Design, synthesis and biochemical evaluation of novel ethanoanthracenes and related compounds to target burkitt’s lymphoma

Article abstract of DOI:10.3390/ph13010016

Lymphomas (cancers of the lymphatic system) account for 12% of malignant diseases worldwide. Burkitt’s lymphoma (BL) is a rare form of non-Hodgkin’s lymphoma in which the cancer starts in the immune B-cells. We report the synthesis and preliminary studies on the antiproliferative activity of a library of 9,10-dihydro-9,10-ethanoanthracene based compounds structurally related to the antidepressant drug maprotiline against BL cell lines MUTU-1 and DG- 75. Structural modifications were achieved by Diels-Alder reaction of the core 9-(2- nitrovinyl)anthracene with number of dienophiles including maleic anhydride, maleimides, acrylonitrile and benzyne. The antiproliferative activity of these compounds was evaluated in BL cell lines EBV^? MUTU-1 and EBV⁺ DG-75 (chemoresistant). The most potent compounds 13j, 15, 16a, 16b, 16c, 16d and 19a displayed IC50 values in the range 0.17–0.38 μM against the BL cell line EBV^? MUTU-1 and IC50 values in the range 0.45–0.78 μM against the chemoresistant BL cell line EBV⁺ DG- 75. Compounds 15, 16b and 16c demonstrated potent ROS dependent apoptotic effects on the BL cell lines which were superior to the control drug taxol and showed minimal cytotoxicity to peripheral blood mononuclear cells (PBMCs). The results suggest that this class of compounds merits further investigation as antiproliferative agents for BL.

Full text of DOI:10.3390/ph13010016

pharmaceuticals
Article
Design, Synthesis and Biochemical Evaluation of
Novel Ethanoanthracenes and Related Compounds to
Target Burkitt’s Lymphoma
1
2
1
3
Andrew J. Byrne , Sandra A. Bright , James P. McKeown , John E. O’Brien ,
3
2
2
1,
Brendan Twamley , Darren Fayne , D. Clive Williams and Mary J. Meegan *
1
School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute,
Trinity College Dublin, 152-160 Pearse St, Dublin 2, D02 R590, Ireland; byrnea40@tcd.ie (A.J.B.);
mckeowjp@tcd.ie (J.P.M.)
School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin,
2
1
52-160 Pearse St, Dublin 2, D02 R590, Ireland; brights@tcd.ie (S.A.B.); FAYNED@tcd.ie (D.F.);
Clive.Williams@tcd.ie (D.C.W.)
3
School of Chemistry, Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160 Pearse St,
Dublin 2, D02 R590, Ireland; jeobrien@tcd.ie (J.E.O.); TWAMLEYB@tcd.ie (B.T.)
Correspondence: mmeegan@tcd.ie; Tel.: +353-1-896-2798; Fax: +353-1-8962793
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 19 December 2019; Accepted: 10 January 2020; Published: 17 January 2020
Abstract: Lymphomas (cancers of the lymphatic system) account for 12% of malignant diseases
worldwide. Burkitt’s lymphoma (BL) is a rare form of non-Hodgkin’s lymphoma in which the cancer
starts in the immune B-cells. We report the synthesis and preliminary studies on the antiproliferative
activity of a library of 9,10-dihydro-9,10-ethanoanthracene based compounds structurally related
to the antidepressant drug maprotiline against BL cell lines MUTU-1 and DG-75. Structural
modifications were achieved by Diels-Alder reaction of the core 9-(2-nitrovinyl)anthracene with
number of dienophiles including maleic anhydride, maleimides, acrylonitrile and benzyne. The
−
+
antiproliferative activity of these compounds was evaluated in BL cell lines EBV MUTU-1 and EBV
DG-75 (chemoresistant). The most potent compounds 13j 15 16a 16b 16c 16d and 19a displayed
,
,
,
,
,
−
IC50 values in the range 0.17–0.38
range 0.45–0.78
µ
M against the BL cell line EBV MUTU-1 and IC values in the
M against the chemoresistant BL cell line EBV DG-75. Compounds 15
50
+
µ
,
16b and 16c
demonstrated potent ROS dependent apoptotic effects on the BL cell lines which were superior to the
control drug taxol and showed minimal cytotoxicity to peripheral blood mononuclear cells (PBMCs).
The results suggest that this class of compounds merits further investigation as antiproliferative
agents for BL.
Keywords: Burkitt’s lymphoma; DG-75; MUTU-1; anthracene; nitrostyrene; maprotiline;
9
,10-dihydro-9,10-ethanoanthracene; apoptosis
1
. Introduction
Burkitt’s lymphoma (BL) is an aggressive non-Hodgkin lymphoma occurring with high incidence
in developing areas such as equatorial Africa and Papua New Guinea [
of high incidence (40–50 per 10 ), BL accounts for approximately 50% of childhood cancers and up
1
]. Within these tropical regions
6
to 90% of diagnosed childhood lymphomas [
the development of new, safe and cost-effective therapeutics are of current interest. For developed
countries, the sporadic form represents 1–2% of adult lymphomas [ ]. In contrast, the endemic BL
is commonly associated with infection by the oncogenic Epstein-Barr virus (EBV), which interrupts
cellular pathways regulating cell proliferation and thus prevents apoptosis [ ]. Treatment of BL
2]. The treatment of BL in these regions is difficult and
3
4
Pharmaceuticals 2020, 13, 16; doi:10.3390/ph13010016
www.mdpi.com/journal/pharmaceuticals

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includes the monoclonal antibody rituximab which binds to the cell surface protein CD20 of malignant
and normal B lymphocytes [ ]. Chemotherapy drugs used in combination with rituximab include
vincristine, methotrexate, doxorubicin and cyclophosphamide, which result in approximately 90%
survival rates in children [ ]. However, due to reported development of resistance to chemotherapy
5,6
7
drugs and increased incidences of immunodeficient HIV-associated BL [
selective and potent treatments for BL is required.
8,9], the development of
Among the compounds with reported biochemical effects in BL are a series of
N-heteroarylhydrazones (e.g., compound , EPH116) which demonstrated antiproliferative effects and
inhibition of ribonucleotide reductase in BL cells [10], Figure 1. The sesquiterpene -elemene effectively
1
β
2
inhibited the growth and induced the apoptosis of BL cells through upregulation of p53 upregulated
modulator of apoptosis (PUMA) expression and modulating PUMA related apoptotic signalling
pathway [11]. A phase I clinical trial of lenalidomide
in treating patients with relapsed non-Hodgkin lymphoma including relapsed and refractory Burkitt
lymphoma is currently in progress [12]. The natural naphthoquinone shikonin suppressed cellular
3 (G M arrest) and blinatumomab combination
2
4
proliferation and induced caspase-dependent apoptosis in BL cells with inhibition of c-MYC and
suppression of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin
(
PI3K/AKT/mTOR) pathway [13]. Phenothiazines such as chlorpromazine
thioridazine were noted to both suppress proliferation and induce apoptosis in BL cells [14], while the
novel indole based compound NecroX-7 is a reactive oxygen species scavenger and has been shown
to induce G /M arrest in BL cell lines [15 16]. Amidinopiperidine-based serine protease inhibitor has
5, trifluoroperazine and
6
,
7
2
been reported as a selective inducer of apoptosis in BL cells [17]. The functional overexpression and
the pathogenetic role of the MYC proto-oncogene in BL is established [18], indicating the potential role
of direct and indirect MYC inhibitors as new experimental therapies [19].
Our previous research reported the antidepressant drug maprotiline
anti-proliferative and pro-apoptotic agent in BL cell lines MUTU-I and DG-75 [20
8
(Figure 1) as an
21]. The
,
serotonin transporter (SERT) has been identified in B-cell malignancies; subsequently antidepressants
and structurally related compounds were investigated for potential antileukemia/antilymphoma
activity [22]. Induction of apoptosis was demonstrated by the selective serotonin reuptake inhibitor
(
SSRI) citalopram and the antidepressants imipramine and clomipramine in HL-60 acute myeloid
leukaemia, and human T-lymphocytes [23 25]. Although these compounds act as non-selective
SERT ligands, the pro-apoptotic activity of these drugs appear to be independent of SERT. In
addition, fluoxetine [20 22], 3,4-methylenedioxymethamphetamine (MDMA) and analogues [22 26],
fenfluramine [22], clomipramine [22] and the norepinephrine transporter (NET) targeting maprotiline
and analogues have demonstrated proapoptotic effects in BL cell lines [20 21 27]. Our subsequent work
–
–
,
,
,
involved the generation of a compound library structurally related to the tetracyclic antidepressant
maprotiline. A biological screen of this library identified a number of lead compounds in BL cell lines
(
MUTU-I and DG-75) [27]. From this study we identified the 9,10-dihydro-9,10-ethanoanthracene
scaffold e.g., compound as favourable for anti-proliferative activity in these cell lines while the
E)-9-(2-nitrovinyl)anthracene was also shown to elicit a potent pro-apoptotic effect [27]. The
9
(
ethanoanthracene scaffold was previously reported to demonstrate activity as L-type Ca2 channel
blockers [28], glucocorticoid and cannabinoid receptor modulators [29 30], inhibitors of drug efflux in
multidrug resistance [31 32] together with antimicrobial [33] antimalarial [34], anti-inflammatory [35
and cytotoxic activity [36].
+
,
,
]
Nitrostyrenes and related nitrovinyl compounds induce anti-cancer effects and stimulate an
apoptotic response in cancer cell lines e.g., oral and colon cancers, osteosarcoma and Erlich ascetic
tumour cell lines [37
species (ROS) mediated pathways [43
phospholipase A2 and tubulin have been demonstrated for simple nitrostyrenes, while antimicrobial,
anti-inflammatory and immunosuppressive effects are also reported [37 46 47]. Nitrostyrenes also act as
Retinoid X receptor alpha (RXR ) ligands which inhibit tumor necrosis factor alpha (TNF ) activation
–
42], modulating tumorigenesis in colon and breast cancer via reactive oxygen
–45]. Inhibition of telomerase, protein tyrosine phosphatase (PTP),
,
,
α
α

Pharmaceuticals 2020, 13, 16
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of nuclear factor kappa B (NF–
κ
B) [48]. We have previously investigated the synthesis, characterisation
and biochemical evaluation of a series of structurally diverse nitrostyrenes (e.g., compound 10a, Figure 1)
and related nitrovinyl compounds in the MUTU-I and DG-75 cell lines and identified the potent
apoptotic effect induced by (E)-1,3-bis(aryl)-2-nitro-1-propenes (e.g., compound 10b, Figure 1) [49] and
related heterocycles containing the nitrovinyl pharmacophore e.g., 3-nitro-2-phenyl-2H-chromene (10c),
(
Figure 1) with antiproliferative effects superior to the cancer therapeutics fludarabine and taxol [50].
A subset of these lead nitrostyrene compounds were also shown to elicit a potent anti-proliferative and
pro-apoptotic response towards a range of malignant cell lines e.g., MCF-7 (ER positive breast cancer),
HL-60 (acute promyelocytic leukemia) and HeLa (human cervical cancer) [51]. We have also reported
the pro-apoptotic effect of selected examples of these nitrostyrene compounds in chronic lymphocytic
leukemia (CLL) cell lines and also in ex vivo CLL patient samples [51].
Figure 1. Chemical structures of compounds with reported activity against Burkitt’s lymphoma:
compounds
1–7, maprotiline 8, ethanoanthracene 9 and nitrostyrene lead compounds 10a–c with target
ethanoanthracene structure.

