, m, -CH2-), 1.62 (4H, m, -CH2-), 1.87 (2H, m, -CH2-), 2.07 (2H, m, -CH2-), 3.06 (4H,
m, -CH2-), 3.19 (2H, m, C3-H), 3.52 (2H, m, C10-H), 3.70 (2H, m, C2-H, C6-H), 4.23 (1H, m,
C20-H), 6.88 (4H, m, Ar-H), 7.16 (6H, m, Ar-H), 9.99 (1H, br.s, -NH+); 13CNMR:
δ 9.0, 13.5,
18.9, 19.4, 24.5, 42.8, 43.6, 52.3, 52.7, 55.2, 59.2, 62.6, 127.4, 127.9, 129.1, 133.5, 141.7, 176.0,
176.4, 198.9; HRMS (m/z): calculated value for [M + H+] 571.7688; found 571.3530.
4-[2-hydroxy-3-(piperidin-1-yl)propyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6
]
dec-8-ene-3,5,10-trione (2f)
M.W. = 526.6658 (free amine); C33H38N2O4*HCl; Yield: 55%; white powder, m.p. 190–
195 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
/ppm): 0.89 (6H, m,
δ
-CH3), 1.36 (2H, m, -CH2-), 1.74 (4H, m, -CH2-, -CH2-pip), 1.87 (2H, m, -CH2-pip), 2.07 (2H,
m, -CH2-pip), 2.90 (4H, m, C30-H, -CH2-pip), 3.16 (2H, m, -CH2-pip), 3.46 (1H, m, C6-H),
3.49 (2H, C10-H, C2-H), 4.26 (1H, m, C20-H), 6.87 (4H, m, Ar-H), 7.16 (6H, m, Ar-H), 10.08
(1H, br.s, -NH+); 13CNMR:
δ 9.0, 18.8, 21.2, 22.1, 42.7, 43.7, 52.1, 53.3, 58.8, 59.2, 62.3, 127.4,
128.0, 129.1, 133.5, 141.7, 176.0, 176.3, 199.01; HRMS (m/z): calculated value for [M + H+]
527.6732; found 527.2904.

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4-[-2-hydroxy-3-(morpholin-4-yl)propyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6
]
dec-8-ene-3,5,10-trione (2g)
M.W. = 528.6386 (free amine); C32H36N2O5*HCl; Yield: 54%; white powder, m.p. 226–
◦
1
228 C (from MeOH/(Et)2O); HNMR (300 MHz, DMSO-d6+ TMS, δ/ppm): 0.89 (6H,
m, -CH3), 1.87 (2H, m, -CH2-), 2.06 (2H, m, -CH2-), 3.1 (3H, m, C3-H, C6-H), 3.36 (3H,
m, -CH2-morpholin, C2-H), 3.47 (4H, m, -CH2-morpholin), 3.89 (4H, m, -CH2-morpholin,
C10-H), 4.30 (1H, m, C20-H), 6.01 (1H, br.s, -OH), 6.87 (4H, m, Ar-H), 7.68 (6H, m, Ar-H),
10.57 (1H, br.s, -NH+); 13CNMR:
δ 9.0, 18.8, 39.5, 43.7, 51.0, 52.5, 58.8, 59.2, 62.1, 62.9, 127.4,
128.0, 129.1, 133.5, 141.7, 176.0, 176.3, 199.0; HRMS (m/z): calculated value for [M + H+]
529.6460; found 529.2697.
