Antiproliferative activity and mode of action analysis of novel amino and amido substituted phenantrene and naphtho[2,1-b]thiophene derivatives

Article abstract of DOI:10.1016/j.ejmech.2019.111833

Herein we present and describe the design and synthesis of novel phenantrene derivatives substituted with either amino or amido side chains and their biological activity. Antiproliferative activities were assessed in vitro on a panel of human cancer cell lines. Tested compounds showed moderate activity against cancer cells in comparison with 5-fluorouracile. Among all tested compounds, some compounds substituted with cyano groups showed a pronounced and selective activity in the nanomolar range of inhibitory concentrations against HeLa and HepG2. The strongest selective activity against HeLa cells was observed for acrylonitriles 8 and 11 and their cyclic analogues 15 and 17 substituted with two cyano groups with a corresponding IC₅₀ = 0.33, 0.21, 0.65 and 0.45 μM, respectively. Compounds 11 showed the most pronounced selectivity being almost non cytotoxic to normal fibroblasts. Additionally, mode of biological action analysis was performed in silico and in vitro by Western blot analysis of HIF-1-α relative expression for compounds 8 and 11.

Full text of DOI:10.1016/j.ejmech.2019.111833

Journal Pre-proof
Antiproliferative activity and mode of action analysis of novel amino and amido
substituted phenantrene and naphtho[2,1-b]thiophene derivatives
Nataša Perin, Valentina Rep, Irena Sović, Štefica Juričić, Danijel Selgrad, Marko
Klobučar, Nataša Pržulj, Chhedi Lal Gupta, Noël Malod-Dognin, Sandra Kraljević
Pavelić, Marijana Hranjec
PII:
S0223-5234(19)30985-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.111833
EJMECH 111833
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 September 2019
Revised Date: 28 October 2019
Accepted Date: 28 October 2019
Please cite this article as: Nataš. Perin, V. Rep, I. Sović, Š. Juričić, D. Selgrad, M. Klobučar, Nataš.
Pržulj, C.L. Gupta, Noë. Malod-Dognin, Sandra.Kraljević. Pavelić, M. Hranjec, Antiproliferative
activity and mode of action analysis of novel amino and amido substituted phenantrene and
naphtho[2,1-b]thiophene derivatives, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111833.
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Graphical Abstract

Antiproliferative activity and mode of action analysis of novel amino and amido
substituted phenantrene and naphtho[2,1-b]thiophene derivatives
Nataša Perin1, Valentina Rep1, Irena Sović1, Štefica Juričić1, Danijel Selgrad2, Marko
Klobučar2, Nataša Pržulj 3,4,5, Chhedi Lal Gupta3, Noël Malod-Dognin3,4,
Sandra Kraljević Pavelić2* and Marijana Hranjec1*
1 Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Marulićev trg 19, HR-10000 Zagreb, Croatia; 2 Department of
Biotechnology, Centre for High-throughput technologies, University of Rijeka, Radmile
Matejčić 2, HR-51000 Rijeka, Croatia; 3 Department of Computer Science, University College
London, London, UK; 4 Department of Life Science, Barcelona Supercomputing Center
(BSC), Barcelona, Spain, 5 ICREA, Pg. Lluis Companys 23, 08010 Barcelona, Spain
Corresponding authors: Dr. Marijana Hranjec. Full Prof., Department of Organic Chemistry,
Faculty of Chemical Engineering and Technology, University of Zagreb, Marulićev trg 20,
P.O. Box 177, HR-10000 Zagreb, Croatia, Phone No. +38514597245; Fax No.
+38514597250; e-mail: mhranjec@fkit.hr; Dr. Sandra Kraljević Pavelić, Associate Prof.,
Department of Biotechnology, University of Rijeka, Trg braće Mažuranića 10, HR-51000
Rijeka, Croatia, Phone No. +38551584569; e-mail: sandrakp@biotech.uniri.hr
1

Abstract
Herein we present and describe the design and synthesis of novel phenantrene derivatives
substituted with either amino or amido side chains and their biological activity.
Antiproliferative activities were assessed in vitro on a panel of human cancer cell lines.
Tested compounds showed moderate activity against cancer cells in comparison with 5-
fluorouracile. Among all tested compounds, some compounds substituted with cyano groups
showed a pronounced and selective activity in the nanomolar range of inhibitory
concentrations against HeLa and HepG2. The strongest selective activity against HeLa cells
was observed for acrylonitriles 8 and 11 and their cyclic analogues 15 and 17 substituted with
two cyano groups with a corresponding IC50 = 0.33, 0.21, 0.65 and 0.45 µM, respectively.
Compounds 11 showed the most pronounced selectivity being almost non cytotoxic to normal
fibroblasts. Additionally, mode of biological action analysis was performed in silico and in
vitro by Western blot analysis of HIF-1-α relative expression for compounds 8 and 11.
Keywords: amines, amides, antiproliferative activity, phenatrenes, mode of action analysis,
naphtho[2,1-b]thiophenes
2

1. Introduction
Natural products may serve as drugs or templates for design of novel molecules and
thus, play a crucial role in the drug discovery and development process. This fact is again
gaining interest among researchers in recent years. For example, phenanthrene is a polycyclic
aromatic naturally occurring ring system of many biologically active compounds that occur in
more than 10 plant families.1 Hence, natural differently substituted phenantrene derivatives
were isolated from Combretaceae, Orchidaceae, Dioscoreaceae and Betulaceae families
possessing various biological activities including antitumor,2-4 antibacterial5 or anti-
inflammatory activities.6 Most natural phenanthrenes occur in the monomeric form consisting
of more than 200 compounds substituted mainly with hydroxy and methoxy groups placed at
different positions on the tetracyclic skeleton. Pettit et al.7 have isolated and structurally
characterized a series of active phenanthrenes present in the African willow tree Combretum
caffrum. Their antitumor activity was confirmed on murine P388 lymphocytic leukaemia cell
(Fig. 1, I). Furthermore, Lusianthridin (Fig. 1, II) and Denbinobin which were isolated from
Dendrobium nobile were found to exert cytotoxic effects both in vitro and in vivo on several
cancer cells.8 Phenantrenes isolated from D. thyrsiflorum which has been used in Chinese
ethnomedicine, also showed antitumor activity on several cancer cell.9 Additionally,
derivatives isolated from Domohinea perrieri displayed significant activity towards cancer
cells with some cell-type selectivity (Fig. 1, III). Obtained results revealed that unsubstituted
methyl group placed at the C-7 position is very important for the antitumor activity.10
Figure 1. Biologically active phenathrene derivatives
Phenantrene derivatives could be usually prepared by oxidative coupling of the aromatic rings
of stilbene precursors. Besides, due to the high biologically potential of substituted
phenantrene derivatives and the fact that the synthesis of phenanthrenes is of highly
importance in medicinal chemistry, several synthetic methods were published for the
3

preparation of suchlike compounds. The synthetic strategies for their preparation thus include
benzyne‐alkyne‐benzyne insertion,11 intramolecular cyclizations,12 [4+2] benzannulation
reactions13,14 or Pd catalyzed insertion of alkynes into cyclic diaryliodoniums.15
It is well known that heterocyclic derivatives, both those of natural occurrence and
widely distributed in nature or those of synthetic origin, are essential for many life processes
and have an important role in medicinal chemistry due to the large variety of their possible
chemical, pharmacological and biological properties.16,17 A large number of biologically
important compounds belong to a major class of heterocycles containing sulphur. For
example, thiophenes and its fused derivatives as benzothiophenes, naphthothiophenes ot
thienothiophenes have been classified as highly-privileged structures and valuable building
blocks in organic and medicinal chemistry.18,19 Thiophene nuclei could be found in the
structure of numerous medicinal agents including Raloxifene as selective estrogen receptor
modulator or Zileuton for asthma treatment. Naphthothiophene derivatives display a wide
range of biological activities like antitumor,20 antibacterial,21 antifungal,22 analgestic23 or anti-
inflammantory24 and may be additionally exploited as photographic materials25 or for the
purpose of aqueous cold bleaching of textiles.26 At last, benzothiophenes have been recently
proposed also as MAO-B inhibitors with a potential in treatment of neurodegenerative disease
as well.27
Taking into account that both phenantrene and naphthothiophene derivatives are
promising and expanding groups of potentially biologically active compounds whose
potential has not yet been thoroughly investigated sufficiently, we have designed and
synthesized their novel amino and amido substituted derivatives. As part of the initial
evaluation of their biological activity profile, their antiproliferative activity was determined.
Besides, the structure activity relationships are discussed in this work to correlate between the
substituent effects and the activities that aid in drug design. Furthermore, mode of biological
action analysis was performed based on obtained results in silico and in vitro.
2. Results and Discussion
2.1. Chemistry
All newly prepared compounds were synthesized according to the main experimental
synthetic procedure presented in Scheme 1.
4