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In the present work, a structurally diverse library of 9,10-dihydro-9,10-ethanoanthracene
compounds related in structure to the previous lead nitrostyrene compounds 10a and the tetracyclic
antidepressant maprotiline were synthesised. This approach will facilitate the identification of
–c
8
potent and selective compounds which may be useful in the design of proapoptotic agents. These
compounds are synthesised by Diels-Alder cycloaddition reaction of the required anthracene-diene
system and hence modification of the ethano-bridgehead could be achieved by variation of the
dienophile employed. The dienophiles chosen for the study included maleic anhydride, maleimide
and N-substituted maleimides together with benzyne, acrylate esters and acrylonitrile. Variation
of the anthracene substitution at C-9 from nitrovinyl to alternative double bonded systems such
as cyanovinyl, imine and oxime are also of interest for biological activity, as is the introduction of
maleimide linkers and aryl-substituted maleimides. Structural modifications of the nitrovinyl unit
by reduction and extension of the alkyl chain length at C-2 were also explored, (see target structure,
−
+
Figure 1). The compounds were evaluated in the EBV MUTU-1 cell line and chemoresistant EBV
DG-75 cell line to establish the structure-activity relationships for these ethanoanthraenes and to
optimize the antiproliferative and proapoptotic effects in BL cell lines.
2
. Chemistry
The synthesis of the required compounds was achieved by Diels-Alder cycloaddition reaction of
the 9-(2-nitrovinyl)anthracene dienes with selected dienophiles. The maleimide-based dienophiles
were first prepared by reaction of maleic anhydride with a series of amines (alkyl, benzyl, aryl
amines and a diamine) and involved generation of the corresponding amic acid intermediates,
(
step (a), Scheme 1). Subsequent intramolecular cyclisation of the amic acid with sodium acetate
and acetic anhydride, results in the formation of the related maleimide or N-substituted maleimide
compounds 11a , (step (b), Scheme 1). The (E)-9-(2-nitrovinyl)anthracenes 12a were prepared
–
s
–f
by Henry-Knoevenagel condensation of the nitroalkane with various 9-anthraldehydes giving
access to a number of diverse diene systems (step (c), Scheme 1). 9-(2-Nitroethyl)anthracene
1
2g, was obtained by reduction of 12a with sodium borohydride, (step (d), Scheme 1). A series
of (E)-11,12-substiuted-9-(2-nitrovinyl)-9,10-dihydro-9,10-ethanoanthracenes was synthesised by
Diels-Alder reaction of the (E)-9-(2-nitrovinyl)anthracenes 12a–f and the dienophiles 11a–s.
A summary of these Diels-Alder reactions is shown in Schemes 2–5. The novel adducts synthesised
are arranged as follows:
Series I (Schemes 2 and 3, Table 1): (E)-9-(2-Nitrovinyl)-9,10-dihydro-9,10-ethanoanthracenes
1
3a–n including maleic anhydride and maleimide Diels-Alder adducts and dimer 15.
Series II (Scheme 2, Table 1): 9-(2-Nitroethyl)-9,10-dihydro-9,10-ethanoanthracenes 14a–c.
Series III (Scheme 2, Tables 2–5): (E)-9-(2-Nitrovinyl)-9,10-dihydro-9,10-ethanoanthracenes (16a
–
n,
Series IIIA and 17a
–
n, Series IIIB) including substituted aryl and benzyl maleimide Diels-Alder adducts
and triazole adduct 18.
Series IV (Scheme 4, Table 6): (E)-9-(2-Nitrovinyl)-9,10-dihydro-9,10-ethanoanthracenes 19a–f
,
substituted at C-11 and C-12.
Series V (Scheme 5, Table 7): (E)-9-(2-Nitrovinyl)-9,10-dihydro-9,10-[1,2]benzenoanthracenes
0a–g.
Series VI (Scheme 5, Table 8): 9,10-Dihydro-9,10-ethanoanthracene Diels-Alder adducts 21a
substituted at C-9.
Series VII (Scheme 5, Table 9): 9,10-Dihydro-9,10-ethanoanthracene Diels-Alder adducts 23a
containing acrylonitrile, oxime and imine functional groups at C-9.
2
–
–
k
k

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Table 1. Yields and preliminary cell viability data for compounds 13a–n, 15 (Series I) and 14a–c
(
Series II) in MUTU-1 and DG-75 Burkitt lymphoma cell lines a.
MUTU-1 Cell
Viability %
DG-75 Cell
Viability %
13a–n
14a–c
Compound
X
R1
R2
Yield (%)
10 µM
1 µM
10 µM
1 µM
1
1
1
1
1
1
1
1
3a
3b
3c
3d
3e
3f
3g
3h
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
N(CH3)
H
H
H
Cl
Cl
Cl
H
H
H
Cl
Cl
Cl
H
H
-
H
CH3
80
72
98
15
18
55
50
97
76
25
50
55
55
80
43
45
30
10
-
12.5
7.2
24.4
11.7
64.1
35.6
83.3
85.3
0.47
99.7
91.1
0.70
83.3
88.6
0
62.2
0.47
80.7
102.2
98.2
95.1
11.7
101.4
89.4
5.9
101.4
102.0
0.93
20.4
102.1
101.1
81.4
0
78.5
1.1
CH CH
74.1
43.5
101.1
91.6
7.2
82.6
73.0
0.62
51.6
88.3
7.4
68.0
98.7
78.2
79.9
1.1
106.5
96.3
12.1
78.2
90.0
38.1
74.6
83.0
79.3
75.7
3.8
2
3
3
3
3
H
CH3
CH CH
2
H
CH3
1
1
3i
3j
CH CH
2
H
CH3
1
3k
1
3l
3m
3n
CH CH
2
1
1
H
H
-
-
-
-
-
-
-
N(CH )OH
1.8
0
2
1
4a
O
NH
99.3
95.0
79.2
2.1
73.0
5.9
76.7
58.5
75.5
4.5
93.1
81.9
30.4
1
4b
-
-
-
-
-
-
1
1
4c
5
N(C H )
6
5
Dimer
Maprotiline 8
2a
Taxol
-
-
65.1
14.0
9.9
102.0
94.6
48.8
1
99
-
9.8
a
Cell proliferation of MUTU-1 and DG-75 cells was determined with an alamarBlue assay (seeding density
4
1
–5 × 10 cells/mL per well for 96-well plates). Compound concentrations of either 1
µM or 10 µM for 24 h
(
(
MUTU-1) or 48 h (DG-75) were used to treat the cells (in triplicate) with control wells containing vehicle ethanol
1% v/v). The mean value for three experiments is shown.

Pharmaceuticals 2020, 13, 16
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Scheme 1. Synthesis of maleimides 11a
–
s
and nitrovinylanthracenes 12a
a) RNH , diethyl ether, reflux, 1 h. (b) NaOAc, Ac O, 120 C, 30 min.
2
–f and nitroethylanthracene
o
1
2g. Reagents and conditions: (
2
(
9
15–70%); ( ) Piperidine acetate, excess nitroalkane (CH NO , CH CH NO or CH CH CH NO ),
0 C, N , 1.5 h. (50–99%); (d) NaBH , (CH ) CHOH, CH Cl , RT, 24 h, 85%.
2 4 3 2 2 2
c
3
2
3
2
2
3
2
2
2
o

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Scheme 2. Synthesis of ethanoanthracenes 13a–n (Series I, Table 1), 14a–c (Series II, Table 1), 16a–n
◦
(
Series IIIA, Table 2), 17a–n, 18 (Series IIIB, Table 3): Reagents and conditions (a) Toluene, 90 C, 48 h.

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Table 2. Yields and preliminary cell viability data for compounds 16a–n (Series IIIA) in MUTU-1 and
DG-75 Burkitt lymphoma cell lines.
MUTU-1 Cell
Viability %
DG-75 Cell Viability
%
16a–n
Compound
6a
6b
Substitution
Yield (%)
10 µM
1 µM
10 µM
1.1
1 µM
1.1
1
2
3
4
5
1
R =R =R =R =R =H
30
51
1.4
10.3
5.0
1
2
3
5
R =R =R =R =H,
1
0.38
0.52
5.5
4
R =Cl
1
2
3
5
R =R =R =R =H,
1
6c
6d
6e
6f
6g
6h
70
15
27
10
66
60
40
63
58
40
50
45
1.1
0.41
1.4
0.19
0.77
0.07
1.8
2.1
2.3
2.0
2.1
5.1
2.3
0.46
0.55
0.18
2.7
0.16
0
28.4
13.5
35.4
25.6
28.2
20.8
77.5
33.5
69.0
59.2
35.9
20.8
4
R =OCH
3
1
R =Cl,
1
2
3
4
5
R =R =R =R =H
R =R =R =R =H,
1
2
3
5
1
4
R =F
1
2
R =R =H,
R =R =R =OCH
R =R =R =R =H,
1
1.1
3
4
5
3
1
2
3
5
1
1.1
4
R =Br
1
2
3
5
R =R =R =R =H,
R =(C=O)OCH
R =R =R =R =H,
1
0.79
1.2
4
3
1
2
3
5
1
6i
4
R =(C=O)C H
6
5
1
2
4
R =R =R =H,
1
6j
5.4
4.2
2.7
2.3
2.0
3
5
R =R =OCH
3
1
2
4
5
R =R =R =R =H,
1
6k
0.84
0.61
1.2
3
R =Cl
1
3
4
5
R =R =R =R =H,
1
6l
6m
6n
0
2
R =OH
1
2
4
5
R =R =R =R =H,
R =O(C=O)CH
R =R =R =R =H,
1
0
3
3
1
2
3
5
1
3.7
1.7
0.18
4
R =NH2
Maprotiline 8
Taxol
-
-
-
-
73.0
9.8
93.1
30.4
65.1
9.9
102.0
48.8
Cell proliferation of MUTU-1 and DG-75 cells was determined with an alamarBlue assay (seeding density
4
1
(
(
–5 × 10 cells/mL per well for 96-well plates). Compound concentrations of either 1
µM or 10 µM for 24 h
MUTU-1) or 48 h (DG-75) were used to treat the cells (in triplicate) with control wells containing vehicle ethanol
1% v/v). The mean value for three experiments is shown.