4-[3-(ethylamino)-2-hydroxypropyl]-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-
3,5-dione (3b)
M.W. = 568.7040 (free amine); C38H36N2O3*HCl; Yield: 45%; white powder, m.p. 254–
255 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ
/ppm): 1.19 (3H, t,
J = 7.2 Hz, -CH3), 2.34 (1H, m, C10-H), 2.78 (1H, m, C10-H), 2.91 (2H, m, -CH2-) 3.05 (1H, m,
0
0
0
C2-H), 3.16 (1H, m, C6-H), 3.30 (2H, m, C3 -H), 3.46 (1H, s, C2 -H), 4.29 (2H, m, C1 -H), 5.85
(1H, s, -OH), 6.51 (4H, m, Ar-H), 6.88 (6H, m, Ar-H), 7.32 (6H, m, Ar-H), 7.76 (4H, m, Ar-H),
8.60 (1H, br.s, -NH2+), 8.94 (1H, br.s, -NH2+); 13CNMR:
δ 10.7, 42.2, 49.4, 51.9, 63.0, 63.4,
65.1, 126.5, 126.8, 127.2, 127.8, 129.2, 129.5, 134.4, 139.7, 145.1, 176.6, 176.9; HRMS (m/z):
calculated value for [M + H+] 569.7119; found 569.2798.
4-[3-(tert-butylamino)-2-hydroxypropyl]-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-
8-ene-3,5-dione (3c)
M.W. = 596.7575 (free amine); C40H40N2O3*HCl; Yield: 60%; white powder, m.p. 177–
179 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 1.28 (9H, s,
-C(CH3)3), 2.36 (1H, d, J = 8.7 Hz C6-H), 2.76 (1H, m, C10-H), 3.06 (1H, m, C10-H), 3.27 (1H,
m, C2-H), 3.35 (4H, m, C30-H, C10-H), 3.52 (1H, m, C20-H), 4.21 (1H, s, -OH), 6.51 (4H, m,
Ar-H), 6.88 (6H, m, Ar-H), 7.31 (6H, m, Ar-H), 7.76 (4H, m, Ar-H), 8.41 (1H, br.s, -NH2+), 9.06
(1H, br.s, -NH2+); 13CNMR:
δ 24.9, 42.7, 44.6, 51.7, 52.0, 56.2, 63.0, 63.7, 65.1, 126.4, 126.5,
127.2, 127.8, 129.1, 129.5, 134.4, 139.7, 145.0, 145.1, 176.5, 177.0; HRMS (m/z): calculated
value for [M + H+] 597.7651; found 597.3117.
4-[3-(diethylamino)-2-hydroxypropyl]-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-
ene-3,5-dione (3d)
M.W. = 596.7575 (free amine); C40H40N2O3*HCl; Yield: 62%; white powder, m.p. 240–
247 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 1.20 (6H, t,
J = 7.2 Hz, -CH3), 2.33 (1H, d, J = 8.4 Hz, C6-H), 3.16 (6H, m, -CH2-, -CH2- C30-H, C10-H),
3.38 (1H, m, C2-H), 3.50 (1H, m, C20-H), 4.26 (2H, m, C10-H), 5.98 (1H, s, -OH), 6.52 (4H,
m, Ar-H), 6.88 (6H, m, Ar-H), 7.31 (6H, m, Ar-H), 7.77 (4H, m, Ar-H), 9.93 (1H, br.s, -NH+);
13CNMR:
δ 8.4, 42.7, 46.7, 47.4, 51.9, 52.0, 54.2, 62.5, 63.0, 65.1, 126.5, 126.8, 127.2, 127.8,
129.2, 129.5, 134.4, 139.7, 145.0, 145.1, 176.6, 177.0; HRMS (m/z): calculated value for [M +
H+] 597.7651; found 597.3117.
4-[3-(dibutylamino)-2-hydroxypropyl]-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-
ene-3,5-dione (3e)
M.W. = 652.8635 (free amine); C44H48N2O3*HCl; Yield: 64%; white powder, m.p. 237–
238 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 0.90 (6H, t,
J = 7.2 Hz, -CH3), 1.29 (4H, m, -CH2-), 1.62 (4H, m, -CH2-), 2.37 (1H, m, C6-H), 3.16 (4, m,
-CH2-), 3.34 (8H, m, C2-H, C10-H, C20-H, C30-H, C10-H), 5.94 (1H, s, -OH), 6.51 (4H, m,
Ar-H), 6.88 (6H, m, Ar-H), 7.31 (6H, m, Ar-H), 7.74 (4H, m, Ar-H), 9.71 (1H, br.s, -NH+);
13CNMR:
δ 13.5, 19.4, 24.6, 48.5, 51.8, 52.0, 52.3, 52.7, 55.4, 62.6, 63.0, 65.1, 126.5, 126.8, 127.3,
127.8, 129.2, 129.5, 134.4, 139.7, 145.0, 145.1, 176.6, 177.0; HRMS (m/z): calculated value for
[M + H+] 653.8714; found 653.3743.