R
MeOHabs.
MeONa
Ar
Ar
CN
CHO
CN
3 R = F
4 R = H
5 R = CN
R
6 R = F
7 R = H
1 Ar =
2 Ar =
Ar =
Ar =
8 R = CN
S
9 R = F
10 R = H
11 R = CN
S
h
EtOHabs.
iodine
Ar
R
CN
14 R = H
12 R = F, Ar =
13 R = F, Ar =
Ar =
Ar =
15 R = CN
S
MW
S
16 R = H
17 R = CN
CH3CN
amine
2N NaOH
Ar
R
Ar
R
CN
18 R = NHCH2CH(CH3)2
COOH
19 R =
20 R =
N
Ar =
Ar =
24 R = H, Ar =
N
NH
S
25 R = H
26 R = COOH
Ar =
21 R = NHCH2CH(CH3)2
SOCl2,
S
22 R =
23 R =
N
tolueneabs.
N
NH
Ar
R
COCl
27 R = H, Ar =
S
28 R = H
29 R = COCl
Ar =
CH2Cl2abs.
amine
Ar
R2
R1
30 R1= CONHCH2CH(CH3)2, R2 = H, Ar =
31 R1 = CONHCH2CH(CH3)2, R2 = H
32 R1 = CONH(CH2)3N(CH3)2, R2 = H
S
Ar =
33 R1 = R2 = CONHCH2CH(CH3)2
34 R1 = R2 = CONH(CH2)3N(CH3)2
Scheme 1. Synthesis of phenantrene and naphtho[2,1-b]thiophene derivatives
5

Acyclic precursors 6–11 were prepared in the reaction of aldol condensation of corresponding
benzaldehydes 3–5 and benzylcyanide 1 or 2-(thiophen-2-yl)acetonitrile 2 in absolute
methanol using sodium methoxide as a base. Cyclic derivatives of phenanthrene 12–14 and
naphtho[2,1-b]thiophene 15–17 were prepared by photochemical dehydrocyclization in
ethanolic solution with the addition of small amount of iodine, using 400 W high-pressure
mercury lamp and Pyrex filter for 2–37 hours. The photochemical dehydrocyclization reaction
was monitored by UV/Vis spectroscopy. Targeted amino substituted phenanthrenes 18–20
and naphtho[2,1-b]thiophenes 21–23 were prepared from main precursors, namely 3-
fluorophenanthrene-9-carbonitrile 12 or 8-fluoronaphtho[2,1-b]thiophene-4-carbonitrile 15 by
uncatalyzed microwave assisted amination with an excess of added corresponding amine. The
reaction was conducted in acetonitrile at 170 °C. Within the aqueous basic hydrolysis of
cyano substituted derivatives 14–15 and 16–17 in 2N NaOH, the corresponding carboxylic
acids 24–26 were obtained. Carboxylic acids gave in the reaction with thionyl-chloride
corresponding acyl-halogenides 27–29 as the main precursors for the synthesis of designed
amides. Amide substituted derivatives 30–34 were obtained by reaction of acyl-halogenides
27–29 and an excess amount of the corresponding amine in absolute dichloromethane.
The structures of all prepared compounds were determined by NMR spectroscopy (1H
13
and C) based on the analysis of H-H coupling constants as well as chemical shifts and by
elemental analysis. Cyclization of acyclic precursors 6–11 into phenantrene and naphtho[2,1-
b]thiophene derivatives resulted in the disappearance of signals for two H protons confirming
thus the formation of tricyclic skeleton. The NMR spectra of carboxylic acids 24-26 showed
one additional signal in comparison to spectra of cyano substituted derivatives 14 and 16–17.
Introduction of amino substituents into the structure of derivatives 18–23 has been confirmed
by appearance of the signals related to protons of amino side chains in the aliphatic part in
1
both H and 13C NMR spectra. Additionally, the formation of amide derivatives was
confirmed by signal related to the proton of the amide group in the 1H NMR spectra as well as
signals related to the protons of amide side chains in aliphatic part of both spectra.
2.2. Antiproliferative activity in vitro
Antiproliferative activities of acrylonitrile 6-11, phenantrene 12-15, 18-20 and 30 and
naphtho[2,1-b]thiophene derivatives 16-17, 21-23 and 31-34 were assessed on five human
tumor cells in vitro; HeLa (cervical carcinoma), SW620 (colorectal adenocarcinoma,
metastatic), HepG2 (hepatocellular carcinoma), CFPAC-1 (ductal pancreatic adenocarcinoma)
6

and A549 (lung adenocarcinoma) as well as on normal skin fibroblasts HFF. 5-Fluorouracil
was used as a standard drug.
Table 1. In vitro antiproliferative activity (aIC50) of tested derivatives.
IC50a (µM)
Comp.
A549
CFPAC-1
HeLa
HepG2
SW620
HFF
6
7
45.76
64.60
8.39
46.92
88.19
6.70
3.44
48.88
0.33
7.73
81.51
0.52
38.45
>100
9.18
52.70
93.73
6.07
8
9
40.84
58.95
47.70
9.57
34.24
97.84
52.35
13.22
50.98
80.05
8.20
3.59
5.22
36.41
>100
61.39
7.80
86.89
84.33
>100
3.43
10
11
12
13
14
15
16
17
18
19
20
21
22
23
30
31
32
33
34
5-FU
19.99
0.21
43.43
0.30
8.31
18.33
32.11
48.75
10.85
54.28
7.22
49.91
72.37
3.30
11.63
30.31
0.65
42.42
>100
6.80
16.35
>100
1.78
50.43
5.53
55.65
8.19
29.93
0.45
70.31
6.04
84.61
5.13
31.10
58.91
5.49
35.46
>100
6.19
23.63
59.37
3.33
33.56
81.78
19.44
47.63
62.01
20.91
93.35
75.40
23.19
86.08
43.36
>100
4.62
52.91
>100
23.82
98.42
62.01
37.46
>100
96.82
23.97
0.74
31.01
61.30
7.09
18.57
25.72
3.66
18.56
32.22
2.38
41.46
64.14
2.58
64.23
63.90
20.23
27.74
5.57
68.58
97.52
17.46
>100
28.42
39.59
14.38
10.76
61.32
>100
17.67
0.90
6.59
0.14
4.21
8.81
16.23
8.9
4.40
0.08
5.83
-
a IC50 values are the concentrations that cause 50% inhibition of cancer cell growth (µM).
7

According to the obtained results presented in the Table 1, it could be concluded that the
majority of tested derivatives showed moderate activity at micromolar range of inhibitory
concentrations with some selectivity among tested cell lines. The strongest antiproliferative
activity was observed for dicyano substituted acrylonitriles 8 and 11 and their cyclic
analogues 15 and 17. Thus, both acyclic derivatives showed the strongest inhibitory effect and
selectivity towards HeLa and HepG2 cell lines in the submicromolar range of inhibitory
concentrations. Their cyclic analogues, dicyano substituted phenantrene 15 and naphtho[2,1-
b]thiophene 17 derivatives displayed selective activity against HeLa cells in submicromolar
range of IC50 concentrations (IC50 0.65 and 0.45 µM, respectively). Also, obtained results
revealed that the thiophene nuclei in the structure of acyclic 11 and cyclic 17 derivatives
enhanced the antiproliferative activity in comparison to phenyl analogues 8 and 15. Fluoro
substituted acrylonitriles 6 and 9 showed selective activity against HeLa and HepG2 cells in
comparison to other rested cells. Introduction of piperazine group on phenantrene skeleton
(20) caused enhancement of antiproliferative activity compared to other amino substituted
derivatives, namely compounds 18 and 19 with similar results regarding the amino substituted
naphtho[2,1-b]thiophenes 21-23. Among amido substituted derivatives 30-34, the most
pronounced activity was observed with di-N,N-dimethylaminopropyl substituted naphtho[2,1-
b]thiophene 34. Mono substituted derivative bearing N,N-dimethylaminopropyl amide side
chain showed slightly improvement of activity in comparison to N-isobutyl substituted amide
derivatives 30, 31 and 33.
Regarding the results of non-cancer cell line HFF, obtained results revealed some selective
compounds. Thus, compound 11 showed the most significant selectivity in comparison to the
activity against HeLa (IC50 0.21 µM) and HepG2 (IC50 0.230 µM) cells being non cytotoxic
against normal skin fibroblasts (IC50 > 100 µM).
2.3. Mode of action analysis
2.3.1. Target prediction analysis
PIDGIN analysis of all 23 phenanthrene derivatives against all 1651 potential targets,
resulted in 23x1651 = 37973 compound-target interactions. By applying the set threshold, 277
compound-target interactions between the 23 phenanthrene derivatives and 112 unique targets
were predicted.
8