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Table 3. Yields and preliminary cell viability data for compounds 17a–n, 18 (Series IIIB) in MUTU-1
and DG-75 Burkitt lymphoma cell lines a.
MUTU-1 Cell
Viability %
DG-75 Cell
Viability %
17a–n
18
Compound
7a
7b
7c
7d
7e
7f
7g
7h
n
Substitution
Yield (%)
10 µM
68.6
83.4
77.6
99.9
91.1
84.5
78.5
1 µM
79.8
92.6
78.0
102.0
84.2
86.5
88.0
10 µM
1 µM
78.3
96.4
96.6
96.6
82.8
91.4
85.3
1
3
4
5
6
R =R =R =R =R =H,
1
0
0
0
0
0
0
0
88
82
60
44
65
54
39
88.9
99.9
86.3
78.4
100.4
85.1
84.3
2
R =CH
3
1
3
4
5
6
R =R =R =R =R =H,
1
2
R =CH CH
2
3
1
3
4
6
5
R =R =R =R =H, R =Cl,
1
2
R =CH
3
1
3
4
6
5
R =R =R =R =H, R =Cl,
1
2
R =CH CH
2
3
1
3
4
6
R =R =R =R =H,
R =OCH , R =CH
R =R =R =R =H,
R =OCH , R =CH CH
1
5
2
3
3
1
3
4
6
1
5
2
3
2
3
1
3
4
5
6
R =Cl, R =R =R =R =H,
1
2
R =CH
3
1
3
4
5
6
R =Cl, R =R =R =R =H,
1
0
1
1
38
74
62
96.3
0.86
85.6
106.7
74.9
94.6
90.3
0.67
87.9
89.2
93.1
2
R =CH CH
2
3
1
3
4
5
6
2
1
7i
7j
R =R =R =R =R =R =H
1
3
4
5
6
R =R =R =R =R =H,
1
103.6
2
R =CH
3
1
3
4
5
6
R =R =R =R =R =H,
1
7k
1
1
50
45
94.0
77.3
80.4
66.8
96.0
82.1
74.8
81.9
2
R =CH CH
2
3
1
3
R =R =H,
R =R =R =OCH ,
4
5
6
1
7l
3
2
R =CH
3
1
3
R =R =H,
4
5
6
1
7m
7n
1
R =R =R =OCH ,
54
66.8
82.2
77.8
82.2
3
2
R =CH CH
2
3
1
3
4
5
6
2
1
2
0
-
R =R =R =R =R =R =H
77
68
-
2.0
13.2
73.0
9.8
2.8
0.070
13.2
65.1
9.9
19.9
87.3
102.0
48.8
1
8
-
-
-
75.0
93.1
30.4
Maprotiline 8
Taxol
-
-
a
Cell proliferation of MUTU-1 and DG-75 cells was determined with an alamarBlue assay (seeding density
4
1
–5 × 10 cells/mL per well for 96-well plates). Compound concentrations of either 1
µM or 10 µM for 24 h
(
(
MUTU-1) or 48 h (DG-75) were used to treat the cells (in triplicate) with control wells containing vehicle ethanol
1% v/v). The mean value for three experiments is shown.

Pharmaceuticals 2020, 13, 16
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Scheme 3. Synthesis of ethanoanthracene dimer 15 (Series 1, Table 1): Reagents and conditions:
◦
(
a) Toluene, 90 C, 48 h., (10%).
Scheme 4. Synthesis of ethanoanthracenes 19a
–
f
(Series IV, Table 6)
. Reagents and conditions:
1
2
3
(
a) R R C=CHR , (NCC=CH , CH CH OCOC=CH , CH OCOC=CH , CH OCO(CN)C=CH ),
2
3
2
2
3
2
3
2
◦
Toluene, 90 C, 48 h; (b) H SO , R-OH, (R = CH , CH CH ), reflux, 6 h.
2
4
3
2
3

Pharmaceuticals 2020, 13, 16
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Scheme 5. Synthesis of ethanoanthracenes 20a
–
g
(Series V, Table 7), 21a–k (Series VI, Table 8) and
2
0
3a–k (Series VII, Table 9). Reagents and conditions: (a) Anthranilic acid, CCl CO H, isoamyl nitrite,
3
2
◦
◦ ◦
C then toluene, 90 C, 1 h; (b) NaBH , (CH ) CHOH, CH Cl ; (c) Toluene, 90 C, 48 h.
4 3 2 2 2

Pharmaceuticals 2020, 13, 16
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Table 4. X-ray crystallography data for compounds 16c, 16j, 17l, 17n, 19a and 20a.
d1
(Å)
C-C
d2
(Å)
C-C
Compound
Structure
Ao
Bo
Co
1
6c
122.05
121.71
-
1.546
1.544
1.555
1.563
1
1
6j
7l
125.99
129.35
118.05
119.69
-
-
1.542
1.563
1.557
1.573
1
7n
126.73
120.80
-
-
1
2
9a
0a
126.94
-
1.565
1.400
1.556
1.518
118.50o
121.24o 120.18o
Maprotiline 8
119.46
-
-
1.540
1.546

Pharmaceuticals 2020, 13, 16
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Table 5. Crystal Data and Structure Refinement Parameters for compounds 16c
,
16j, 17l, 17n, 19a
and 20a.
Compound No.
16c
16j
17l
17n
19a
20a
Empirical formula
Formula weight
F(000)
Temperature (K)
Radiation
C27H20N O5 C28H22N O
C31H28N O
C30H26N O
C19H14N O
C22H15NO2
325.35
680.0
100(2)
MoKα
2
2
6
2
6
2
6
2
2
452.45
482.47
540.39
1136.0
100(2)
MoKα
510.53
536.0
100(2)
MoKα
302.32
632.0
100(2)
MoKα
944.0
100(2)
MoKα
1008.0
100(2)
MoKα
0
.170 × 0.150 0.190 × 0.160 0.21 × 0.11 × 0.230 × 0.180 0.270 × 0.140 0.250 × 0.120
Crystal size (mm3)
×
0.100
× 0.120
0.03
× 0.140
× 0.070
× 0.100
monoclinic
Cc
Crystal system
Space group
a (Å)
monoclinic
monoclinic
monoclinic
triclinic
monoclinic
P2 /c
1
P2 /c
1
P2 /c
1
P1
P2 /c
1
8.3650(3)
9.8612(4)
26.0162(11)
90
14.6412(5)
8.5186(3)
18.2016(6)
90
100.5867(12)
90
14.9320(6)
9.6505(4)
18.4287(7)
90
97.566(2)
90
11.2039(3)
12.0105(3)
12.7111(3)
65.1220(10)
70.7630(10)
67.6170(10)
1405.31(6)
2
15.2934(7)
6.8232(3)
15.9060(7)
90
117.8513(18)
90
13.9319(4)
11.3026(4)
10.7959(4)
90
112.9496(12)
90
b (Å)
c (Å)
◦
α ( )
◦
β ( )
90.5702(8)
90
◦
γ ( )
3
Volume (Å )
2145.94(15)
4
2231.51(13)
4
2632.48(18)
4
1467.52(12)
4
1565.44(9)
4
Z
3
ρ
(g/cm )
µ (mm
1.400
1.436
1.363
1.206
1.368
1.380
calc
−
1
)
0.098
0.102
0.097
0.085
0.090
0.089
Reflections collected
Independent reflections
48612
4926
90366
6330
66279
6043
60287
6461
114265
7157
53230
4616
Rint
Rsigma
=
=
0.0306
0.0162
4926/0/308
1.014
0.0482
0.0245
6330/0/327
1.033
0.1354
0.0827
6043/419/500
1.019
0.0331
0.0215
6461/0/345
1.017
0.0257
0.0128
7157/0/208
1.066
0.0200
0.0111
4616/2/226
1.080
Data/restraints/parameters
Goodness-of-fit on F2
R = 0.0414,
R = 0.0421,
2
R = 0.0624,
2
R = 0.0472,
2
R = 0.0359,
2
R = 0.0331,
2
1
1
1
1
1
1
Final R indexes [I ≥ 2σ (I)]
wR = 0.1057 wR = 0.0977 wR = 0.1164 wR = 0.1227 wR = 0.1026 wR = 0.0939
2
R = 0.0516,
R = 0.0654,
R = 0.1372,
R = 0.0709,
R = 0.0438,
R = 0.0353,
1
1
1
1
1
1
Final R indexes [all data]
wR = 0.1124 wR = 0.1099 wR = 0.1422 wR = 0.1368 wR = 0.1080 wR = 0.0964
2
2
2
2
2
2
Diff. peak/hole (e Å 3
Flack parameter
CCDC No.
−
)
0.37/−0.23
0.42/−0.29
0.66/−0.46
0.27/−0.24
0.59/−0.37
0.45/−0.22
0.28(17)
1938155
1938150
1938151
1938152
1938153
1938154
Table 6. Yields and preliminary cell viability data for compounds 19a–f (Series IV) in MUTU-1 and
DG-75 Burkitt lymphoma cell lines a.
MUTU-1 Cell
Viability %
DG-75 Cell
Viability %
1
9a–f
Compound
9a
9b
9c
9d
9e
9f
Substitution
Yield (%)
10 µM
1 µM
10 µM
1 µM
1
2
3
1
1
1
1
1
1
R =CN, R =R =H
30
18
15
35
32
15
-
0
0.69
8.4
0.69
10.7
13.8
73.0
9.8
9.0
0
13.4
1.4
20.6
9.8
1
2
3
R =(C=O)OCH CH , R =R =H
48.9
15.6
48.9
84.7
13.6
93.1
30.4
2
3
1
2
3
R =(C=O)OCH , R =R =H
92.2
99.4
101.8
13.9
102.0
48.8
3
1
2
3
R =O(C=O)CH , R =CN, R =H
1.0
3
1
3
2
R =R =(C=O)OCH , R =H
10.3
13.8
65.1
9.9
3
1
3
2
R =R =(C=O)OCH CH , R =H
2 3
Maprotiline 8
-
-
Taxol
-
a
Cell proliferation of MUTU-1 and DG-75 cells was determined with an alamarBlue assay (seeding density 1–5
×
4
1
0 cells/mL per well for 96-well plates). Compound concentrations of either 1
µM or 10
µM for 24 h (MUTU-1) or
4
8 h (DG-75) were used to treat the cells (in triplicate) with control wells containing vehicle ethanol (1% v/v). The
mean value for three experiments is shown.