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4-[2-hydroxy-3-(piperidin-1-yl)propyl]-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-
ene-3,5-dione (3f)
M.W. = 608.7679 (free amine); C41H40N2O3*HCl; Yield: 54%; white powder, m.p. 268–
270 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 1.35 (1H, m,
C2-H), 1.75 (5H, m, -CH2-pip., C6-H), 2.36 (1H, m, C10-H), 2.88 (2H, m, C10-H), 2.99 (1H,
m, C20-H), 3.40 (9H, m, -CH2-pip, C30-H, C10-H), 5.98 (1H, s, -OH), 6.51 (6H, m, Ar-H),
6.88 (4H, m, Ar-H), 7.31 (6H, m, Ar-H), 7.74 (4H, m, Ar-H), 9.93 (1H, br.s, -NH+); 13CNMR:
δ
21.22, 22.1, 42.6, 51.9, 53.4, 59.0, 62.28, 63.0, 65.1, 126.5, 126.7, 127.2, 127.8, 129.1, 129.5,
+
134.3, 139.7, 144.9, 145.1, 176.6, 176.9; HRMS (m/z): calculated value for [M + H ] 609.7758;
found 609.3117.
4-[-2-hydroxy-3-(morpholin-4-yl)propyl]-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-
8-ene-3,5-dione (3g)
M.W. = 610.7407 (free amine); C40H38N2O3*HCl; Yield: 64%; white powder, m.p. 276–
277 ◦C (from MeOH/(Et)2O); 1HNMR (300 MHz, DMSO-d6+ TMS,
δ/ppm): 2.34 (1H, m,
C6-H), 3.07 (3H, m, -CH2-morpholin, C2-H), 3.29 (5H, m, -CH2-morpholin, C10-H), 3.46
0
0
0
(3H, m, C3 -H, C2 -H), 3.73 (3H, m, C1 -H, C10-H), 3.92 (2H, m, -CH2-morpholin), 6.48 (4H,
m, Ar-H), 6.88 (6H, m, Ar-H), 7.31 (6H, m, Ar-H), 7.74 (4H, m, Ar-H), 10.45 (1H, br.s, -NH+);
13CNMR:
δ 21.22.0, 50.7, 51.9, 52.7, 58.9, 61.9, 63.0, 65.0, 126.5, 126.8, 127.2, 127.8, 129.1, 129.5,
145.1, 176.6, 176.9; HRMS (m/z): calculated value for [M + H+] 611.7486; found 611.2904.
4.1.9. Preparation of Acetate Salt of Derivatives Ic, Ie, II
Alkylamine derivatives were obtained according to the methods published previ-
ously [9,10]. Next, the appropriate derivative (0.01 mol) was dissolved in acetic acid
(60%) and heated to boiling. After cooling, the excess acid was evaporated to give a
crystalline precipitate.
Acetate of 4-[2-(piperidin-1-yl)ethyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]-dec-
8-ene-3,5,10-trione (Ic*CH3COOH)
M.W. = 496.2720 (free amine); C32H36N2O3*CH3COOH; Yield: 64%; white powder, m.p. 85–
◦
89 C (from CH3COOH); 1HNMR (300 MHz, CDCl3,
δ/ppm): 0.99 (6H, t, -CH3, J = 7.5 Hz),
1.46 (2H, m, -CH2-), 1.62 (4H, m, -(CH2)2-), 2.02 (2H, m, -CH2-piper), 2.19 (2H, m, -CH2-),
2.67 (4H, m, -CH2-piper), 3.48 (2H, m, C2-H, C6-H), 3.52 (2H, m, -CH2-piper), 3.74 (2H, m,
-CH2-piper), 6.90 (4H, m, Ar-H), 7.14 (6H, m, Ar-H); 13CNMR:
δ 9.0, 18.9, 23.7, 25.2, 36.2,
43.5, 53.8, 55.2, 59.3, 127.4, 127.9, 129.1, 133.5, 141.5, 176.0, 199.8; HRMS (m/z): calculated
value for [M + H+] 497.2799; found 498.2799.