Tested derivatives were found to be active on around half of the known targets (6 out of 13
known targets viz., Q99714, P00352, Q16236, O75164, P11509, and Q12809) of the original
phenanthrene (Compound ID: CHEMBL46730). As presented in Fig. 2, phenanthrene
derivative 14 has the largest number of predicted targets (49 targets), followed by derivatives
20 and 7 (33 targets), which suggests that these derivatives are less selective towards
biological targets. On the other hand, three derivatives (31, 33, and 34) out of 23 are found to
be inactive with no predicted targets.
Figure 2. The bar plot displays the number of targets (Y-axis) that is predicted for each
derivative (X-axis) when applying the specific threshold (Pa ≥ 0.9).
As presented in Figure 3, PIDGIN predicts target AR (Androgen receptor, Uniprot ID:
P10275) to interact with the largest number of derivatives (10, 43.48% of our input
compounds). The prediction of Androgen receptor as a potential target for our Phenanthrene
derivative is also supported by literature.28 AR abnormalities have been identified in various
diseases such as androgen insensitivity syndrome, spinal bulbar muscular atrophy, benign
prostatic hyperplasia, and prostate cancer.29 A recent study also demonstrates the association
of androgens with depressive symptoms and cognitive status in general populations.30 Thus,
our results open a new insight to explore the therapeutic potential of these derivatives by
targeting Androgen receptors to produce anti-cancer and/or anti-depressant activity.
9

Figure 3. The bar plot showing the number and percentage of Phenathrene derivatives (Y-
axis) found to be active for each predicted biological target (X-axis).
To explore the mode of action of Phenanthrene derivatives on various target groups, we have
identified the protein classes of our predicted potential targets (Fig. 4).
Figure 4. The pie chart shows the percentage distribution of the various target classes for the
112 unique predicted targets of phenanthrene derivatives. The result indicates the presence of
high percentage of kinase targets for phenanthrene derivatives compared to other target
classes.
The largest proportion of predicted targets belongs to protein kinase target class (27%).
Protein kinases play a predominant regulatory role in almost every aspect of cell biology and
have ability to modify the function of a protein in almost every conceivable way.31
10

Protein kinases are an important class of drug target for developing therapeutic agents against
Cancer, inflammatory diseases, autoimmune disease, neurodegenerative and mental
disorders.32 Interestingly, among the predicted protein kinase targets, NUAK1 (NUAK family
SNF1-like kinase 1, Uniprot ID: O60285) and MAP2K1 (Dual specificity mitogen-activated
protein kinase kinase 1, Uniprot ID: Q02750), which acts on the largest proportion of
phenanthrene derivatives (39.13% and 26.08% respectively), were identified. Both have
important role in cancer progression and are potential targets for the treatment of non-small
cell lung cancer.33 In a previous study, Wang et al., has also explored the anti-cancer activity
of a phenathrene derivative (T26) by inhibiting a protein kinase.
2.3.2. Functional analysis of the predicted targets
As presented in Figure 5, the functional analysis revealed that predicted targets for
phenanthrene derivatives are significantly enriched in seven GO-BP terms, ten GO-MF terms,
and five GO-CC terms, which could be categorize into five main terms for both GO-BP &
MF and two main terms for GO-CC ontology’s.
Figure 5. Enriched Gene ontology - Biological Processes (BP), Molecular Function (MF),
and Cellular Component (CC) terms for the predicted targets of Phenanthrene derivatives.
The vertical axis shows the GO (BP, MF, and CC) terms, and the horizontal axis displays the
number of targets per term. The enriched GO terms are presented here with significance level
of ≤ 5% after Bonferroni adjustment. The GO terms belonging to same group (same color).
11

For GO-BP annotation, we find that a large number of the targets are enriched for the
negative regulation of biological process term (80 targets) followed by the cellular aromatic
compound metabolic process term (72 targets). Significant number of targets is enriched for
the serotonin receptor signalling pathway term. For GO-MF annotation, predicted targets of
phenanthrene derivatives are significantly enriched with the serotonin receptor activity, DNA
binding activity as well as protein dimerization activity. In GO-CC annotation, the predicted
targets were found to be enriched with mainly intracellular non-membrane-bounded organelle
and nucleoplasm term. The KEGG pathway enrichment analysis was also done and only
Serotonergic synapse pathway term was significantly enriched with the predicted targets of
tested phenanthrene derivatives (Table 1).
Table 1. Pathway enrichment analysis of the predicted targets of phenanthrene derivatives
against the background data set.
No. of
targets targets
% of
Bonferroni
(Corrected P-value)
Fold
Enrichment
Pathway term
P- value
Serotonergic synapse
Acute myeloid leukemia
cAMP signaling pathway
Cocaine addiction
15
8
13.39
7.14
13.39
5.35
8.03
3.57
6.25
6.59E-0.5
8.57E-03
1.73E-02
2.30E-02
3.75E-02
4.23E-02
4.84E-02
0.0118
0.787
0.957
0.984
0.998
0.999
0.999
3.31
3.32
1.91
3.50
2.26
4.86
2.55
15
6
HIF-1 signaling pathway
Prion diseases
9
4
Chronic myeloid leukemia
7
Disease enrichment analysis was also done by using DAVID v6.8 to explore the involvement
of the predicted targets of phenanthrene derivatives in various diseases. As presented in
Figure 6, predicted targets were significantly enriched in five disease terms, which are mostly
related to mental illness. The bar chart showing the significantly enriched disease terms for
the predicted targets of phenanthrene derivatives. The vertical axis represents the disease
terms, and the horizontal axis represents the number (red color) & percentage (Blue color) of
targets per term.
12

Figure 6. Enriched disease terms for the predicted targets of phenanthrene derivatives.
In summary, we have performed a target prediction analysis of 23 phenanthrene and
naphtho[2,1-b]thiophene derivatives and predicted a total of 277 compound-target interactions
between our derivatives and 112 potential biological targets. Among the predicted targets, the
Androgen Receptor (AR) has predicted to interact with large number of phenanthrene and
naphtho[2,1-b]thiophene derivatives, suggesting anti-cancer and anti-depressant activity.
Additionally, the protein kinase target class are found to be the largest target group predicted
for our derivatives. Furthermore, the functional enrichment analysis of the predicted targets,
suggests the therapeutic role of our phenanthrene and naphtho[2,1-b]thiophene derivatives in
mental disorders (suggesting Serotonin receptor activity as well as association of predicted
targets of our phenanthrene and naphtho[2,1-b]thiophene derivatives in mental disorders).
The largest numbers of targets (31, 27.67% of the predicted targets) were enriched for
Schizophrenia disease followed by Attention deficit hyperactivity disorder and Suicide
disease terms (10, 8.92% of the predicted targets for the both terms) (Fig. 6). In a previous
study, Moreno et al., has suggested the role of phenanthrene type alkaloids in the treatment of
schizophrenia or Parkinson’s disease.34
13

2.4. Western blot analysis of HIF-1-α relative expression
As the in silico analysis suggested some of the pathways known to be important in
tumor development as potential targets of the tested derivatives, we analysed HIF-1-α relative
expression in HeLa and HepG2 cells treated with compounds 8 and 11 due to observed
selective antiproliferative effects on these cell lines. The hypoxia response indeed, proved to
be an important target for anticancer therapy.35 Western blot analysis revealed significant up-
regulation (p<0.05) of the hydroxylated HIF-1 protein after treatment of HeLa cells with
compounds 8 and 11 after 48h and 72h respectively (Fig. 7). A similar trend was observed in
HepG2 cells treated with compounds 8 and 11 but only after 72 h (Fig. 7B). This result is
indicative for increased levels of the protein HIF-1α form tagged for proteasomal
degradation.35
Figure 7. Representative Western blots and summary representation of (A) HIF-1α and (B)
MEK 1/2 relative expression levels in HeLa and HepG2 cells treated with compounds 8 and
11 for 48h and 72h. Results are presented as average relative expression values + standard
error of the mean (SEM) of chemiluminescent signals obtained in three replicate experiments.
Statistically significant changes (Student's t-test, p<0.05) are marked with an asterisk (*).
14

Furthermore, significantly decreased activated MEK1/2 protein levels were observed in HeLa
cells treated with compound 8 for 48 h (Fig. 7A) and compound 11 after 72 h-treatment, while
in HepG2 cells a significant downregulation of MEK 1/2 was observed only after 72 h
treatment with both compounds (Fig. 7B). This result is indicative for deregulation of
Ras/Raf/MEK/ERK signaling cascade involved in enhanced dedifferentiation and
proliferation of tumor cells (U0126, a mitogen-activated protein kinase kinase 1 and 2 (MEK1
and 2) inhibitor, selectively up-regulates main isoforms of CYP3A subfamily via a pregnane
X receptor (PXR) in HepG2 cells; Intrinsically active MEK variants are differentially
regulated by proteinases and phosphatases) (Figures 7A and 7B).
3. Conclusion
Herein we described the design and synthesis of novel phenantrene and naphtho[2,1-
b]thiophene derivatives bearing either different amino or amido side chains. In addition, their
antiproliferative activity and mode of biological action are presented and described.
Targeted cyclic derivatives were obtained by using photochemical dehydrocyclization. Amino
substituted derivatives were prepared by uncatalyzed microwave assisted amination while
amide substituted derivatives obtained by reaction of corresponding acyl-halogenides an
excess amount of the amine. The most prominent antiproliferative activity was observed for
for dicyano substituted acrylonitriles 8 and 11 and their cyclic analogues 15 and 17.
Additionally both acyclic derivatives showed the selectivity towards HeLa and HepG2 cell
lines and were chosen for further structure optimization as a lead compounds for design of
more efficient antiproliferative agents. Furthermore, compound 11 did not show cytotoxic
effect on normal cells compared to its activity against HeLa and HepG2 cells in nanomolar
range of concentrations. The antiproliferative activities were assessed in vitro on a several
human cancer cell lines as well as on normal skin fibroblasts. Tested compounds showed
moderate activity against cancer cells in comparison with 5-fluorouracile. Among the
predicted targets in silico, the androgen receptor (AR) was predicted to interact with large
number of phenanthrene derivatives, suggesting their potential anti-cancer and anti-depressant
activity that should be studied separately. Additionally, the protein kinase target class were
found to be the largest target group predicted for the new derivatives.
The functional enrichment analysis of the predicted targets, suggests a potential
therapeutic role of phenanthrene derivatives in tumor cells signalization, such as for example
hypoxia response that was validated in HeLa cells and HepG2 cells.
15