Pharmaceuticals 2020, 13, 16
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Table 7. Yields and preliminary cell viability data for compounds 20a–g (Series V) in MUTU-1 and
DG-75 Burkitt lymphoma cell lines a.
MUTU-1
Cell
Viability
%
DG-75
Cell
Viability
%
20a–g
Compound
0a
0b
0c
0d
0e
0f
0g
R1
R2
R
Yield (%)
10 µM
1 µM
10 µM
1 µM
2
2
2
2
2
2
2
H
H
H
Cl
H
H
H
-
NV
NV
NV
NV
H
CH3
20
23
32
30
80
21
85
-
1.3
101.8
101.8
0.34
110.9
5.4
102.8
73.0
9.8
6.0
95.2
106.2
0.95
98.9
97.9
101.1
93.1
30.4
0.091
100.0
84.8
0
97.2
81.7
68.7
65.1
9.9
90.2
86.4
95.1
46.0
97.6
72.9
97.0
102.0
48.8
CH CH
2
3
H
-
-
-
-
CH CH NO
2
2
2
CHO
CH OH
2
Maprotiline 8
-
-
Taxol
-
-
-
a
Cell proliferation of MUTU-1 and DG-75 cells was determined with an alamarBlue assay (seeding density 1–5
×
4
1
0 cells/mL per well for 96-well plates). Compound concentrations of either 1
µM or 10
µM for 24 h (MUTU-1) or
4
8 h (DG-75) were used to treat the cells (in triplicate) with control wells containing vehicle ethanol (1% v/v). The
mean value for three independent experiments is shown.
Table 8. Yields and preliminary cell viability data for compounds 21a
–
k
(Series VI) in MUTU-1 and
DG-75 Burkitt lymphoma cell lines a.
21a–k
MUTU-1 Cell
Viability %
Compound
R
X
Yield (%)
DG-75 Cell Viability %
10 µM
1 µM
10 µM
1 µM
2
2
2
2
2
2
2
2
1a
1b
1c
1d
1e
1f
1g
1h
H
H
H
CHO
CHO
CHO
COOH
COOH
COOH
OH
O
NH
90
85
90
40
35
62
81
41
43
55
58
-
93.1
92.8
91.2
88.4
95.1
77.7
88.8
92.0
92.5
100.4
100.1
73.0
9.8
103.2
104.1
93.0
87.4
96.1
102.2
99.2
92.6
103.4
103.4
104.0
93.1
69.9
74.5
71.3
64.7
71.2
72.4
65.4
73.1
67.5
93.0
80.9
65.1
9.9
91.4
82.0
85.7
64.8
76.0
102.0
93.1
86.3
73.2
68.0
65.9
102.0
48.9
N(C H )
6
5
O
NH
N(C H )
6
5
O
NH
2
2
1i
1j
N(C H )
6
5
O
NH
-
-
2
1k
OH
-
-
Maprotiline 8
Taxol
-
30.4
a
Cell proliferation of MUTU-1 and DG-75 cells was determined with an alamarBlue assay (seeding density 1–5
×
4
1
0 cells/mL per well for 96-well plates). Compound concentrations of either 1
µM or 10
µM for 24 h (MUTU-1) or
4
8 h (DG-75) were used to treat the cells (in triplicate) with control wells containing vehicle ethanol (1% v/v). The
mean value for three experiments is shown.

Pharmaceuticals 2020, 13, 16
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Table 9. Yields and preliminary cell viability data for compounds 23a–k (Series VII) in MUTU-1 and
DG-75 Burkitt lymphoma cell lines a.
MUTU-1 Cell
Viability %
DG-75 Cell
Viability %
2
3a–k
Compound
3a
3b
3c
3d
3e
3f
3g
3h
X
Y
R1
R2
R3
Yield (%)
10 µM
1 µM
10 µM
1 µM
2
2
2
2
2
2
2
2
O
NH
C
C
C
C
C
C
N
N
N
N
N
-
H
H
H
H
H
H
H
H
Cl
H
H
-
CN
CN
CN
CN
CN
CN
NH2
NH2
NH2
OH
OH
-
H
H
H
CN
CN
CN
H
H
H
H
H
74
38
20
15
42
44
63
54
34
43
66
-
82.6
79.4
74.1
82.7
83.6
71.8
100.9
78.0
106.2
87.6
83.9
73.0
9.8
82.7
79.4
90.7
99.8
82.9
78.9
95.2
91.1
102.1
92.8
96.9
93.1
30.4
75.8
54.1
89.1
95.7
98.7
81.6
55.0
87.4
18.9
63.9
74.1
65.1
9.9
89.0
101.1
94.4
103.1
85.7
82.0
78.1
85.8
85.8
69.1
81.7
102.0
48.8
N(C H )
6
5
O
NH
N(C H )
6
5
NH
N(C H )
6
5
2
2
3i
3j
NH
NH
2
3k
N(C H )
6
5
Maprotiline 8
Taxol
-
-
-
-
-
-
-
-
a
Cell proliferation of MUTU-1 and DG-75 cells was determined with an alamarBlue assay (seeding density
4
1
–5 × 10 cells/mL per well for 96-well plates). Compound concentrations of either 1
µM or 10 µM for 24 h
(
(
MUTU-1) or 48 h (DG-75) were used to treat the cells (in triplicate) with control wells containing vehicle ethanol
1% v/v). The mean value for three experiments is shown.
The compounds 13a
–
n
and 14a
–
c
(Series I and II) encompasses maleic anhydride, maleimide
17a
and simple maleimide Diels-Alder adducts of 9-(2-nitrovinyl)anthracenes while the 16a
–
n,
–n
products (Series IIIA and IIIB) include substituted phenyl and benzyl maleimide Diels-Alder adducts
of 9-(2-nitrovinyl)anthracenes. All products were prepared by cycloaddition reaction of the anthracene
◦
dienes with the appropriate dienophile at 90 C in toluene for 48 h and afforded compounds 13a
–
n
(15–98%), 14a–c (30–45%), 16a–n (10–70%) and 17a–n (38–88%), Scheme 2. Variation of alkyl chain
length at the C-2 and C-10 substitution was achieved by utilising different nitroalkanes for the
Henry-Knoevenagel condensation with substituted 9-anthraldehydes giving access to a number
of diverse diene systems. 9-(2-Nitroethyl)anthracene 12g was also reacted with maleic anhydride,
maleimide and phenyl-maleimide to give the corresponding saturated Diels-Alder adducts (14a
Table 1, Scheme 2. In the H NMR spectrum of the novel maleic anhydride adduct 13a the signal at
–
c
),
1
3.82 ppm appears as a double doublet (J = 9.16, 3.66 Hz) and is assigned to H-11 due to interaction
with H-10 and H-12 protons which appear as doublets at 4.98 ppm and 4.20 ppm respectively. The
doublets occurring at 8.11 ppm and 8.28 ppm (J = 14.04 Hz) were assigned to the trans coupled
protons of the nitrovinyl unit. The assignments were confirmed from the heteronuclear multiple
bond correlation (HMBC) and carbon-hydrogen correlation spectroscopy (C-H COSY) NMR spectra,
(
(
Supplementary Information). The novel dimer compound 15 was obtained by cycloaddition reaction of
E)-9-(2-nitrovinyl)anthracene 12a and the dimaleimide 11s, Scheme 3. The H NMR spectrum of 15 was
1
analogous with the above data for 13a: the ethano bridge protons at 3.36 ppm (double-doublet), 3.76 ppm
double-doublet) and 4.86–4.92 ppm (multiplet) as expected. High resolution mass spectrometry
(
+
confirmed the required molecular ion for the dimer 775.2771, C H N O [M + H].
46
39
4
8
The (E)-9-(2-nitrovinyl)-13-phenyl-9,10-dihydro-9,10-[3,4]epipyrroloanthracene-12,14-diones
Series IIIA (16a 16n) and Series IIIB (17a 17n) were prepared from the appropriately substituted
E)-9-(2-nitrovinyl)anthracenes via Diels-Alder cycloaddition with substituted benzyl and aryl
–
–
(

Pharmaceuticals 2020, 13, 16
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maleimides with varied aryl substitutions including methoxy, halogen (bromine, chlorine, fluorine),
1
alkyl, hydroxy, ester and amines, (Tables 2 and 3). The characteristic H NMR spectrum of compound
1
6j shows a double doublet at 3.53 ppm (J = 8.55, 3.05 Hz) assigned to H . Doublets occurring at
11
3
.92 ppm (J = 8.55 Hz) and 4.95 ppm (J = 3.05 Hz) were assigned to H12 and H , respectively. The
10
assignments were confirmed from the C-H COSY and DEPT 90 NMR spectra, (See Supplementary
Information).
Single crystal X-ray structure determination was completed on (E)-9-(2-nitrovinyl)-9,10-dihydro-
9,10-[3,4]epipyrroloanthracene-12,14-diones 16c, 16j, 17l and 17n (Figure 2) and selected data is
summarised in Tables 4 and 5. The core dihydroethanoanthracene moiety is rigid, possessing
analogous conformations in the different structures; the packing structure displayed by the products
is centrosymmetric and monoclinic. From the asymmetric unit, it was possible to confirm the
o
(
E)-configuration of the nitrovinyl unit. The angle A was calculated for each compound as the angle
◦
between the centroid of phenyl ring 1 and the centroid of phenyl ring 2—ranging from 122.05 to
29.35 . This angle is 119.46 in the crystal structure of maprotiline [52]. The flat succinimide ring is
fused to the ethano bridge, so that it is tilted through almost exactly 60 with respect to the plane of the
C- (4)-C(18)-C(22)-C(11) bridging atoms. The angle B (maleimide substituent centroid to centroid of
◦
◦
1
◦
o
◦
main axis) was also calculated and ranged from 118.05–121.71 . The distance between the carbons in
the ethano bridge (d ) ranged from 1.542–1.563 Å, comparing well to that of maprotiline 1.54 Å [52] and
1
related inclusion complexes [53]. The distance between the carbon at C-10 and the nearest carbon of the
ethano bridge (d ) for the series was 1.555–1.573 Å, while in maprotiline, this distance is 1.546 Å [52].
2
Reaction of the dienophile 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione 11t with the (E)-9-(2-nitrovinyl)
anthracene 12a afforded the adduct 18 in 68% yield (Scheme 2) and allowed direct comparison
of the nitrogen-nitrogen bridge system in 18 with the carbon-carbon bridge in compound 16a
The substituted simple (E)-9-(2-nitrovinyl)-9,10-dihydro-9,10-ethanoanthracenes 19a , (Series IV)
Scheme 4, Table 6) were prepared from 12a and the corresponding dienophiles (1-cyanovinyl acetate,
methyl acrylate, ethyl acrylate and cyanoacrylate) affording yields of 15–35% (19a ) or via a ring
opening reaction (19e ) in yields of 15–32% (Scheme 4, Table 6). Two products are possible from
.
–
f
(
–
d
,f
the Diels-Alder addition of the above dienophiles to the anthracene diene: an ‘ortho’ and a ‘meta’
addition product, (Scheme 4). The Diels-Alder addition of cyanoacrylate to diene systems such as
9
-anthraldehyde and (E)-3-(anthracen-9-yl)acrylonitrile has been previously documented as forming
the ortho addition adduct only [54]. The preference for the ‘ortho’ adduct is attributed to the stabilising
molecular orbital overlap between the nitrile and the carbonyl group. In the present work, we have
confirmed the exclusive formation of the novel ortho adduct 19a from the reaction of acrylonitrile and
1
(
E)-9-(2-nitrovinyl)anthracene 12a using H NMR and X-ray crystallography, (Table 4, Figure 2). In the
1
H NMR spectrum of 19a, the diastereotopic protons H-12a and H-12b were identified as multiplets
at 1.96 ppm and 2.30 ppm. The bridging protons H-11 and H-10 were identified at 3.69 ppm as a
double doublet (J = 10.68, 3.97 Hz) and 4.63 ppm as a singlet respectively. Interestingly, although
coupling was demonstrated for H-12a with H-10, (J = 2.44 Hz), this is not observed from its singlet
signal. The assignments were confirmed from the C-H COSY and Distortionless Enhancement by
Polarization Transfer (DEPT 90) NMR spectra, see Supplementary Information. The esters 19e and 19f
were prepared from the anhydride 13a using acid catalysed ring opening conditions Scheme 4, [55].
X-ray crystallographic analysis of the novel Diels-Alder adduct (E)-10-(2-nitrovinyl)-9,10-dihydro-
9
,10-ethanoanthracene-11-carbonitrile (19a) confirmed the regioisomer obtained, (Figure 2, Tables 4
o
and 5), and the packing structure of the product was centrosymmetric and monoclinic. The angle A
between the centroid of phenyl ring 1 and the centroid of phenyl ring 2 was determined as 126.94 .
This angle is reported as 119.46 in the crystal structure of maprotiline [52]. The distance between the
o
o
carbons in the ethano bridge (d ) was 1.565 Å, comparing well to that of maprotiline 1.540 Å. The
1
distance between the carbon at C-10 and the nearest carbon of the ethano bridge (d ) was 1.556 Å,
2
in maprotiline this distance is 1.546 Å [52].