Acetate of 4-[2-hydroxy-3-(propan-2-ylamino)propyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo
[5.2.1.02,6]dec-8-ene-3,5,10-trione (Ie * CH3COOH)
M.W. = 500.2669 (free amine); C31H36N2O4*CH3COOH; Yield: 77%; white powder, m.p.
◦
1
156–158 C (from CH3COOH); HNMR (300 MHz, CDCl3, δ/ppm): 0.89 (6H, t, -CH3,
J = 7.5 Hz), 1.24 (6H, d, -CH3, J = 6.3 Hz), 2.00 (2H, m, -CH2-), 2.19 (2H, m, -CH2-), 2.80 (1H,
m, C10-H), 2.89 (1H, m, C10-H), 3.11 (1H, m, -CH-), 3.57 (3H, m, C20-H, C30-H), 3.65 (2H, m,
C2-H, C6-H), 4.17 (1H, m, -OH), 6.87 (4H, m, Ar-H), 7.17 (6H, m, Ar-H), 13CNMR:
δ 9.0, 18.9,
21.7, 43.5, 48.4, 50.0, 59.2, 65.7, 127.3, 127.9, 129.2, 133.5, 141.7, 172.7, 176.2, 198.9; HRMS
(m/z): calculated value for [M + H+] 501.2748; found 501.2748.
Acetate of 4-[2-(dimethylamino)ethyl]-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-
ene-3,5-dione (II*CH3COOH)
M.W. = 538.2614 (free amine); C37H34N2O2*CH3COOH, Yield: 82%; white powder, m.p.
203–205 ◦C (from CH3COOH); 1HNMR (300 MHz, CDCl3,
δ/ppm): 2.32 (6H, m, -CH3),
2.35 (1H, m, C10-H), 2.65 (2H, t, J = 6.3, C10-H), 3.17 (1H, m, C10-H), 3.64 (2H, t, J = 6.3,
0
C2 -H), 4.18 (2H, s, C2-H, C6-H), 6.55 (4H, m, Ar-H), 6.92 (6H, m, Ar-H), 7.27 (6H, m, Ar-H),
7.72 (4H, m, Ar-H); 13CNMR:
δ 21.0, 36.5, 44.8, 51.8, 55.7, 63.0, 65.3, 126.4, 126.7, 127.1, 127.7,

Int. J. Mol. Sci. 2021, 22, 4318
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129.1, 129.5, 134.3, 139.8, 144.9, 176.6; HRMS (m/z): calculated value for [M + H+] 539.2693;
found 539.2693.
4.1.10. Determination of Lipophilicity by Reversed-Phase Chromatography
The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis
was performed on a Merck–Hitachi LaChrom Elite System (Merck, Darmstadt, Germany)
with diode array detector L-2455, thermostat L-2300, pump L-2130, and autosampler
L-2200 and with the Waters XTerra MS RP-18 (3.5
µ
m, 150
×
4.6 mm) chromatographic
column (Waters, Milford, MA, USA) as the stationary phase. The mobile phase consisted of
methanol, water, and 0.1% (v/v) formic acid and was degassed by the use of the built-in
membrane degasser. The methanol gradient grade for HPLC was purchased from Merck
(Merck, Darmstadt, Germany) and formic acid from Polish Reagents (Polish Reagents,
Gliwice, Poland); double-distilled water was used. Then, 5 µL of methanolic solutions
(0.1%; m/v) of selected samples were applied to the chromatographic column by the use
of an autosampler Hitachi L-2200 (LaChrom Elite, Hitachi-Merck, Darmstadt, Germany).