4. Experimental part
4.1. Synthesis
4.1.1. General methods
All chemicals and solvents were purchased from commercial suppliers Aldrich and
Acros. Melting points were recorded on SMP11 Bibby and Büchi 535 apparatus. All NMR
spectra were measured in DMSO-d6 solutions using TMS as an internal standard. The 1H and
13C NMR spectra were recorded on a Varian Gemini 300 or Varian Gemini 600 at 300, 600
and 150 and 75 MHz, respectively. Chemical shifts are reported in ppm (δ) relative to TMS.
All compounds were routinely checked by TLC with Merck silica gel 60F-254 glass plates.
In preparative photochemical experiments, the irradiation was performed at room temperature
with a water-cooled immersion well with “Origin Hanau”, 400 W, high-pressure, mercury arc
lamp using Pyrex glass as a filter. Elemental analysis for carbon, hydrogen and nitrogen were
performed on a Perkin-Elmer 2400 elemental analyzer. Where analyses are indicated only as
symbols of elements, analytical results obtained are within 0.4% of the theoretical value.
4.1.2. General method for the synthesis of compounds 6–11
Sodium was dissolved in 10 mL of absolute methanol and benzyl cyanide or 2-(thiophen-2-
yl)acetonitrile was added. The mixture was stirred at room temperature for 20 minutes, after
that the appropriate aldehyde was added and heated at reflux. The cooled reaction mixture
was filtered and, if necessary, recrystallized from the appropriate solvent.
(Z)-3-(4-fluorophenyl)-2-phenylacrylonitrile 6
The reaction of benzyl cyanide 1 (0.98 mL, 8.50 mmol) and 4-fluorobenzaldehyde 3 (0.62
mL, 8.50 mmol) after refluxing for 4 hours and recrystallization from ethanol gave 0.71 g
(37%) white powder product; Mp = 114–118 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.06 (s, 1H, Harom,), 8.04–7.99 (m, 2H, Harom), 7.79–
7.74 (m, 2H, Harom), 7.56–7.45 (m, 3H, Harom), 7.44–7.37 (m, 2H, Harom); 13C NMR (150
MHz, DMSO) (δ/ppm): 163.0 (d, J = 249.8 Hz), 141.7, 133.6, 131.6 (d, J = 8.8 Hz), 130.3 (d,
J = 3.2 Hz), 129.3, 129.2 (2C), 125.7 (2C), 117.8, 116.1 (d, J = 21.9 Hz), 110.0; Found: C,
80.52; H, 4.70; N, 6.35. Calc. for C15H10FN: C, 80.70; H, 4.52; N, 6.27%.
16

(Z)-2,3-diphenylacrylonitrile 7
The reaction of benzyl cyanide 1 (0.98 mL, 8.50 mmol) and benzaldehyde 4 (0.86 mL, 8.50
mmol) after refluxing for 2 hours and recrystallization from ethanol gave 1.69 g (97%) of
white crystalline product; Mp = 88–92 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.06 (s, 1H, Harom.), 7.95 (d, J = 7.7 Hz, 2H, Harom),
7.78 (d, J = 7.1 Hz, 2H, Harom), 7.59–7.50 (m, 5H, Harom), 7.48–7.43 (m, 1H, Harom); 13C NMR
(150 MHz, DMSO) (δ/ppm): 142.9, 133.7, 130.6, 129.3 (2C), 129.2 (2C), 129.1 (2C), 128.9
(2C), 125.8, 117.9, 110.3; Found: C, 87.97; H, 5.20; N, 6.82. Calc. for C15H11N: C, 87.77; H,
5.40; N, 6.82%.
(Z)-4-(2-cyano-2-phenylvinyl)benzonitrile 8
The reaction of benzyl cyanide 1 (0.98 mL, 8.50 mmol) and 4-cyanobenzaldehyde 5 (1.12 g,
8.50 mmol) after refluxing for 1.5 hours and recrystallization from ethanol gave 1.89 g (96%)
of white powder product; Mp = 174–178 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.16 (s, 1H, Harom.), 8.08 (d, J = 8.4 Hz, 2H, Harom),
8.02 (d, J = 8.6 Hz, 2H, Harom), 7.81 (dd, J1 = 8.2 Hz, J2 = 1.4 Hz, 2H, Harom), 7.59–7.46 (m,
3H, Harom); 13C NMR (150 MHz, DMSO) (δ/ppm): 140.9, 138.1, 133.1, 132.8 (2C), 129.9,
129.6 (2C), 129.3 (2C), 126.1 (2C), 118.4, 117.3, 113.5, 112.3; Found: C, 83.26; H, 4.50; N,
12.24. Calc. for C16H10N2: C, 83.46; H, 4.38; N, 12.17%.
(E)-3-(4-fluorophenyl)-2-(thiophen-2-yl)acrylonitrile 9
The reaction of thiophene-2-acetonitrile 2 (0.86 mL, 8.10 mmol) and 4-fluorobenzaldehyde 3
(0.87 mL, 8.10 mmol) after refluxing for 4 hours and recrystallization from methanol gave
1.51 g, 97%) of a yellow powder product; Mp = 80–86 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 7.98 (dd, J1 = 8.7 Hz, J2 = 5.6 Hz, 2H, Harom), 7.82 (s,
1H, Harom), 7.70 (dd, J1 = 5.0 Hz, J2 = 0.9 Hz, 1H, Harom), 7.45 (dd, J1 = 3.6 Hz, J2 = 0.9 Hz,
1H, Harom), 7.39 (t, J = 8.9 Hz, 2H, Harom), 7.18 (dd, J1 = 5.0 Hz, J2 = 3.7 Hz, 1H, Harom); 13C
NMR (150 MHz, DMSO) (δ/ppm): 163.8 (d, J = 248.5 Hz), 139.0, 138.2, 131.4 (d, J = 8.7
Hz), 129.9 (d, J = 3.2 Hz), 128.4 (2C), 127.8, 126.7 (2C), 116.8, 116.2 (d, J = 21.9 Hz),
104.6; Found: C, 68.25; H, 3.43; N, 6.20. Calc. for C13H8FNS: C, 68.10; H, 3.52; N, 6.11%.
(E)-3-phenyl-2-(thiophen-2-yl)acrylonitrile 10
The reaction of thiophene-2-acetonitrile 2 (0.86 mL, 8.10 mmol) and benzaldehyde 3 (0.83
mL, 8.10 mmol) after refluxing for 4 hours and recrystallization from methanol gave 0.79 g
(46%) of a yellow powder product; Mp = 79–83 °C.
17