Pharmaceuticals 2020, 13, 16
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1
6c
16j
17l
17n
19a
20a
Figure 2. X-ray crystallographic molecular structures of 16c
,
16j, 17l, 17n, 19a and 20a with atomic
displacement shown at 50% probability. Only major disordered moiety shown for 17l. Non-hydrogen
atoms labelled where possible.
To investigate the effect of increased rigidity on the ethanoanthracene bridge, the
(
E)-9-(2-nitrovinyl)anthracenes (12a
–
12d) were converted to the corresponding (E)-9-(2-nitrovinyl)-9,10-
) in modest yields (Scheme 5, Series V, Table 7). The
dihydro-9,10-[1,2]benzenoanthracenes (20a
–
d
Diels-Alder reaction was achieved with benzyne as the dienophile generated in situ by thermal
decomposition of benzenediazonium-2-carboxylate, (prepared from anthranilic acid and isoamyl

Pharmaceuticals 2020, 13, 16
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nitrite) [56–60]. Diphenyl and triphenyl side products have been reported using in situ benzyne
generation [57], but these were not isolated during the current work. The benzyne adduct 20f was
similarly prepared from anthracene carboxaldehyde. Sodium borohydride reduction of 20a and
2
0f afforded the alcohol and nitroalkane analogues 20e and 20g, respectively. The library of novel
1
triptycene analogues synthesised are summarised in Table 7. The H NMR spectrum of compound 20d
shows two multiplets occurring at 7.58–7.69 ppm and 7.72–7.82 ppm both integrating for three protons
each. These signals were assigned to the two groups of three equivalent aromatic protons H-1, H-8,
H-3” and H-4, H-5, H-6” respectively. The assignments were confirmed from the C-H COSY, HMBC
and DEPT 90 NMR spectra, (see Supplementary Information).
The structure of 20a was confirmed by X-ray crystallographic analysis (Figure 2, Tables 4 and 5),
showing that the packing structure assumed by the product was monoclinic and confirming the
(
E)-configuration of the nitrovinyl unit. The angles between the centroids of each of the three phenyl
o
rings and the centroid of the central axis were calculated from the asymmetric unit, these were A
(
o
o
o
o
o
o
118.50 ), B (121.24 ) and C (120.18 ). This angle is 119.46 in the crystal structure of maprotiline.
The distance between the carbons in the ethano bridge (d ) was 1.400 Å, in maprotiline this distance is
1
1.540 Å [52]. The distance between the carbon at C-10 and the nearest carbon of the ethano bridge
(d2) was 1.518 Å, in maprotiline this distance is 1.546 Å [52], hence the X-ray structures indicate a
comparable configuration for 20a and maprotiline.
Diels-Alder cycloaddition reactions with substituted anthracenes using maleic anhydride and
maleimide dienophiles were further investigated to determine how the replacement of the nitrovinyl
group by other functionalities at C-9 would affect the potency of the products. The 9-substituted
Diels-Alder adducts 21a
anthraldehyde and anthrone in yields of 35–90%, (Series VI, Scheme 5, Table 8). Similar Diels-Alder
reaction with anthracene malononitrile (22a), acrylonitrile (22b) [27 61], hydrazines (22c 22d) and
oxime (22e) dienes afforded the cycloaddition products 23a in yields of 15–74%, (Series VII, Scheme 5,
Table 9). We now report the first direct Diels-Alder reaction of (E)-(anthracen-9-ylmethylene)hydrazine
22c) and (E)-anthracene-9-carbaldehyde oxime (22e) with dienophiles maleic anhydride, maleimide
and N-phenylmaleimide. Previous routes to these compounds required the reaction of Diels-Alder
adducts of 9-anthraldehyde (e.g., 20f) with hydroxylamine [27 62 64] or hydrazine [65 66]. In the
–k were obtained using with diene systems anthracene carboxylic acid,
,
,
–k
(
,
–
,
current work, the synthesis of the novel 2-(anthracen-9-ylmethylene)malononitrile Diels-Alder adducts
1
2
3d
–f
was also achieved. In the H NMR spectrum of the hydrazine 23g the characteristic signals for
H-12 and H-11 were identified as doublets at 3.23 ppm and 3.44 ppm (J = 8.55 Hz) while the singlet at
4
.72 ppm accounted for H-9, (see Supplementary Information).
A preliminary stability study of the representative ethanoanthracene compound 16a was carried
out at acidic, neutral and basic conditions (pH 4, 7.4 and 9) using HPLC. The half-life (t
1
) was
2
determined to be 11 h at pH 4, 10.5 h at pH 7.4 and greater than 24 h at pH 9. Based on this stability
study the compound would be suitable for further preclinical investigation.
3
. Biochemistry
3
.1. Preliminary Evaluation of In Vitro Anti-Proliferative Activity of the Ethanoanthracenes in Burkitt
−
+
Lymphoma EBV MUTU-1 and EBV DG-75 (Chemoresistant) Cell Lines
The panel of compounds synthesised (Series I–VII) based on the 9,10-dihydro-9,10-
ethanoanthracene scaffold was then initially screened at two concentrations (1
for antiproliferative activity in the BL EBV MUTU-1 and EBV DG-75 (chemoresistant) cell lines to
determine the structure-activity relationship for these maprotiline analogues. The results obtained
µM and 10 µM)
−
+
from this preliminary screen in the MUTU-I and DG-75 cell lines (at 10
Tables 1–3 and Tables 6–9, with maprotiline and taxol used as the control drugs. Maprotiline induced
a modest anti-proliferative effect at 10 M in the MUTU-I and DG-75 BL cell lines, while taxol was
more effective with 10% cell viability at 10 M in both cell lines. The results obtained for these novel
ethanoanthracene compounds (Series I-VII) are discussed by structural type.
µM and 1 µM) are displayed in
µ
µ

Pharmaceuticals 2020, 13, 16
19 of 58
3
.1.1. Series I and II, Compounds 13a–n, 14a–c and 15
The initial lead 9-(2-nitrovinyl)anthracene 12a demonstrated activity (< 14% cell viability) in both
BL cell lines at 10 M, (Table 1). The effects of the maleic anhydride adducts 13a and maleimide
adducts (13g ) of the lead nitrovinyl anthracene compound 12a on cell viability were first investigated
Table 1). The 9-(2-nitrovinyl)-9,10-dihydro-9,10-ethanoanthracenes 13a 13b produced a significant
anti-proliferative effect at both 1 and 10 M concentrations in the MUTU-I cell line (7–24% cell viability),
Table 1). In the DG-75 cell line the maleic anhydride derivative 13a gave slightly improved results
µ
–f
–n
(
,
µ
(
compared to maprotiline at both concentrations. C-10 chloro substitution e.g., 13d resulted in decreased
activity in both cell lines. The anhydride 13b and the imide 13g demonstrated potent antiproliferative
effects in DG-75 cell line (1% cell viability at 1
3a. The 9-chlorosubstituted imide 13j, N-methylimide 13m and alcohol 13n demonstrated potent
antiproliferative effects in the MUTU-1 cell line (< 1% cells remaining at 1 M). Increasing alkyl chain
length at the C-2 position decreased the anti-proliferative effect across most examples of the series
e.g., 13f 13i 13l). Reduction of the C-9 nitrostyrenes 13a 13g to afford the C-9 nitroalkane substituted
4a and 14b (Series II) resulted in significant reduction in activity, indicating the essential requirement
µM), with the imide 13g more potent than the anhydride
1
µ
(
,
,
,
1
of the nitrostyrene functional group for activity. The dimer compound 15 was identified as a potential
lead compound with significant activity in both cell lines (< 5% viable cells at 1 µM).
3
.1.2. Series IIIA, Compounds 16a–n
Compounds 16a (Series IIIA, Table 2) were designed to investigate the effect of N-arylsubstitution
halogen, ether, phenol, ketone, ester, amine) on antiproliferative activity of the lead ethanoanthracene
scaffold structure 13a. All analogues in the series elicited a very potent anti-proliferative action in the
MUTU-I cell line at 1 M (<5% cell viability, except 16a, 10%). In the DG-75 cell line, the most potent
activity was produced by compounds 16a and 16b, with <6% viable cells remaining at 1 M. The
-chloro compound 16d also produced a promising result at 1 M with <14% cell viability. Significantly
reduced activity was observed when comparing compounds 16b (p-Cl) and 16k (m-Cl) in the DG-75 cell
line. In a study of para phenyl substitution of this series of compounds 16a 16n in the DG-75 cell line,
the unsubstituted compound 16a and p-chloro compound 16b were found to elicit more favourable
–
n
(
µ
µ
9
µ
–
antiproliferative effects than the bromo (16g), fluoro (16e), amino (16n), ketone (16m) and methoxy (16c
analogues (Table 2). Maleimide (13g) and phenyl maleimide (16a) adducts produce superior activity in
the two BL cell lines than the maleic anhydride adduct 13a. C-9 chloro substituted compounds 13j and
)
1
6d possessed significant anticancer effects in both cell lines (0.4–13.5% cell viability).
3
.1.3. Series IIIB, Compounds 17a–n
Compounds 17a were designed to investigate the effect of the alkyl substitution on the
–
n
nitrostyrene group, and also to investigate the introduction of a benzyl or phenylethylamine substitution
on the heterocyclic nitrogen (Table 3). The deactivating effect of extended alkyl chain length (methyl and
ethyl) at C-2 on anticancer activity can once again be observed (70–90% cell viability). With the
exception of compounds 17i (N-benzyl) and 17n (N-phenylethylamine), these analogues elicited
poor anti-proliferative action in both MUTU-I and DG-75 cell lines confirming that alkyl substitution
(methyl or ethyl) on the nitrostyrene group dramatically reduces activity e.g., comparing adducts 16a
with 17a and 17b in both DG-75 and MUTU-I cell lines at 10 M, (Table 3). Compound 18 was less potent
µ
at the lower concentration in both BL lines than 16a, indicating that the ethanoanthracene structure
was more favourable than the triazole-anthracene bridged system for the desired anticancer effect.
3
.1.4. Series IV, Compounds 19a–f
The effect of ethano-bridge substitution on antiproliferative effects was assessed in compounds
1
9a
–f using a range of different dienophiles for the Diels-Alder reaction, together with the ring opening
reaction of the anhydride 13a to introduce nitrile and ester groups on the ethano-bridge, Table 6. All