The analysis was performed with the flow rate of 0.7 mL min−1 in isocratic mode using
various concentrations of organic modifier (methanol) in binary polar mobile phases;
percentages of methanol in water were 45–70% (%, v/v) and changed by 5% per step.
◦
Chromatograms were detected at 254 nm and the temperature of the column was 25 C. All
experiments were repeated in triplicate, and the final results were taken to be the arithmetic
means. Dead time was measured by the use of uracil (Calbiochem. Merck, Darmstadt,
Germany). Statistical and regression analyses were performed using Statistica (ver. 13.3
for Windows). The chromatographic lipophilicity parameters (logkW) for selected samples
were obtained by the extrapolation of the retention parameter logk to pure water, according
to Equation (1):
logk = logkw − S·φ;
(1)
where logkW is the value of the retention factor of a substance in pure water,
S
is the slope
of the regression curve, and
φ is the concentration of the organic modifier [33]. The values
of logk were calculated based on the raw HPLC data using the formula (2):
ꢀ
ꢁ
tR − t0
logk = log
;
(2)
t0
where tR is the retention time and t0 is the dead retention time (determined for uracil).
4.2. Anticancer Activity
4.2.1. Cells and Cytotoxicity Assay
Human umbilical vein endothelial cells (Life Technologies, Carlsbad, CA, USA) were
cultured (according to the manufacturer’s instructions) in Medium 200 containing low
serum growth supplement; 1
Roskilde, Denmark). The HeLa (human cervix carcinoma), K562, and MOLT-4 (leukemia)
cells were cultured in RPMI 1640 medium supplemented with antibiotics and 10% fetal
×
104 cells were seeded on each well on a 96-well plate (Nunc,
calf serum in a 5% CO2–95% air atmosphere; 7
×
103 cells were seeded on each well on a
96-well plate (Nunc). Twenty-four hours later, cells were exposed to the test compounds
for additional 48 hours. Stock solutions of test compounds were freshly prepared in
DMSO. The final concentrations of the compounds tested in the cell cultures were 2 × 10−1
,
1 × 10−1, 5
×
10−2, 1
×
10−2, 1
×
10−3, and 1
×
10−4 mM. The concentration of DMSO in
the cell culture medium was 1%. The values of IC50 (the concentration of test compound
required to reduce the cell survival fraction to 50% of the control) were calculated from
dose–response curves and used as a measure of cellular sensitivity to a given treatment.
The cytotoxicity of all compounds was determined by the MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO, USA] assay. Briefly, after 24 or
48 h of incubation with drugs, the cells were treated with the MTT reagent, and incubation
was continued for 2 h. MTT-formazan crystals were dissolved in 20% SDS and 50% DMF

Int. J. Mol. Sci. 2021, 22, 4318
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at pH 4.7, and absorbance was read at 570 and 650 nm on a microplate reader FLUOstar
Omega (BMG Labtech, Offenburg, Germany). As a control (100% viability), we used cells
grown in the presence of the vehicle (1% DMSO) only.
4.2.2. Analysis of Cell Apoptosis by Caspase-3/7 Activity Assay
First, 20
×
103 K562 cells were seeded on each well of a 96-well plate in RPMI 1640
medium supplemented with 10% fetal calf serum and antibiotics. Cells were grown for
24 h at 37 ◦C and 5% CO2. Test dicarboximides were dissolved in DMSO and added to
cell culture to the final concentration of 5
×
IC50. Cells treated with 1% DMSO served
as a negative control, while cells incubated with staurosporine (Sigma, St. Louis, MO,
USA)◦were used as a positive control. Cells were exposed to test compounds for 18 h
at 37 C and 5% CO2. Subsequently, the activity of caspase 3 and 7 was measured by
Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega, Madison, WI, USA) according
to the manufacturer’s instructions. Briefly, the cells were lysed and incubated for 1.5 h
with a profluorescent substrate for caspase 3 and 7. Next, the fluorescence was read at
an excitation wavelength of 485 nm and emission of 520 nm with a FLUOStar Omega
microplate reader (BMG-Labtech, Offenburg, Germany).