1H NMR (300 MHz, DMSO) (δ/ppm): 7.91 (d, J = 8.0 Hz, 2H, Harom), 7.82 (s, 1H, Harom),
7.70 (dd, J1 = 5.1 Hz, J2 = 1.0 Hz, 1H), 7.57–7.48 (m, 3H, Harom), 7.47 (dd, J1 = 3.6 Hz, J2 =
1.0 Hz, 1H, Harom), 7.19 (dd, J1 = 5.0 Hz, J2 = 3.7 Hz, 1H, Harom); 13C NMR (75 MHz, DMSO)
(δ/ppm): 140.7, 138.8, 133.8, 131.1, 129.5 (2C), 129.4 (2C), 128.9, 128.4, 127.3, 117.4,
105.4; Found: C, 74.00; H, 4.10; N, 6.70. Calc. for C13H9NS: C, 73.90; H, 4.29; N, 6.63%.
(E)-4-(2-cyano-2-(thiophen-2-yl)vinyl)benzonitrile 11
The reaction of thiophene-2-acetonitrile 2 (0.86 mL, 8.10 mmol) and 4-cyanobenzaldehyde 5
(1.06 g, 8.10 mmol) after refluxing for 4 hours and recrystallization from methanol gave 1.04
g, 53%) of a light green crystalline product; Mp = 162–165 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.05 (d, J = 8.5 Hz, 2H, Harom), 7.99 (d, J = 8.6 Hz,
2H, Harom), 7.91 (s, 1H, Harom), 7.77 (dd, J1 = 5.0 Hz, J2 = 0.9 Hz, 1H, Harom), 7.53 (dd, J1 =
3.6 Hz, J2 = 0.8 Hz, 1H, Harom ), 7.21 (dd, J1 = 5.0 Hz, J2 = 3.8 Hz, 1H, Harom); 13C NMR (150
MHz, DMSO) (δ/ppm): 137.8, 137.8, 137.7, 132.7 (2C), 129.4 (2C), 129.0, 128.6, 127.9,
118.4, 116.3, 112.2, 107.9; Found: C, 71.02; H, 3.50; N, 11.60. Calc. for C14H8N2S: C, 71.16;
H, 3.41; N, 11.86%.
4.1.3. General method for the synthesis of compounds 12 - 17
Ethanolic solutions of acrylonitriles 6 – 11 and small amount of iodine (5%) were irradiated at
room temperature with 400 W, high-pressure mercury lamp using a Pyrex filter for 2–37
hours, until the UV spectra showed that the reaction of photochemical dehydrocyclization was
completed. The air was bubbled through the solution. The solutions were concentrated under
reduced pressure and resulting product was filtered off.
3-fluorophenanthrene-9-carbonitrile 12
Compound 12 was prepared from 6 0.30 g (1.30 mmol) in ethanol (400 mL) after irradiation
for 4 hours to yield 0.14 (47%) of light yellow powder. Mp = 159–170 °C
1H NMR (300 MHz, DMSO) (δ/ppm): 8.96 (dd, J1 = 7.1 Hz, J2 = 2.1 Hz, 1H, Harom), 8.76 (dd,
J1 = 11.4 Hz, J2 = 2.3 Hz, 1H, Harom), 8.71 (s, 1H, Harom), 8.25 (dd, J1 = 8.9 Hz, J2 = 6.1 Hz,
1H, Harom), 8.18 (dd, J1 = 6.9 Hz, J2 =2.3 Hz, 1H, Harom), 7.95–7.83 (m, 2H, Harom), 7.70 (td, J1
13
= 8.6 Hz, J2 = 2.5 Hz, 1H, Harom); C NMR (75 MHz, DMSO) (δ/ppm): 163.6 (d, J = 248.4
Hz), 136.2, 133.8 (d, J = 9.5 Hz), 133.1 (d, J = 9.7 Hz), 129.7, 129.6 (d, J = 4.2 Hz), 128.9,
128.7, 126.9, 125.6, 124.9, 118.0, 117.6 (d, J = 24.3 Hz), 109.3 (d, J = 23.2 Hz), 107.8;
Found: C, 81.14; H, 3.84; N, 6.39. Calc. for C15H8FN: C, 81.44; H, 3.65; N, 6.33%.
18

8-fluoronaphtho[2,1-b]thiophene-4-carbonitrile 13
Compound 13 was prepared from 9 0.30 g (1.31 mmol) in ethanol (400 mL) after irradiation
for 26 hours to yield 0.12 (37%) of brown powde; Mp = 170–176 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.64 (s, 1H, Harom), 8.47 (dd, J1 = 10.51 Hz, J2 = 2.63
Hz, 1H, Harom), 8.38 (d, J = 5.42 Hz, 1H, Harom), 8.27 (dd, J1 = 9.01 Hz, J2 = 5.93 Hz, 1H,
13
Harom), 8.16 (d, J = 5.42 Hz, 1H, Harom), 7.63 (td, J1 = 8.83 Hz, J2 = 2.64 Hz, 1H, Harom); C
NMR (151 MHz, DMSO) (δ/ppm): 163.4 (d, J = 247.8 Hz), 136.4 (d, J = 6,5 Hz), 132.6 (d, J
= 9,87 Hz), 132.2, 131.7 (d, J = 10,3 Hz), 128.9, 126.6, 123.8, 117.2, 116. (d, J = 24,8 Hz),
108.9 (d, J = 22,3 Hz), 103.0;
Found: C, 68.51; H, 2.75; N, 6.20. Calc. for C13H6FNS: C, 68.71; H, 2.66; N, 6.16%.
Phenanthrene-9-carbonitrile 14
Compound 14 was prepared from 7 0.30 g (1.50 mmol) in ethanol (400 mL) after irradiation
for 2.5 hours to yield 0.12 (41%) of orange powder; Mp = 110–116 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 9.02–8.96 (m, 1H, Harom), 8.94 (d, J = 8.4 Hz, 1H,
Harom), 8.70 (s, 1H, Harom), 8.23–8.13 (m, 2H, Harom), 7.95–7.85 (m, 3H, Harom), 7.80 (t, J = 7.5
Hz, 1H, Harom); 13C NMR (75 MHz, DMSO) (δ/ppm): 136.7, 131.7, 130.8, 130.2, 130.0,
129.9, 129.1, 129.0, 128.5, 128.4, 125.6, 124.3, 123.7, 118.1, 108.5; Found: C, 88.76; H,
4.35; N, 6.89. Calc. for C15H9N: C, 88.64; H, 4.46; N, 6.89%.
Phenanthrene-3,9-dicarbonitrile 15
Compound 15 was prepared from 8 0.50 g (1.90 mmol) in ethanol (400 mL) after irradiation
for 4 hours to yield 0.30 (69%) of yellow crystals; Mp = 279–284 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 9.52 (s, 1H, Harom), 9.13–9.06 (m, 1H, Harom), 8.77 (s,
1H, Harom), 8.31 (d, J = 8.3 Hz, 1H, Harom), 8.25–8.18 (m, 1H, Harom), 8.12 (d, J = 8.3 Hz, 1H,
13
Harom), 7.99–7.90 (m, 2H, Harom); C NMR (75 MHz, DMSO) (δ/ppm): 135.7, 132.6, 132.1,
131.4, 131.3, 130.8, 130.5, 130.0, 129.7, 129.4, 129.4, 128.8, 128.5, 128.0, 125.8, 124.8,
119.2, 117.5, 112.9, 111.7; Found: C, 83.89; H, 3.75; N, 12.36. Calc. for C16H8N2: C, 84.17;
H, 3.53; N, 12.27%.
Naphtho[2,1-b]thiophene-4-carbonitrile 16
Compound 16 was prepared from 10 0.50 g (2.37 mmol) in ethanol (800 mL) after irradiation
for 9 hours to yield 0.12 (23%) of brown powder; Mp = 126–132 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.64–8.62 (m, 2H, Harom), 8.39 (d, J = 5.4 Hz, 1H,
Harom), 8.18 (d, J = 8.9 Hz, 1H, Harom), 8.16 (d, J = 5.5 Hz, 1H, Harom), 7.87 (t, J = 7.1 Hz, 1H,
19

13
Harom), 7.73 (t, J = 7.6 Hz, 1H, Harom); C NMR (75 MHz, DMSO) (δ/ppm): 137.2, 135.9,
132.9, 130.6, 130.5, 129.9, 129.9, 129.4, 127.4, 124.7, 123.9, 117.8, 104.1; Found: C, 74.35;
H, 3.21; N, 6.60. Calc. for C13H7NS: C, 74.16; H, 3.37; N, 6.69%.
Naphtho[2,1-b]thiophene-4,8-dicarbonitrile 17
Compound 17 was prepared from 11 0.30 g (1.27 mmol) in ethanol (400 mL) after irradiation
for 2 hours to yield 0.26 (90%) of pink crystals; Mp = >300 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 9.30 (s, 1H, Harom), 8.72 (s, 1H, Harom), 8.51 (s, 1H,
Harom), 8.39–8.23 (m, 2H, Harom), 8.02 (d, J = 7.0 Hz, 1H, Harom);
13C NMR (150 MHz, DMSO) (δ/ppm): 136.8, 131.8, 131.2, 130.7, 130.5, 130.3, 129.2, 127.7,
123.8, 118.7, 116.8, 111.9, 106.6; Found: C, 71.65; H, 2.65; N, 12.01. Calc. for C14H6N2S: C,
71.78; H, 2.58; N, 11.96%.
4.1.4. General method for preparation of compounds 18–23
Compounds 18–23 were prepared using microwave irradiation, at optimized reaction time
with power 800 W and 40 bar pressure, from compound 12 or 15 in acetonitrile (10 mL) with
excess of added corresponding amine. After cooling, the resulting product was separated by
column chromatography on SiO2 using dichloromethane/methanol as eluent.
3-(N-isobutylamino)phenanthrene-9-carbonitrile 18
Compound 18 was prepared using above described method from 12 (0.10 g, 0.46 mmol) and
i-butylamine (1.50 mL, 15.80 mmol) after 29 hours of irradiation to yield 0.06 g (45%) of
brown oil.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.76 (d, J = 7.4 Hz, 1H, Harom), 8.34 (s, 1H, Harom),
8.03 (d, J = 7.2 Hz, 1H, Harom), 7.79 (d, J = 8.8 Hz, 1H, Harom), 7.76–7.69 (m, 2H, Harom), 7.66
(d, J = 1.5 Hz, 1H, Harom), 7.14 (dd, J1 = 8.8 Hz, J2 = 2.0 Hz, 1H, Harom), 6.72 (t, J = 5.5 Hz,
1H, NH), 3.12 (t, J = 6.6 Hz, 2H, CH2), 1.96 (p, J = 6.7 Hz, 1H, CH), 1.02 (d, J = 6.6 Hz, 6H,
13
CH3); C NMR (75 MHz, DMSO) (δ/ppm): 151.6, 136.5, 134.3, 131.3, 129.6, 128.9, 128.5,
127.5, 125.2, 124.3, 121.3, 119.3, 117.4, 101.0, 100.3, 50.7, 27.9, 20.9 (2C); Found: C, 83.01;
H, 6.74; N, 10.25. Calc. for C19H18N2: C, 83.18; H, 6.61; N, 10.21%.
3-(N-piperidinyl)phenanthrene-9-carbonitrile 19
Compound 19 was prepared using above described method from 12 (0.20 g, 0.92 mmol) and
piperidine (1.80 mL, 17.39 mmol) after 14 hours of irradiation to yield 0.07 g (8%) of brown
oil.
20