Pharmaceuticals 2020, 13, 16
20 of 58
but one of these simpler Diel-Alder adducts possessed enhanced anti-proliferative activity compared
to the parent nitrovinylanthracene compound 12a. The most promising compounds identified from
this cohort were 19a
viability of 0–14% at 10
reduced the potency observed for compound 19a, (Table 6).
,
19c and 19f showing good anti-proliferative effects in both cell lines with cell
µM. The inclusion of the ester group on the ethano-bridged system in 19d
3
.1.5. Series V, Compounds 20a–g
A series of triptycene based Diel-Alder adducts were prepared to assess the effect of the rigid
triptycene moiety on the anti-proliferative activity (Table 7). The C-9 functionalities such as nitrovinyl
0a 20b 20c 20d, nitroalkane 20e, aldehyde 20f and alcohol 20g were included to assess their impact
2
,
,
,
on the anticancer properties of the series. The most promising results were obtained by nitrovinyl
compounds 20a and 20d (10-chloro) having potent effects (<6% cell viability) in the MUTU-I cell line.
A potent effect was also observed for 20d in DG-75 at 10 µM with no viable cells detected. Reduction of
the nitrovinyl bond was once again detrimental to anti-proliferative effects in both cell lines (e.g., 20e).
Alkyl substituent at C-2 of the nitrovinyl unit resulted in diminished activity demonstrated for methyl
(20b) and ethyl (20c) compounds. The aldehyde based triptycene (20f) exhibited good activity at the
higher concentration in the MUTU-I cell line (<5%), while alcohol (20g) was inactive (Table 7).
3
.1.6. Series VI, Compounds 21a–21k
The 9,10-dihydro-9,10-ethanoanthracenes 21a–k which do not contain the nitrovinyl unit at
C-9 demonstrated greatly reduced antiproliferative activity in both cell lines (>60% viability),
indicating the C-9 nitrovinyl substitution was essential for enhanced anti-proliferative effect, Table 8.
The 9,10-dihydro-9,10-ethanoanthracenes assessed include C-9 unsubstituted 21a
aldehydes 21d , alcohols 21j and acids 21g , Table 8. Although these compounds possess anticancer
activity exceeding the activity of maprotiline at 1 M in the DG-75 cell line, the antiproliferative effect
in the MUTU-I cell line was minimal (>80% cell viability).
–c, C-9 substituted
–f
,
k
–i
µ
3
.1.7. Series VII, Compounds 23a–k
A group of C-9 substituted 9,10-dihydro-9,10-ethanoanthracenes 23a–k derived from maleic
anhydride, maleimide and phenyl maleimide were next investigated for antiproliferative activity,
to assess the effect of variation of the C-9 unsaturated substituent, Table 9. Of the C-9 acrylonitrile
compounds 23a
comparable to maprotiline in the MUTU-I (79% cell viability) and DG-75 (54% cell viability) cell lines,
Table 9. Further C-9 substituted 9,10-dihydro-9,10-ethanoanthracenes included hydrazones (23g
and oximes (23j ). The most potent compound in the DG-75 cell line was identified as the hydrazone
3i, (19% cell viability). In summary, introduction of alternative double bond functionality to replace
the nitrovinyl e.g., cyanovinyl (23a ), oxime (23j ) and hydrazone (23g ) were found to reduce the
–f, the most promising was identified as compound 23b which demonstrated activities
–i)
,k
2
–
f
,
k
–i
observed antiproliferative effects when compared with the C-9 nitrovinyl compounds 13a, 13f and 16a
respectively, (Table 9).
In this initial evaluation of the ethanoanthracenes (Series I–VII) for antiproliferative activity,
a number of key structural requirements were identified. The nitrovinyl pharmacophore was
demonstrated to be critical for antiproliferative effect, epipyrrolo bridgeheads were also found to
exert a more potent effect than simple ethano and furan-based bridgeheads. Introduction of a chloro
substituent at C-9 of the anthracene core also contributed to potency for many compounds.
3
.2. In Vitro Anti-Proliferative Activity of Selected Potent Ethanoanthracenes
Based on the results obtained from the cell viability study above, the following potent
compounds were identified for further investigation in the MUTU-I cell line: maleimide 13j
,
,
N-hydroxymethylmaleimide 13n, and maleimide dimer 15 (Series I), N-arylmaleimides 16a 16j 16m
–
,
1
6n (Series IIIA), N-benzylmaleimide 17n (series IIIB), acrylonitrile adduct 19a, acrylate ester adduct

Pharmaceuticals 2020, 13, 16
21 of 58
1
9c and diester 19f (Series IV) and triptycene 20d (Series V). The IC50 values were determined in the
sub-micromolar range (0.09–0.55 µM), with compound 15 identified as the most potent (IC50 = 0.09 µM),
Table 10. The MUTU-1 IC50 values combined with the data provided by the preliminary screen in the
DG-75 cell line were used to select the following compounds for subsequent IC50 determination in
the chemoresistant DG-75 cell line: 13j, 15 (Series I), 16a, 16b, 16c, 16d (Series II) and 19a (Series IV),
Table 11.
Table 10. IC50 values for selected ethanoanthracenes in the MUTU-I cell line (24 h) a.
Compound
IC50 (µM)
Compound
IC50 (µM)
Compound
IC50 (µM)
Taxol
Maprotiline
0.32
15.8
2.57
0.25
0.32
0.09
0.21
0.17
16c
16d
16e
16f
16g
16h
16i
0.20
0.22
0.25
0.24
0.22
0.20
0.24
0.26
16m
16n
17n
19a
19c
19f
0.23
0.30
0.26
0.38
0.54
0.55
0.28
1
2a
3j
3m
1
1
1
5
1
6a
20d
1
6b
16j
a
4
Cell proliferation of MUTU-1 cells was determined with an alamarBlue assay (seeding density 1–5
×
10 cells/mL
per well for 96-well plates). The compounds (concentration range 0.01 nM to 50 µM) were added (in triplicate) and
the cells retained for 24 h, with control wells containing vehicle ethanol (1% v/v). The values represent the mean for
three experiments. IC50 value is the concentration required for 50% growth inhibition of MUTU-1 cells.
Table 11. IC50 values of selected compounds 12a, 13j, 15, 16a–d, 19a in BL cell lines DG-75 and MUTU-I
Burkitt lymphoma cell lines.
logP b
Compound
IC50 (µM) a
IC50 (µM) a
MUTU-I 24 h
DG-75 48 h
1
1
1
2a
3j
5
2.57
0.25
0.09
0.21
0.17
0.20
0.22
0.38
0.32
15.8
2.08
0.78
0.24
0.71
0.45
0.56
0.59
0.69
1.32
37.5
4.763
3.310
5.060
4.616
5.269
4.625
4.960
4.275
7.380
4.602
1
1
1
1
6a
6b
6c
6d
9a
1
Taxol
Maprotiline 8
a
Cell proliferation of MUTU-1 and DG-75 cells was determined with an alamarBlue assay (seeding density
4
1
–5 × 10 cells/mL per well for 96-well plates). The compounds (concentration range 0.01 nM to 50
µM) were added
(
in triplicate) and the cells retained for 24 h (MUTU-1) or 48 h (DG-75), with control wells containing vehicle ethanol
(
1% v/v). The values represent the mean for three experiments. IC50 value is the concentration required for 50%
b
growth inhibition of MUTU-1 cells. log P values calculated from ChemDraw Ultra, version 12.0. (PerkinElmer,
Waltham, MA, USA).
The IC50 values of all the selected compounds 13j
than the lead 9-nitrovinylanthracene based compound 12a (MUTU-I; IC50 2.57
and maprotiline (MUTU-I; 15.8 M, DG-75; 37.5 M) in both BL cell lines but also these compounds
exerted a more potent effect than taxol (MUTU-I; 0.32 M, DG-75; 1.32 M) with submicromolar IC50
,
15
,
16a
–d
and 19a were not only more potent
µ
M, DG-75; 2.08 M)
µ
µ
µ
µ
µ

Pharmaceuticals 2020, 13, 16
22 of 58
values of 0.09–0.38
µM in the MUTU-I cell line and 0.24–0.78
µM in the chemoresistant DG-75 cell line.
(Table 11). These novel compounds were selected for further investigation also based on analysis of
their drug-like properties (Lipinski) from a Tier-1 profiling screen, together with predictions of blood
brain barrier partition, permeability, plasma protein binding, metabolic stability and human intestinal
absorption properties which confirmed that these compounds are moderately lipophilic-hydrophilic
drugs and are suitable candidates for further investigation (Tables S1 and S2, Supporting information).
Compounds 12a, 13j, 16a–d and 19a were found to satisfy all the Lipinski rule of five criteria with logP
values in the range 3.31–5.26, indicating their potential as lead compounds for further development.
The dimeric compound 15 was identified as the most potent analogue evaluated with an IC50 value of
0
.09 µM in the MUTU-I cell line and an IC50 of 0.24 µM in the DG-75 cell line.
Examples of the potent compounds 13j 16a 16b and 19a (displayed as yellow in their
respective overlays) were flexibly aligned with the lead compound maprotiline (cyan) using MOE
Molecular Operating Environment) 2016.V8, (Figure 3). The close correspondence between overlays of
these compounds with maprotiline highlights the presence of three main shared molecular features: the
E)-configuration nitrovinyl pharmacophore located at C-9, the 9,10-dihydroanthracene core structure
,
,
8
(
(
and the presence of the 9,10-ethanoanthracene bridge, unsubstituted as in maprotiline, having a nitrile
substituent as in the acrylonitrile adduct 19a or forming part of the heterocyclic structure as in the
maleimide adducts 13j, 16a and 16b.
13j
16a
16b
19a
Figure 3. Overlay of 9,10-dihydroethanoanthracene compounds 13j
,
16a
,
16b and 19a (yellow) with
maprotiline
8
(cyan). The atoms are coloured by element type: oxygen = red, nitrogen = blue,
chlorine = green.
3
.3. Investigations into the Pro-Apoptotic Effect of the Most Potent Ethanoanthracenes-FITC Annexin V/PI
FACS Analysis
The potential pro-apoptotic effects of the most potent maprotiline analogues were determined by
Annexin V/PI FACS analysis of a subset of the most potent compounds identified e.g., compounds
2a 13j 15 16a and 19a. The study was carried out over a concentration range (0.2–10 M) in
both the BL cell lines (DG-75 and MUTU-I), Figure 4. Taxol was used as a positive control. Taxol
was found to elicit a pro-apoptotic effect in the MUTU-I at both 10 M (87%) and 1 M (63%). The
initial anthracene-nitrostyrene compound 12a demonstrated potent apoptotic activity at 10 M (90%),
with little effect at 1 M [27] (Figure 4A). Compound 16a induced over 80% apoptosis at 10 M, 1
and 0.5 M with 46% apoptosis at 0.2 M (Figure 4A). The p-chlorophenyl compound 16b induced
1
,
,
,
–
d
µ
µ
µ
µ
µ
µ
µM
µ
µ
a more potent effect than the unsubstituted compound 16a with a response of >80% apoptosis at all
concentrations in the MUTU-I cell line. The p-methoxyphenyl compound 16c induced >90% apoptosis
at 0.5
µM. Similar activity was observed for the 10-chloro compound 16d with 63% apoptosis at 0.2
µM,
(Figure 4B). Compounds 13j and 19a were also found to possess potent apoptotic activity at 0.5 µM
>
90%. The most potent compound investigated was the dimer 15, demonstrating > 90% apoptosis
M, (Figure 4C). In summary, compounds 15 and 16b not only possessed the most potent
at 0.2
µ
antiproliferative activity but were found to induce the most favourable pro-apoptotic response in the
MUTU-I cell line.