4.2.3. Gene Expression Analysis with DNA Microarray
The expression of apoptotic genes was studied using predesigned 384-well microflu-
idic cards (TLDA TaqMan®human apoptosis array, Life Technologies, Applied Biosystems,
Foster City, CA, USA). The RT-PCR experiments were executed twice, each on three sepa-
rate TLDA plates. K562 tumor cells were seeded on a six-well plate (Nunc) in an amount of
3
×
106 cells/well and were exposed to the test compounds at a concentration of 5
×
IC50
for another 18 h. The control cells were exposed to 1% DMSO or 1
µM staurosporine. The
total RNA pool was isolated from the cell lysates using TriPure Isolation Reagent (Roche,
Basel, Switzerland) according to the manufacturer’s instruction. The RNA quality was
checked with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA). Two micrograms of total RNA were reverse transcribed using a High Capacity
cDNA Reverse Transcription kit (Applied Biosystems, Life Technologies). The 384-well
TLDA (the qRT-PCR based TaqMan low-density array) cards were configured into four
identical 96-gene sets consisting of 93 human genes and three endogenous controls: 18S,
ACTB, and GAPDH. The primers (TaqMan Gene Expression Assay, Applied Biosystems)
were selected to produce amplicons not longer than 100 bp (a mean of 74 bp). Those
93 genes were categorized into multiple classes including intrinsic, extrinsic, regulatory,
and execution apoptosis traits. A reaction mixture with a cDNA template (100 ng) and an
equal volume of TaqMan® universal master mix (Applied Biosystems) was immediately
loaded into each line of the TLDA microfluidic card. Each card was spun twice (each time
for 1 min) at 1200 rpm, then was sealed and loaded into an ABI 7900HT fast real-time PCR
system◦(Applied Bios◦ystems, Waltham, MA, USA), operating for 2 min at 50 ◦C, 10 min
◦
at 94.5 C, 30 s at 97 C, and 1 min at 59.7 C (40 cycles). The results were statistically
analyzed using the Student’s t-test, assuming a level of significance value of p < 0.05. The
MetaCoreTM software (Thomson Reuters, from GeneGo) was used to perform the pathway
analysis of the differentially expressed genes.
4.2.4. MAPKs Immunoblotting
First, 2
×
106 K562 cells per well were seeded on a six-well plate in the complete
M JNK kinase inhibitor SP600125 or 10 µM
medium (RPMI 1640 + 10% FBS). Then, 20
µ
p38 inhibitor SB203580 (Cell Signaling Technology, Danvers, MA, USA) was added for 24 h.
Alternatively, K562 cells were exposed to 1% DMSO (control sample), test compound Ic
(6
µM), II (3
µM), or staurosporine (1
µM) for 18 h. Cells exposed to staurosporine served
as a positive control in ERK1/2 experiments. K562 cells treated with 20
µM anisomycin
(BioShop, Burlington, ON, Canada) for 30 min served as a positive control in JNK and p38
◦
experiments. After incubation, cells were centrifuged (600 rpm, 6 min, 24 C), washed

Int. J. Mol. Sci. 2021, 22, 4318
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once with PBS, and lysed in the buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl,
and 1% Triton X-100, containing protease (complete protease inhibitor cocktail, Roche)
and phosphatase inhibitors (PhosSTOP phosphatase inhibitor cocktail, Roche). Cells were
lysed for 10 min on ice and centrifuged (12,000 rpm, 10 min, 4 ◦C), and supernatants were
collected. Total protein concentration in cell lysates was measured with DC Protein Assay
(Bio-Rad, Hercules, CA, USA). Cell lysates containing 25
µg of total protein were mixed
with Laemli buffer supplemented with SDS and
β-Mercaptoethanol, denatured (10 min,
95 ◦C), and subjected to SDS-PAGE electrophoresis (4% stacking and 10% resolving gels).