1H NMR (300 MHz, DMSO) (δ/ppm): 8.94 (d, J1 = 7.5 Hz, 1H, Harom), 8.43 (s, 1H, Harom),
8.10–8.04 (m, 2H, Harom), 7.92 (d, J = 9.0 Hz, 1H, Harom), 7.82–7.70 (m, 2H, Harom), 7.49 (dd,
J1 = 9.0 Hz, J2 = 2.2 Hz, 1H, Harom), 3.54 (s, 4H, CH2), 1.66 (s, 6H, CH2); 13C NMR (75 MHz,
DMSO) (δ/ppm): 152.7, 136.3, 133.6, 131.3, 129.3, 128.7, 127.8, 125.3, 124.6, 122.2, 119.0,
117.9, 105.2, 102.9, 48.9 (2C), 25.5 (2C), 24.5; Found: C, 83.65; H, 6.50; N, 9.85. Calc. for
C20H18N2: C, 83.88; H, 6.34; N, 9.78%.
3-(N-piperazinyl)phenanthrene-9-carbonitrile 20
Compound 20 was prepared using above described method from 12 (0.10 g, 0.46 mmol) and
piperazine (0.56 g, 7.28 mmol) after 14 hours of irradiation to yield 0.07 g (48%) of orange
oil. 1H NMR (600 MHz, DMSO) (δ/ppm): 8.97 (d, J = 8.0 Hz, 1H, Harom), 8.47 (s, 1H, Harom),
8.11 (s, 1H, Harom), 7.96 (d, J = 9.0 Hz, 1H, Harom), 7.83–7,74 (m, 2H, Harom), 7.55–7.49 (m,
1H, Harom), 3.54 (s, 4H, CH2), 3.04 (s, 4H, CH2); 13C NMR (75 MHz, DMSO) (δ/ppm): 152.5,
136.3, 133.5, 131.3, 129.4, 128.8, 127.9, 125.3, 124.7, 122.8, 118.9, 117.9, 105.7, 103.6, 47.5
(2C), 44.9 (2C); Found: C, 79.71; H, 5.90; N, 14.38. Calc. for C19H17N3: C, 79.41; H, 5.96; N,
14.62%.
8-(N-isobutylamino)naphtho[2,1-b]thiophene-4-carbonitrile 21
Compound 21 was prepared using above described method from 15 (0.10 g, 0.48 mmol) and
i-butylamine (1.18 mL, 11.95 mmol) after 37 hours of irradiation to yield 0.04 g (36%) of
light brown powder. Mp = 136–138 °C
1H NMR (300 MHz, DMSO) (δ/ppm): 8.23 (s, 1H, Harom), 8.18 (d, J = 5.4 Hz, 1H, Harom),
7.94 (d, J = 5.4 Hz, 1H, Harom), 7.79 (d, J = 8.9 Hz, 1H, Harom), 7.28 (d, J = 1.8 Hz, 1H, Harom),
7.11 (dd, J1 = 8.9, J2 = 2.1 Hz, 1H, Harom), 6.72 (t, J = 5.5 Hz, 1H, NH), 3.12–3.04 (t, J = 5.9
13
Hz, 2H, CH2), 1.99–1.90 (m, 1H, CH), 1.01 (d, J = 6.6 Hz, 6H, CH3); C NMR (75 MHz,
DMSO) (δ/ppm): 151.2, 135.1, 133.5, 132.7, 130.8, 126.9, 124.0, 122.0, 118.9, 117.9, 99.6,
96.9, 50.7, 27.9, 20.9 (2C); Found: C, 72.96; H, 5.60; N, 9.90. Calc. for C17H16N2S: C, 72.82;
H, 5.75; N, 9.99%.
8-(N-piperidinyl)naphtho[2,1-b]thiophene-4-carbonitrile 22
Compound 22 was prepared using above described method from 15 (0.10 g, 0.48 mmol) and
piperidine (0.94 mL, 9.56 mmol) after 31 hours of irradiation to yield 0.05 g (41%) of yellow
powder; Mp = 147–149 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.34 (d, J = 5,6 Hz, 1H, Harom), 8.32 (s, 1H, Harom),
7.98 (d, J = 5.4 Hz, 1H, Harom), 7.93 (d, J = 9.2 Hz, 1H, Harom), 7.72 (d, J = 2.2 Hz, 1H, Harom),
21

7.46 (dd, J1 = 9.2 Hz, J2 = 2.4 Hz, 1H, Harom), 3.51 (bs, 4H, CH2), 1.65 (bs, 6H, CH2);
13C NMR (75 MHz, DMSO) (δ/ppm): 152.3, 135.8, 132.8, 132.5, 130.9, 127.4, 124.3, 122.8,
118.7, 117.8, 105.1, 104.7, 98.7, 48.8 (2C), 25.5 (2C), 24.5; Found: C, 73.77; H, 5.38; N,
9.73. Calc. for C18H16N2S: C, 73.94; H, 5.52; N, 9.58%.
8-(N-piperazinyl)naphtho[2,1-b]thiophene-4-carbonitrile 23
Compound 23 was prepared using above described method from 15 (0.10 g, 0.48 mmol) and
piperazine (0.58 mg, 6.69 mmol) after 30 hours of irradiation to yield 0.06 g (23%) of light
brown powder; Mp = 147–149 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.41 (s, 1H, Harom), 8.39 (d, J = 5.5 Hz, 1H, Harom),
8.05 (d, J =4.0 Hz, 1H, Harom), 8.03 (d, J = 7.8 Hz, 1H, Harom), 7.86 (d, J = 2.1 Hz, 1H, Harom),
7.53 (dd, J1 = 9.1 Hz, J2 = 2.4 Hz, 1H, Harom), 3.79–3.61 (m, 4H, CH2), 3.33–3.27 (m, 4H,
13
CH2); C NMR (75 MHz, DMSO) (δ/ppm): 151.1, 136.6, 136.0, 132.5, 132.4, 131.2, 128.0,
124.2, 123.8, 118.5, 118.0, 106.3, 100.0, 45.3 (2C), 43.2 (2C); Found: C, 69.79; H, 5.01; N,
14.50. Calc. for C17H15N3S: C, 69.60; H, 5.15; N, 14.32%.
4.1.5. General method for the synthesis of compounds 24–26
2 N aqueous solution of sodium hydroxide and compounds 13, 16 and 17 were refluxed for 24
hours. Cooled reaction mixture was poured into ice, and resulting product was filtered off.
Phenanthrene-9-carboxylic acid 24
Compound 24 was prepared using above described method, from phenanthrene-9-carbonitrile
13 (0.20 g, 0.98 mmol) and 2 N aqueaus solution of sodium hydroxide (19.0 mL) to yield 0.15
g (71%) of grey powder; Mp 208–216 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.87 (t, J = 8.8 Hz, 2H, Harom), 8.33 (d, J = 7.7 Hz, 1H,
Harom), 8.12 (s, 1H, COOH), 8.06 (d, J = 8.0 Hz, 1H, Harom), 7.99 (s, 1H, Harom), 7.80–7.63 (m,
13
4H, Harom); C NMR (75 MHz, DMSO) (δ/ppm): 171.1, 134.0, 130.6, 130.6, 130.4, 129.5,
128.8, 128.3, 127.7, 127.5, 127.5, 126.8, 126.3, 123.6, 123.3; Found: C, 81.33; H, 4.67. Calc.
for C15H10O2: C, 81.07; H, 4.54%.
Naphtho[2,1-b]thiophene-4-carboxylic acid 25
Compound 25 was prepared using above described method, from naphtho[2,1-b]thiophene-4-
carbonitrile 16 (0.14 g, 0.65 mmol) and 2 N aqueaus solution of sodium hydroxide (13.0 mL)
to yield 0.13 g (91%) of grey powder; Mp 283–286 °C.
22