Pharmaceuticals 2020, 13, 16
23 of 58
In the chemoresistant DG-75 cell line, taxol was found to elicit a pro-apoptotic effect at both 10
73%) and 1 M (21%), (Figure 5A). Compound 12a elicits similar effects to taxol at both 10 M (82%)
and 1 M (10%). The unsubstituted maleimide adduct 16a shows a potent apoptotic effect at 10
92%) but is inactive at lower concentrations (<5% apoptosis). The p-chlorophenylmaleimide 16b and
µM
(
µ
µ
µ
µM
(
p-methoxyphenylmaleimide compound 16c exhibits improved pro-apoptotic activity when compared
to the unsubstituted compound 16a in the DG-75 cell line. Compound 16b and 16c induce 61% and
2
9% apoptosis at 1
and a modest 15% at 1
9a induced 90% and 87% apoptosis at 10
apoptosis at all concentrations assayed with 95% (10
compounds 15 16b and 16c were shown to induce a superior potent pro-apoptotic response in both
µ
M respectively. The 10-chloro compound 16d induced 44% apoptosis at 10
M (Figure 5B). The maleimide Diels-Alder adduct 13j and acrylonitrile adduct
M. The leading compound 15 induced dose dependent
M) in the DG-75 cell line, Figure 5C. Overall
µM
µ
1
µ
µ
,
the MUTU-I and DG-75 BL cell lines than other selected compounds in this grouping and merit
further study.
A
MUTU-I cell line (24 h)
MUTU-I cell line (24 h)
MUTU-I cell line (24 h)
1
00
100
80
60
40
20
0
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
H
M
M
H
M
M
H
M
M
M
M

E


E


E



V
1
0
V
1
0
V
0.2
0.5
1
0
1
1
1
Concentration
Concentration
Concentration
Taxol
B
MUTU-I cell line (24 h)
MUTU-I cell line (24 h)
MUTU-I cell line (24 h)
1
00
100
80
60
40
20
0
1
00
8
0
0
0
0
0
8
6
4
2
0
0
0
0
0
6
4
2
H
M
M
M
M
H
M
M
M
M
E




H
M
M
M
M
VE
V
.
2
.
5
1
0
E
µ
µ
µ
µ
2
5
1
V
0.2
0.5
1
0
1
0.
0.
10
0
0
1
Concentration
Concentration
Concentration
Figure 4. Cont.

Pharmaceuticals 2020, 13, 16
24 of 58
C
MUTU-I cell line (24 h)
MUTU-I cell line (24 h)
MUTU-I cell line (24 h)
1
00
100
80
60
40
20
0
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
H
M
M
M
M
H
M
M
M
M
H
M
M
M
M
E




E




E
µ
µ
µ
µ
V
.
2
.
5
1
0
V
.
2
.
5
1
0
V
2
.
5
0
1
0
.
1
0
0
1
0
0
1
0
Concentration
Concentration
Concentration
Figure 4. Ethanoanthracene nitrostyrene compounds 16a
–d
,
13j, 19a, 15 induce apoptosis in Burkitt’s
lymphoma MUTU-1 cell line. Induction of apoptosis in Burkitt’s lymphoma MUTU-1 cell line following
treatment for 24 h with selected ethanoanthracene nitrostyrene compounds (0.2–10 M) or a comparative
control (taxol) in MUTU-1 cell line is determined using Annexin V and propidium iodide staining/FACS.
) Taxol, 12 16a; ( 16b 16c 16d; ( 13j 19a 15. Ethanol was used as the vehicle. Values represent
the mean of three independent experiments.
µ
(
A
,
B
)
,
,
C
)
,
,
A
DG-75 cell line (48 h)
DG-75 cell line (48 h)
DG-75 cell line (48 h)
1
00
100
80
60
40
20
0
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
H
M
M
H
M
M
H
M
M
M
M

E


E


E



V
1
0
V
1
0
V
0.2
0.5
1
0
1
1
1
Concentration
Concentration
Concentration
Taxol
Figure 5. Cont.

Pharmaceuticals 2020, 13, 16
25 of 58
B
DG-75 Cell line (48 h)
DG-75 Cell line (48 h)
DG-75 Cell line (48 h)
1
00
100
80
60
40
20
0
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
H
M
M
M
M
H
M
M
M
M
H
VE
M
M
M
M
E




E
µ
µ
µ
µ
V
.
2
.
5
1
0
V
2
.
5
0
1
0
2
5
1
1
.
1
0.
0.
10
0
0
0
Concentration
Concentration
Concentration
C
DG-75 Cell line (48 h)
DG-75 Cell line (48 h)
DG-75 Cell line (48 h)
100
1
00
100
8
6
4
0
0
0
8
6
4
2
0
0
0
0
0
80
60
40
20
0
20
0
H
M
M
M
M
H
M
M
M
M
H
M
M
M
M
µ
E




E




E
µ
µ
µ
V
.
2
.
5
1
0
V
.
2
.
5
0
1
0
V
0.2
0.5
1
0
1
0
0
1
0
1
Concentration
Concentration
Concentration
Figure 5. Ethanoanthracene nitrostyrene compounds 16a
lymphoma DG-75 cell line. Induction of apoptosis in Burkitt’s lymphoma DG-75 cell line following
treatment for 48 h with selected ethanoanthracene nitrostyrene compounds (0.2–10 M) or a comparative
control (taxol) in DG-75 cell line is determined by Annexin V and propidium iodide staining/FACS:
) Taxol, 12 16a; ( 16b 16c 16d; ( 13j 19a 15. Ethanol was used as the vehicle. Values represent
the mean of three independent experiments.
–d, 13j, 19a, 15 induce apoptosis in Burkitt’s
µ
(
A
,
B
)
,
,
C
)
,
,
3
.4. Evaluation of In Vitro Cytotoxicity of Ethanoanthracenes
Compounds 15 16b and 16c were found to elicit the most potent anti-proliferative and
,
pro-apoptotic activity of the series. In vitro cytotoxicity of these compounds was evaluated using a
lactate dehydrogenase (LDH) assay. LDH is released during mechanisms of cell death associated
with loss of cell membrane integrity (necrosis). The MUTU-I and DG-75 BL cell lines were treated at
1
0
µ
M and 1
LDH release, (Figure 6). In the MUTU-I cell line low levels of LDH release were obtained (2–17%) at
M and 1 M concentration, indicating low cytotoxicity. The lowest LDH release was observed by
compound 16b with 2% and 5% cytotoxicity (at 10 M and 1 M concentrations respectively). The
µM for the desired treatment period and the results are presented as percentage of total
10
µ
µ
µ
µ

Pharmaceuticals 2020, 13, 16
26 of 58
compounds showed low to moderate cytotoxicity in the DG-75 cell line at 10
µM (25–41%) and 1
µM
(
27–30%). The lowest LDH release was observed for compound 16c with 25% (10
µM) and 17% (1 µM).
Figure 6. Lactate dehydrogenase (LDH) assay of ethanoanthracenes 15
with ethanoanthracene compounds 15 16b and 16c (1 M and 10 M) for 24 or 48 h. Induction of necrotic
cell death was determined by measuring LDH release in MUTU-I ( ) and DG-75 cell lines ( ), with
, 16b and 16c. Cells were treated
,
µ
µ
A
,C
B,D
control lysis solution (100% necrosis). Values are shown the mean of three independent experiments.
3
.5. Effect of Compounds 15, 16b and 16c on the Viability of PBMCs
The 2-nitrovinyl-9,10-dihydro-9,10-ethanoanthracenes compounds 15, 16b and 16c were evaluated
for their toxicity on peripheral blood mononuclear cells (PBMCs) to determine the selective toxicity of
these compounds on malignant BL cell lines over normal lymphatic cells. Compounds were evaluated
at 1
in the PBMCs at 0.5
comparison, compounds 15
µ
M and 0.5
µ
M over a 24 h treatment time. Compounds 15
M (~99% viable cells) and low toxicity at 1
16b and 16c induced a potent anti-proliferative effect in the MUTU-I at
,
16b and 16c elicited minimal toxicity
µM (72–81% viable cells), Figure 7. In
µ
,

Pharmaceuticals 2020, 13, 16
27 of 58
1
µM and 0.5
µM, (2–7% viable cells remaining). Compounds
5,
16b and 16c also induced a potent
anti-proliferative effect in the DG-75 at 1
µM (6–28%) and 0.5 µM (18–57%). Comparing the results
obtained from the BL cell lines and the PBMCs, the results suggest compounds 15
selectively toxic effect on BL cell lines.
, 16b and 16c exert a
A
PBMC (24 h)
1
00
8
6
4
2
0
0
0
0
0
)
)
)
)
)
)
M
M
M
M
M
M
µ
µ
µ
µ
µ
µ
1
0.5
1
0.5
1
(
0.5
(
(
(
b
(
c
(
5
6
6
c
1
5
1
1
b
1
6
6
1
1
Compound (Treatment Concentration)
B
C
DG-75 cell line (48 h)
MUTU-I cell line (24 h)
100
1
00
8
6
4
2
0
0
0
0
0
8
6
4
2
0
0
0
0
0
)
)
)
)
)
)
)
)
)
)
)
)
M
M
M
M
M
M
M
M
M
M
M
M
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
1
0.5
1
0.5
1
(
0.5
(1
0.5
(1
0.5
(1
0.5
(
(
(
b
(
6
c
(
5
(
b
(
c
6
1
(
5
6
b
1
c
1
5
6
b
c
1
5
1
1
6
6
1
1
6
6
1
1
1
1
Compound (Treatment Concentration)
Compound (Treatment Concentration)
Figure 7. In Vitro antiproliferative effect of compounds 15, 16b and 16c on (A) PBMCs (24 h), (B) MUTU-1
cell line (24 h) and (C) DG-75 cell line (48 h) at 1 µM and 0.5 µM.
3
.6. Effect of Pre-Treatment with Antioxidants on Cellular Viability
Reactive oxygen species (ROS) are short lived diffusible entities containing oxygen such as hydroxy,
nitroxyl, alkoxy, superoxide or peroxyl radicals. ROS are generated as metabolites of oxygen and are
utilised for signalling events for essential cell functions. ROS are often associated with the induction of
cell death and apoptosis. A potential role for ROS in the mechanism of cell death induced by selected
potent compounds 15
, 16b and 16c was investigated. A viability assay was used to investigate the
effects of pre-incubation with an antioxidant on ROS levels in DG-75 BL cells with the compound of