Resolved proteins were electrotransferred (semi-dry, 90 min, 1.5 mA/cm2) to nitrocellulose
membranes (0.45 µm, ThermoScientific). Membranes were blocked for 2 h in TBST buffer
(20 mM Tris-HCl pH 7.5, 0.9% NaCl, 0.1% Tween 20) containing 5% BSA (BioShop) and
probed with primary antibodies (overnight at 4 ◦C) diluted in TBST buffer with 0.5% BSA.
Tubulin served as a loading control for IB experiments. Anti-phospho ERK1/2 (rabbit
polyclonal, dilution 1:1000) and anti-ERK1/2 (rabbit polyclonal, dilution 1:1000) were from
Cell Signaling Technology. Anti-phospho JNK (mouse polyclonal, dilution 1:250) and
anti-JNK (mouse polyclonal, dilution 1:250) were from Santa Cruz Biotechnology (Dallas,
TX, USA). Anti-phospho p38 (mouse polyclonal, dilution 1:250) and anti-p38 (mouse
polyclonal, dilution 1:250) were from BD Transduction Laboratories (San Diego, CA, USA).
Anti-α-tubulin (mouse monoclonal IgG, 1:2000) was from Sigma. After extensive washing
with the TBST buffer, membranes were probed with secondary goat anti-rabbit (1:5000 in
TBST buffer) or goat anti-mouse HRP-conjugated IgG (1:5000 in TBST buffer, from Santa
Cruz Biotechnology) for 45 min at room temperature. Membranes were washed in the
TBST buffer, and the chemiluminescent signal was developed with ECL Plus Western
Blotting Substrate (Thermo Scientific, Waltham, MA, USA) and visualized in a Syngene
G:Box detection system.
4.2.5. Digestion of Plasmid DNA with BamHI Restriction Nuclease
First, 0.5 µg of plasmid DNA (pcDNAHisC, total length 5.5 kbp) containing a unique
BamHI restriction site was dissolved in a 1
×
BamHI reaction buffer and incubated overnight
at 37 ◦C with the test compounds or daunorubicin. Daunorubicin, a strong intercalating
agent, was used as a positive control. The concentration of the test compounds and
daunorubicin samples was 100
with BamHI restriction endonuclease (2 U/
was 10 L. Products of the reaction were subjected to the 1% agarose gel electrophoresis
µ
M. In the next step, the reaction mixtures were digested
µ
L) for 3 h at 37 ◦C. The total reaction volume
µ
in TBE buffer. The gel was stained with ethidium bromide, and DNA fragments were
visualized under a UV lamp (GBox, Syngene).
Supplementary Materials: Supplementary Materials are available online at https://www.mdpi.
com/article/10.3390/ijms22094318/s1.
Author Contributions: Conceptualization: M.C., M.N., and B.N.; methodology: M.C. and M.N.;
validation: M.C., M.N., and I.W.; investigation: M.C., M.N., J.K.-B., K.K.-G., I.W., and A.H.; resources:
M.C., M.N., B.N., and I.W.; data curation: M.C., M.N., and I.W.; writing—original draft preparation:
M.C. and M.N.; writing—review and editing: M.C., M.N., and B.N.; visualization: M.C. and M.N.;
supervision: M.C., M.N., and B.N.; project administration: M.C., M.N., and B.N.; funding acquisition:
M.N. and B.N. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Science Centre in Poland, project number UMO-
2014/15/B/NZ7/00966 (for B.N. and M.N.), by statutory funds of the Centre of Molecular and
Macromolecular Studies, Polish Academy of Sciences, and by Warsaw Medical University.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Int. J. Mol. Sci. 2021, 22, 4318
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Acknowledgments: The cytotoxicity, activation of caspases, and DNA interaction studies were per-
formed in the Screening Laboratory at the Department of Bioorganic Chemistry, Centre of Molecular
and Macromolecular Studies of the Polish Academy of Sciences (CMMS PAS). The cost of this publi-
cation was covered in part by the statutory funds of CMMS PAS. The authors wish to thank Markus
Düchler for revising the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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