1H NMR (300 MHz, DMSO) (δ/ppm): 8.56 (d, J = 8.2 Hz, 1H, Harom), 8.51 (s, 1H, Harom),
8.35 (s, 1H, COOH), 8.23 (d, J = 5.6 Hz, 1H, Harom), 8.06 (d, J = 8.1 Hz, 1H, Harom), 7.96 (d, J
= 5.5 Hz, 1H, Harom), 7.75 (t, J = 7.6 Hz, 1H, Harom), 7.64 (t, J = 7.0 Hz, 1H, Harom); 13C NMR
(75 MHz, DMSO) (δ/ppm): not enouht soluble; Found: C, 68.70; H, 3.26. Calc. for C13H8O2S:
C, 68.40; H, 3.53%.
Naphtho[2,1-b]thiophene-4,8-dicarboxylic acid 26
Compound 26 was prepared using above described method, from naphtho[2,1-b]thiophene-
4,8-dicarbonitrile 17 (0.30 g, 0.613 mmol) and 2 N aqueaus solution of sodium hydroxide
(20.0 mL) to yield 0.30 g (86%) of light yellow powder; Mp >300 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 13.52 (s, 2H, COOH), 9.13 (s, 1H, Harom), 8.69 (s, 1H,
Harom), 8.39 (d, J = 5.6 Hz, 1H, Harom), 8.36 (d, J = 8.7 Hz, 1H, Harom), 8.14–8.08 (m, 2H,
Harom); 13C NMR (150 MHz, DMSO) (δ/ppm): 167.3, 166.9, 137.7, 135.3, 132.3, 131.1,
130.7, 130.4, 129.8, 128.0, 125.7, 125.4, 124.9, 121.8; Found: C, 61.95; H, 2.72. Calc. for
C14H8O4S: C, 61.76; H, 2.96%.
4.1.6. General method for the synthesis of compounds 27–29
A mixture of corresponding carboxylic acids 26–26 and thionyl chloride in absolute toluene
was refluxed for 19 hours. Toluene and excess of thionyl chloride was removed under
reduced pressure. The crude product was washed 3 times with absolute toluene to obtained
powdered product.
Phenanthrene-9-carbonyl chloride 27
Compound 27 was prepared using above described method, from phenanthrene-9-carboxylic
acid 24 (0.11 g, 0.51 mmol), absolute toluene (10 mL) and 0.37 mL thionyl chloride to yield
0.11 g (91%) of yellow powder.
1H NMR (600 MHz, DMSO) (δ/ppm): 9.00–8.99 (m, 1H, Harom), 8.95 (d, J = 8.4 Hz, 1H,
Harom), 8.89 (d, J = 8.0 Hz, 1H, Harom), 8.72 (s, 1H, Harom), 8.20–8.19 (m, 1H, Harom), 8.17 (d, J
= 7.9 Hz, 1H, Harom), 7.92 (t, J = 7.6 Hz, 1H, Harom), 7.80 (t, J = 7.4 Hz, 1H, Harom), 7.76–7.72
(m, 1H, Harom); 13C NMR (75 MHz, DMSO) (δ/ppm): not enouht soluble; Found: C, 74.70; H,
3.98. Calc. for C15H9ClO: C, 74.85; H, 3.77%.
Naphtho[2,1-b]thiophene-4-carbonyl chloride 28
Compound 28 was prepared using above described method, from naphtho[2,1-b]thiophene-4-
carboxylic acid 25 (0.10 g, 0.44 mmol), absolute toluene (10 mL) and 0.37 mL thionyl
23

chloride to yield 0.10 g (96%) of yellow powder.
1H NMR (600 MHz, DMSO) (δ/ppm): 8.63 (s, 1H, Harom), 8.58 (d, J = 8.3 Hz, 1H, Harom),
8.28 (d, J = 5.5 Hz, 1H, Harom), 8.23 (d, J = 8.0 Hz, 1H, Harom), 8.01 (d, J = 5.5 Hz, 1H, Harom),
7.80–7.76 (m, 1H, Harom), 7.66–7.62 (m, 1H, Harom); 13C NMR (75 MHz, DMSO) (δ/ppm):
167.6, 137.5, 131.1, 130.5 (2C), 129.6, 129.2, 126.6, 124.4, 123.5, 122.3, 120.9; Found: C,
63.50; H, 2.75; N, 6.35. Calc. for C13H7ClOS: C, 63.29; H, 2.86%.
Naphtho[2,1-b]thiophene-4,8-dicarbonyl dichloride 29
Compound 29 was prepared using above described method, from naphtho[2,1-b]thiophene-
4,8-dicarboxylic acid 26 (0.50 g, 1.83 mmol), absolute toluene (10 mL) and 1.32 mL thionyl
chloride to yield 0.49 g (86%) of yellow powder.
1H NMR (600 MHz, DMSO) (δ/ppm): 9.13 (s, 1H, Harom), 8.69 (s, 1H, Harom), 8.39 (d, J = 5.5
Hz, 1H, Harom), 8.36 (d, J = 8.5 Hz, 1H, Harom), 8.13–8.08 (m, 2H, Harom); 13C NMR (75 MHz,
DMSO) (δ/ppm): 167.2, 166.8, 137.7, 135.3, 132.3, 131.1, 130.7, 130.4, 129.8, 128.00, 125.7,
125.4, 124.9, 121.7; Found: C, 54.55; H, 2.06; N, 6.35. Calc. for C14H6Cl2O2S: C, 54.39; H,
1.96%.
4.1.7. General method for the synthesis of compounds 30–34
A mixture of carbonyl chlorides 27–29 and excess of corresponding amine in dry
dichloromethane was stirred at room temperature for 2 hours. The mixture was washed with
10 mL of 20% Na2CO3 and 10 mL water. After drying over MgSO4, the organic layer was
concentrated at reduced pressure.
N-isobutylphenanthrene-9-carboxamide 30
Compound 30 was prepared using above described method, from phenanthrene-9-carbonyl
chloride 27 (0.37 g, 1.52 mmol), dry dichloromethane (20 mL) and 0.91 mL (9.15 mmol) i-
butylamine to obtain 0.04 g (10%) of white powder. Mp = 177–179 °C
1H NMR (300 MHz, DMSO) (δ/ppm): 8.88 (t, J = 9.2 Hz, 2H, Harom), 8.68 (t, J = 5.7 Hz, 1H,
NH), 8.20 (d, J = 7.8 Hz, 1H, Harom), 8.08 (d, J = 7.6 Hz, 1H, Harom), 7.92 (s, 1H, Harom), 7.79–
7.65 (m, 4H, Harom), 3.20 (t, J = 6.4 Hz, 2H, CH2), 1.96–1.88 (m, 1H, CH), 0.98 (d, J = 6.7
Hz, 6H, CH3); 13C NMR (75 MHz, DMSO) (δ/ppm): 169.1, 134.6, 130.7, 130.5, 130.4, 129.5,
128.9, 128.3, 127.7, 127.5, 127.5, 126.6, 126.1, 123.7, 123.3, 47.0, 28.7, 20.7 (2C); Found: C,
82.36; H, 6.98; N, 4.87. Calc. for C19H19NO: C, 82.28; H, 6.90; N, 5.05%.
24

N-isobutylnaphtho[2,1-b]thiophene-4-carboxamide 31
Compound 31 was prepared using above described method, from naphtho[2,1-b]thiophene-4-
carbonyl chloride 28 (0.10 g, 0.41 mmol), dry dichloromethane (10 mL) and 0.24 mL (2.43
mmol) i-butylamine to obtain 0.06 g (49%) of white powder; Mp = 148–152 °C.
1H NMR (300 MHz, DMSO) (δ/ppm): 8.88 (t, J = 5.7 Hz, 1H, NH), 8.56 (d, J = 8.2 Hz, 1H,
Harom), 8.47 (s, 1H, Harom), 8.24 (d, J = 5.6 Hz, 1H, Harom), 8.10 (d, J = 7.8 Hz, 1H, Harom),
7.97 (d, J = 5.5 Hz, 1H, Harom), 7.75 (t, J = 6.9 Hz, 1H, Harom ), 7.65 (t, J = 8.0 Hz, 1H, Harom ),
3.20 (t, J = 6.4 Hz, 2H, CH2), 2.00–1.89 (m, 1H, CH), 0.96 (d, J = 6.7 Hz, 6H, CH3);
13C NMR (75 MHz, DMSO) (δ/ppm): 166.5, 137.3, 135.1, 130.8, 130.5, 130.3, 129.7, 128.6,
127.1, 126.5, 124.4, 124.3, 121.8, 47.3, 28.6, 20.7 (2C); Found: C, 72.30; H, 6.35; N, 4.70.
Calc. for C17H17NOS: C, 72.05; H, 6.05; N, 4.94%.
N-(3-(N',N'-dimethylamino)propyl)naphtho[2,1-b]thiophene-4-carboxamide 32
Compound 32 was prepared using above described method, from naphtho[2,1-b]thiophene-4-
carbonyl chloride 28 (0.14 g, 0.55 mmol), dry dichloromethane (12 mL) and 0.41 mL (3.28
mmol) N,N-dimethylaminopropyl-1-amine to obtain 0.07 g (41%) of yellow oil.
1H NMR (600 MHz, DMSO) (δ/ppm): 8.95 (t, J = 5.4 Hz, 1H, NH), 8.57 (d, J = 8.1 Hz, 1H,
Harom), 8.44 (s, 1H, Harom), 8.24 (d, J = 5.5 Hz, 1H, Harom), 8.09 (d, J = 8.0 Hz, 1H, Harom),
7.98 (d, J = 5.5 Hz, 1H, Harom), 7.76–7.72 (m, 1H, Harom), 7.66–7.63 (m, 1H, Harom), 3.41 (q, J
= 6.6 Hz, 2H, CH2), 2.43 (t, J = 7.0 Hz, 2H, CH2), 2.25 (s, 6H, CH3), 1.80–1.75 (m, 2H, CH2);
13C NMR (150 MHz, DMSO) (δ/ppm): 165.9, 136.8, 134.5, 130.4, 130.0, 129.8, 129.2, 128.2,
126.5, 126.0, 123.8, 123.7, 121.3, 56.6, 44.8 (2C), 37.7, 26.8; Found: C, 69.01; H, 6.56; N,
8.25. Calc. for C18H20N2OS: C, 69.20; H, 6.45; N, 8.97%.
N4,N8-diisobutylnaphtho[2,1-b]thiophene-4,8-dicarboxamide 33
Compound 33 was prepared using above described method, from naphtho[2,1-b]thiophene-
4,8-dicarbonyl dichloride 29 (0.23 g, 0.72 mmol), dry dichloromethane (20 mL) and 0.43 mL
(4.31 mmol) i-butylamine to obtain 0.02 g (4%) of white powder; Mp = 187–190 °C.
1H NMR (600 MHz, DMSO) (δ/ppm): 9.03 (s, 1H, Harom), 8.95 (t, J = 5.6 Hz, 1H, NH), 8.78
(t, J = 5.6 Hz, 1H, NH), 8.49 (s, 1H), 8.29 (d, J = 5.6 Hz, 1H, Harom), 8.16 (d, J = 8.5 Hz, 1H,
Harom), 8.08–8.04 (m, 2H, Harom), 3.21–3.18 (m, 4H, CH2), 1.94–1.90 (m, 2H, CH), 0.96 (d, J
13
= 6.0 Hz, 6H, CH3), 0.95 (d, J = 6.0 Hz, 6H, CH3); C NMR (150 MHz, DMSO) (δ/ppm):
166.0, 165.9, 137.4, 135.0, 133.7, 131.3, 131.0, 129.3, 128.9, 127.9, 124.5, 123.2, 122.7,
121.4, 46.9, 46.8, 28.2, 28.1, 20.3, 20.3; Found: C, 69.19; H, 6.70; N, 7.15. Calc. for
25