Pharmaceuticals 2020, 13, 16
28 of 58
interest. DG-75 cells were pre-treated with N-acetylcysteine (NAC), a known ROS scavenger and
subsequently treated with the selected compounds 15 16b and 16. Viability was monitored using the
alamarBlue assay, (Figure 8). From the results, obtained it is evident that in the presence of the reactive
oxygen species inhibitor NAC—the anti-proliferative effects of compounds 15 16b and 16 at 1
was reduced. Overall the anti-proliferative effects previously observed by compounds 16b 15 and 16c
,
,
µM
,
increased from 6–23% viable cells to 73–83% viable cells in the presence of 5 mM NAC, indicating that
ROS may be involved in the mechanism of cell death induced by these compounds, (Figure 8).
DG-75 Cell line (48 h)
100
80
60
40
20
0
)
)
(
)
)
)
)
M
M
M
M
M
µ
M
µ
m
µ
m
1
)
5
1
)
5
1
m
(
(
(
(
(
5
(
b
c
5
6
C
6
C
1
C
1
A
1
A
A
N
N
N
+
+
+
)
M
M
M
µ
µ
µ
1
1
1
(
(
c
b
6
5
1
6
1
1
Compound (Treatment Concentration) ± NAC (5 mM)
Figure 8. Effect of antioxidant pre-treatment on viability of DG-75 cells treated with compounds 15
6b and 16c.
,
1
Cell proliferation of MUTU-1 and DG-75 cells was determined with an alamarBlue assay
4
(
seeding density 1–5
×
10 cells/mL per well for 96-well plates). Compound concentrations of
either 1 M or 0.5
µ
µ
M for 24 h (MUTU-1) or 48 h (DG-75) were used to treat the cells (in triplicate) with
control wells containing vehicle ethanol (1% v/v). The mean value for three independent experiments
is shown.
Cell proliferation of DG-75 cells was determined with an alamarBlue assay (seeding density
4
2
× 10 cells/mL per well for 96-well plates), with vehicle control ethanol 1% (v/v). Cells were retained
for 24 h and then pre-treated with NAC (5 mM) for 1 h, followed by compounds 15
, 16b and 16c at 1 µM
for 48 h. Cell viability was measured by alamarBlue assay (mean of three independent experiments).
4
. Conclusions
A series of 9,10-dihydro-9,10-ethanoanthracene based maprotiline analogues were synthesised
and evaluated for potential antiproliferative activity in the MUTU-I and chemoresistant DG-75 BL
cell lines. Substitution at C-9 and C-10 of the 9,10-dihydro-9,10-ethanoanthracene compounds was
achieved by modification of the diene system to include functionalities such as nitrovinyl, nitroalkyl,
aldehyde, imine, carboxylic acid, alcohol, oxime, cyanovinyl and hydrazone on the anthracene scaffold.
The effect of a number of 9,10-dihydro-9,10-ethanoanthracene structural modifications on activity was
also investigated; these modifications included ethano bridge modifications, phenyl substitutions,

Pharmaceuticals 2020, 13, 16
29 of 58
maleimide substitutions and extension of alkyl chain length at C-2 of the nitrovinyl unit. The most
promising 9,10-dihydro-9,10-ethanoanthracene based maprotiline analogues were identified and
all included a nitrovinyl substituent at C-9. The structure-activity relationships for the series of
ethanoanthracenes synthesised in this study are summarised in Figure 9. The preliminary screen of the
9
,10-dihydro-9,10-ethanoanthracenes identified the maleimide compounds 15
compounds from this study. The dimer compound 15 displayed potent anticancer activity, in both BL
lines with IC50 values of 0.09 M (MUTU-I) and 0.24 M (DG-75), while both compounds 16b and 16c
, 16b and 16c as the lead
µ
µ
elicited significant anti-proliferative activity in the BL cell lines. The present work has demonstrated
the selectively toxic effect of compounds 15 and 16c towards the MUTU-I and DG-75 cell lines over
PBMCs. These compounds were shown to induce a significant pro-apoptotic effect in MUTU-I cell
and DG-75 BL cell lines and may target the stress response to ROS in DG-75 BL cell lines. The results
suggest that this class of compounds merits further investigation as antiproliferative agents for BL.
Further studies are in progress to investigate the role of these compounds in ROS mediated cell death
and in the reversal of drug efflux in multidrug resistant cancer cells.
Figure 9. Summary of SAR for 9,10-dihydro-9,10-ethanoanthracenes.
5
. Experimental Section
5
.1. Chemistry
All commercially available reagents were used without further purification. Solvents were dried
prior to use; tetrahydrofuran (THF) by distillation from sodium/benzophenone under nitrogen, toluene
was distilled from sodium, dichloromethane was distilled from calcium hydride. Melting points were
recorded on a Gallenkamp SMP 11 melting point apparatus and are uncorrected. Infra-red (IR) spectra
1
13
were obtained on a Perkin Elmer FT-IR Spectrum 100 spectrometer. H and C NMR spectra were
1
obtained on a Bruker Avance DPX 400 spectrometer operating at 400.13 MHz, ( H) and 100.61 MHz
13
◦
C) at 20 C in either CDCl or DMSO-d with appropriate solvent peaks as reference standards.
3 6
(
Mass spectrometry (ESI-MS) was performed on a Micromass LCT instrument with mass measurement
accuracies of < 5 ppm. Low resolution mass spectra (LRMS) were obtained on a Hewlett-Packard 5973
±
MSD GC-MS system. Preparative separations were performed using flash column chromatography
on silica gel (Merck Kieselgel 60, particle size 0.040–0.063 mm). Chromatographic separations were
also performed on Biotage SP4 instrument. All reactions and products were monitored on thin layer
chromatography (TLC) using Merck silica gel 60 F254. HPLC was used to determine the purity of the
compounds (2487 Dual Wavelength Absorbance detector (Waters), 1525 binary HPLC pump, In-Line
Degasser AF and Waters 717plus Autosampler), together with a Varian Pursuit XRs C18 reverse phase

Pharmaceuticals 2020, 13, 16
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1
50
×
4.6 mm chromatography column. Samples were detected using a wavelength of 254 nm. Details
for the preparation of compounds 11a–11n, 11p–11r, 20a, 20f, 20g, 21a–j, 22a–c, 22e is contained in the
Supplementary Information.
5
.2. General Procedure 1: Preparation of Phenyl and Benzyl Maleimides (11a–11s)
To a solution of maleic anhydride (20 mmol) in diethyl ether (25 mL) was added the appropriate
benzyl or aryl amine (20 mmol) in diethyl ether (10 mL). The reaction mixture was stirred under reflux
◦
at 20 C for 1 h. The precipitated solid was filtered and washed with diethyl ether. This solid was
immediately used in the next step and placed in a conical flask containing sodium acetate (8.5 mmol,
◦
0
.7 g) and acetic anhydride (10 mL). The mixture was heated at 90 C for 0.5 h, then poured over ice
water (100 mL). The solid was filtered and recrystallised from ethanol.
5
.2.1. 1-(3,4,5-Trimethoxyphenyl)-1H-pyrrole-2,5-dione (11o)
Compound 11o was prepared from maleic anhydride (20 mmol) and 3,4,5-trimethoxyaniline
(
20 mmol) following the general procedure 1. The precipitated solid filtered and recrystallised from
◦
ethanol, to afford the product as yellow crystals, 3.17 g (60%), Mp. 120–128 C. IR
2
(KBr): 3085,
Vmax
−
1 1
964 (C-H), 1707 (C=O), 1509, 1470 (C=C), 1598 (C=C), 1127 (CN) cm . H NMR (400 MHz, CDCl )
3
13
δ
3.86 (s, 9 H, 3 x OCH ), 6.54 (s, 2 H, 2 x ArH), 6.84 (s, 2 H, 2 x =CH). C NMR (101 MHz, CDCl3)
3
ppm 56.2 (OCH ), 60.8 (OCH ), 104.0 (CH), 126.6, 134.1 (CH), 137.7, 153.4, 169.5 (C=O). HRMS (APCI)
3
3
+
calculated for C H NO [M + H] 264.0872: found 264.0867.
1
3
14
5
0
5
.2.2. 1,1 -(Hexane-1,6-diyl)bis(1H-pyrrole-2,5-dione) (11s)
Compound 11s was prepared from maleic anhydride (20 mmol) and 1,4-diaminobenzene (20 mmol)
◦
following the general procedure 1. The product was obtained as yellow crystals (60%), Mp. 130–136 C
◦
(
lit. M.p. 136–141 C [67]). IRVmax (ATR): 3103, 2939 (C-H), 1688 (C=O), 1587, 1453 (C=C), 1227 (CN)
δ 1.09–1.34 (m, 4 H, 2 x CH ), 1.34–1.56 (m, 4 H, 2 x CH ), 3.39
2 2
t, J = 7.26 Hz, 4 H, 2 x CH ), 6.60 (s, 3 H, 2 x =CH). C NMR (101 MHz, CDCl ) ppm 25.9 (CH ), 28.1
−1
1
cm . H NMR (400 MHz, CDCl )
3
13
(
(
[
2
3
2
CH ), 37.4 (CH ), 133.9 (=CH), 169.7 (C=O), 170.7 (C=O). HRMS (APCI) calculated for C H N O
4
2
2
14 17
2
+
M + H] 277.1188: found 277.1183.
5
.3. General Procedure 2: Preparation of Nitrovinyl Anthracenes 12a–12f
To a solution of 9-anthraldehyde (2 g, 9.7 mmol) in the appropriate nitroalkane
(
nitromethane, nitroethane, nitropropane) (15 mL) was added piperidinium acetate (1.5 g, 10.3 mmol).
(
Piperidinium acetate was prepared from piperidine 6.6 mL and acetic acid 3 mL). The solution was
◦
heated at 90 C for 1.5 h under nitrogen for 1 h, then cooled to room temperature and poured onto
1
00 mL of ice cold H O. Following DCM extraction, the organic layers were combined, dried (Na SO )
2 2 4
and solvent removed. The product was recrystallised from an appropriate solvent.
5
.3.1. (E)-9-(2-nitrovinyl)anthracene (12a)
Compound 12a was prepared from 9-anthraldehyde (9.7 mmol, 2 g) and nitromethane (15 mL) as
outlined in the general procedure 2. The product was recrystallized from methanol and diethyl ether
◦
◦
as red crystals 2.41 g (99%), Mp. 145–147 C (lit. M.p. 142 C [68]). IRVmax (KBr): 3050, 2948 (C-H),
−
617, 1553 (C=C), 1498, 1330 (NO ), 1250 (C-N) cm . H NMR (400 MHz, CDCl ) δ 7.40–7.66 (m, 5 H,
2 3
1 1
1
4
8
x ArH, CH= x 1), 7.80–8.05 (m, 2 H, 2 x ArH), 8.05–8.25 (m, 2 H, 2 x ArH), 8.45 (br. s., 1 H, H10),
.90 (d, J = 13.43 Hz, 1 H, CH= x 1). 13C NMR (101 MHz, CDCl ) ppm 124.3 (C9), 125.7 (CH), 127.5
3
0
(CH), 129.2 (CH), 129.2, 129.8, 130.4 (CH), 131.1, 135.6 (CH), 142.6 (C2 ). HRMS (APCI) calculated for
+
C H NO [M + H] 250.0868: found 250.0879.
16
12
2