C22H26N2O2S: C, 69.08; H, 6.85; N, 7.32%.
N4,N8-bis(3-(N',N'-dimethylamino)propyl)naphtho[2,1-b]thiophene-4,8-dicarboxamide 34
Compound 34 was prepared using above described method, from naphtho[2,1-b]thiophene-
4,8-dicarbonyl dichloride 29 (0.23 g, 0.72 mmol), dry dichloromethane (20 mL) and 0.54 mL
(4.31 mmol) 3-N,N-dimethylaminopropyl-1-amine to obtain 0.11 g (23%) of white powder;
Mp = 170–172 °C.
1H NMR (600 MHz, DMSO) (δ/ppm): 9.03 (s, 1H, Harom), 9.00 (t, J = 5.4 Hz, 1H, NH), 8.82
(t, J = 5.4 Hz, 1H, NH), 8.45 (s, 1H, Harom), 8.28 (d, J = 5.5 Hz, 1H, Harom), 8.16 (d, J = 8.5
Hz, 1H, Harom), 8.7–8.05 (m, 2H, Harom), 3.42–3.34 (m, 4H, CH2), 2.32 (q, J = 7.1 Hz, 4H,
CH2), 2.17 (d, J = 3.3 Hz, 12H, CH3), 1.76–1.71 (m, 4H, CH2); 13C NMR (75 MHz, DMSO)
(δ/ppm): 166.4, 166.3, 137.9, 135.4, 134.1, 131.8, 131.6, 129.9, 129.5, 128.3, 124.9, 123.7,
123.2, 121.8, 57.4, 45.6 (2C), 38.4, 27.6, 27.5; Found: C, 65.21; H, 7.50; N, 12.51. Calc. for
C24H32N4O2S: C, 65.42; H, 7.32; N, 12.72%.
4.2. Antiproliferative activity in vitro
4.2.1. Cell culturing
The newly prepared compounds were tested on five human cell lines HeLa (cervical
carcinoma), SW620 (colorectal adenocarcinoma, metastatic), HepG2 (hepatocellular
carcinoma), CFPAC-1 (ductal pancreatic adenocarcinoma) and A549 (lung adenocarcinoma)
cells cultured as monolayers. The cell lines were maintained in Dulbecco's modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100
U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37
°C.
4.2.2. Proliferation assays
The experiments were implemented according to the standard previously published
experimental procedure. Commercial drug 5-fluorouracile was chosen as a control substance
for comparison of tested effects. The calculation of IC50 and LC50 values for each compound
was performed using mathematical analysis described earlier.
26

4.2.3. Western blot analysis
The HeLa and HepG2 cells were seeded in six well plate, 3x105 cells/well, and treated
with compounds 8 and 11 at concentration of 2xIC50 value for 48h and 72h respectively.
Protein lysates were prepared using a buffer containing 50 mM Tris HCl (pH 8), 150 mM
NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease and phosphatase
inhibitor cocktail (Roche, Switzerland). A total of 50 µg of proteins were resolved on 12%
SDS polyacrylamide gels using the Mini-protean cell (Bio-Rad, USA).
The membranes were incubated with primary antibodies raised against hydroxy-HIF-1α
(Pro564) (HIF-1α ,1:1000, rabbit mAb, Cell Signaling Technology, NL) and phospho-
MEK1/2 (Ser271/221) (MEK1/2, 1:1000, rabbit mAb, Cell Signaling Technology, NL) at 4°C
overnight. Secondary antibody linked to anti-mouse (1:1000, Dako, USA) was used.
The signal was visualized by Western Lightening Chemiluminescence Reagent Plus Kit
(Perkin Elmer, USA) on the ImageQuant LAS500 (GE Healthcare, USA). The signal was
visualized by Western Lightening Chemiluminescence Reagent Plus Kit (Perkin Elmer, USA)
on the ImageQuant LAS500 (GE Healthcare, USA) and α- tubulin (1:1000, mouse mAb,
Sigma, USA) was used as a loading control. The signal intensities of particular bands were
normalized with the intensity of the loading control and compared in Quantity One software
(Bio-Rad, USA). The values are expressed as the average ± SEM. Differences in protein
relative expression status obtained by Western blot analysis were analyzed by two-tailed
paired t-test (p<0.05 ) in Statistica software package (v.12.0).
4.3. Mode of action analysis
4.3.1. Dataset and pre-processing
A target/functional analysis was performed on the phenanthrene and naphtho[2,1-
b]thiophene derivatives. Before performing our target prediction analysis, the SMILES of
these
derivatives
were
standardized
using
ChemAxon
(v.16.5.2.0,
2016,
http://www.chemaxon.com) with options “Remove Fragment” (keep largest), “Neutralize”,
“RemoveExplicitH”, “Clean2D”, “Mesomerize”, and “tautomerize” for structure
normalization.
4.3.2. Target prediction analysis
To predict the potential biological targets of our derivatives, PIDGIN target prediction
algorithm was used. It is a Bernoulli Naïve Bayes (BNB) based method that returns, for each
27

input compound and for each of 1,651 potential human protein targets, the probability that the
compound will have an activity on the given target (probability of activity, Pa). These
potential human protein targets used for PIDGIN model training were extracted from
ChEMBL database and because of its BNB training process PIDGIN only considered the
1651 proteins having 10 or more compounds that target them. PIDGIN was applied on of all
23 derivatives against all 1,651 potential targets, resulting in 23x1,651 = 37,973 compound-
target interactions. To only consider the most probable compound-target interactions, a
probability of activity (Pa) threshold of 0.9 (90% confidence) was chosen for the study. By
using this threshold, 277 compound-target interactions between the 23 derivatives and 112
unique targets were predicted.
4.3.3. Functional analysis of the predicted targets
Further, to understand the biological role of predicted targets of our phenanthrene and
naphtho[2,1-b]thiophene derivatives, ClueGO tool was used (to perform functional
enrichment analysis against the background set of 1,651 human targets from PIDGIN
model).37 ClueGO is a plug-in of Cytoscape, which extracts representative functional
information for a list of genes/proteins based on the latest publically available data from
multiple annotation and ontology resources.38 Here, ClueGO was used for Gene ontology
(GO) Biological Process (BP), Molecular Function (MF), Cellular Component (CC) and
pathway enrichment analysis. It was considered that the predicted targets are enriched in a
given annotation term if the corresponding enrichment p-value (after Bonferroni correction
for multiple hypothesis testing) is ≤ 0.05.
Acknowledgements
We greatly appreciate the financial support of the Croatian Science Foundation under
the projects 4379 entitled Exploring the antioxidative potential of benzazole scaffold in the
design of novel antitumor agents. ects We would like to thank Croatian Government and the
European Union (European Regional Development Fund—the Competitiveness and Cohesion
Operational Programme - KK.01.1.1.01) for funding this research through project
Bioprospecting of the Adriatic Sea (KK.01.1.1.01.0002) granted to The Scientific Centre of
Excellence for Marine Bioprospecting-BioProCro.
28