Novel 2′-alkoxymethyl substituted klavuzon derivatives as inhibitors of Topo I and CRM1

Article abstract of DOI:10.1016/j.bioorg.2020.104162

In this work, 2′-alkoxymethyl substituted klavuzon derivatives were prepared starting from 2-methyl-1-naphthoic acid in eight steps. Anticancer potencies of the synthesized compounds were evaluated by performing MTT cell viability test over cancerous and

Full text of DOI:10.1016/j.bioorg.2020.104162

BioorganicChemistry103(2020)104162
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel 2′-alkoxymethyl substituted klavuzon derivatives as inhibitors of
Topo I and CRM1
Hakkı Çetinkayaa, Mehmet S. Yıldızb, Meltem Kutluerb, Aylin Alkanc, Hasan Ozan Otaşc,
⁎
Ali Çağıra,
a İzmir Institute of Technology, Faculty of Science, Department of Chemistry, Urla 35430, İzmir, Turkey
b İzmir Institute of Technology, Biotechnology and Bioengineering Graduate Program, Urla 35430, İzmir, Turkey
c İzmir Institute of Technology, Faculty of Science, Department of Molecular Biology and Genetics, Urla 35430, İzmir, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this work, 2′-alkoxymethyl substituted klavuzon derivatives were prepared starting from 2-methyl-1-naph-
thoic acid in eight steps. Anticancer potencies of the synthesized compounds were evaluated by performing MTT
cell viability test over cancerous and healthy pancreatic cell lines, along with CRM1 inhibitory properties in
HeLa cells by immunostaining and Topo I inhibition properties by supercoiled DNA relaxation assay. Their
cytotoxic activities were also presented in hepatocellular carcinoma cells (HuH-7) derived 3D spheroids. Among
the tested klavuzon derivatives, isobutoxymethyl substituted klavuzon showed the highest selectivity of cyto-
toxic activity against pancreatic cancer cell line. They showed potent Topo I inhibition while their CRM1 in-
hibitory properties somehow diminished compared to 4′-alkylsubstituted klavuzons. The most cytotoxic 2′-
methoxymethyl derivative inhibited the growth of the spheroids derived from HuH-7 cell lines and PI staining
exhibited time and concentration dependent cell death in 3D spheroids.
Klavuzon
CRM1
Topoisomerase I
Pancreatic cancer
Hepatocellular carcinoma
3D spheroid
Anticancer agent
1. Introduction
adenocarcinoma) cell lines while it is less cytotoxic in ST3 (mouse skin
fibroblast) and HF (human fibroblast) healthy cell lines. [4] Later Al-
Chemotherapy has a central role in the treatment of cancer. Despite
the recent advances in targeted therapies, discovery of novel anticancer
agents that selectively inhibit the growth of cancer cells is one of the
strategy to develop novel anticancer drug candidates. To this end, we
present the development of novel klavuzon derivatives that possess
selective cytotoxic activity in pancreatic cancer cell line (MIA PaCa-2)
compared to healthy one (HPDEC).
Qubaisi and coworkers presented its selective cytotoxic activity over
Hepatoblastoma HepG2 cells in 2011 [5].
In the recent decades, many studies were conducted to assess an-
ticancer properties of goniothalamin. Cytotoxic activitiy of (R)- and (S)-
goniothalamin derivatives have been reported in these studies [5–12].
Similar therapeutical benefits of (R)-goniothalamin were also shown in
in vivo studies. When healthy rats are treated with (R)-goniothalamin
(300 μg, 14 days), it causes no changes in body weight, hematological
and biochemical parameters [13]. When anticancer agent 1 was applied
to Sprague-Dawley rats having cancer developed by 7,12-di-
methylbenzanthracene (DMBA) it stopped the cancer growth and
caused the accumulation of p53 protein [14]. In another in vivo study,
racemic and enantiomerically pure forms of goniothalamin stops the
growth of Ehrlich solid tumor model in vivo [10]. Among these studies
Chromosome Maintenance 1 (CRM1) and Topoisomerase I (Topo I)
proteins have been found as the intramolecular target for GTN so far
[9,15].
Pancreatic cancer and hepatocellular carcinoma are two of the most
deadly diseases and there is still need to develop novel anticancer
agents for the treatment or stop the progress of these illnesses. As it is a
known fact that molecules isolated from the natural sources inspire the
scientist to develop novel drug molecules. In recent years, we especially
interested in to develop the conformationally constrained analogues of
goniothalamin (GTN) [1] as a member of Styryllactone family [2].
Goniothalamin (GTN) is a natural product and its R-enantiomer
((R)-1) was first extracted from the bark of Cryptocarya caloneura [3]
and its selective cytotoxic activity over cancer cell lines was first re-
ported in 2005 by Chatchai and coworkers. They showed that (R)-go-
niothalamin is more cytotoxic in COR-L23 (large cell lung carcinoma),
LS174T (human colon adenocarcinoma), and MCF-7 (human breast
Klavuzons have similar pharmacophore unit with GTN. Syntheses
and anticancer properties of compounds 4′-methylklavuzon and 2′-
methylklavuzon ((R)-2 and (R)-3) were first described by our group in
⁎ Corresponding author.
E-mail address: alicagir@iyte.edu.tr (A. Çağır).
https://doi.org/10.1016/j.bioorg.2020.104162
Received 30 December 2019; Received in revised form 4 July 2020; Accepted 2 August 2020
Availableonline25August2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

H. Çetinkaya, et al.
BioorganicChemistry103(2020)104162
for pancreatic cancerous and healthy cell lines (MIA PaCa-2 and
HPDEC) [16].
Similar to GTN, klavuzon derivatives also inhibits CRM1 and Topo I
activities [15,16]. Both proteins are valuable intracellular targets to
develop novel anticancer agents [17–19]. In the current study, a list of
racemic 2′-alkoxymethyl substituted klavuzon derivatives have been
synthesized and their anticancer activity potentials are presented.
Among the tested compounds 2′-isobutoxymethyl substituted klavuzon
has been found as the most selective klavuzon derivative in the litera-
ture.
2. Results and discussion
The route for these racemic syntheses of novel 2′-alkoxymethyl
substituted klavuzon derivatives (16a-i) have been summarized in
Tables 1 and 2. Transformation of 2-methyl-1-naphthoic acid (7) to
ethyl 1-naphthoate ester (8) was the starting point for these syntheses.
This conversion has been performed quantitatively by the treatment of
2-methyl-1-naphthoic acid by ethyl iodide in the presence of potassium
carbonate. This ester (8) was also prepared by refluxing the compound
7 in ethanol in the presence of catalytic amount of sulfuric acid but this
procedure gave only 48% yield.
Fig. 1. Structures of (R)-goniothalamin ((R)-1) and klavuzon derivatives ((R)-2,
(R)-3, and 4–6).
2008 and it was found that they are more cytotoxic in cancer cells
compared to GTN. Especially 4′-methyl substituted klavuzon had re-
markable cytotoxic activity (Fig. 1) [1]. Later structure activity re-
lationship studies indicated that exchange of methyl groups (4 and 5)
with methoxide decreases the activity of the klavuzons, while com-
pound 6 showed a remarkable cytotoxic activity in pancreatic cancer
cell line [15,16]. Structure activity relationship of a list of 4′-alkyl
substituted klavuzon derivatives revealed that the size of the sub-
stituent is inversely related with potency of the klavuzon derivatives.
On the other hand, selectivity of these derivatives was not so promising
In the next step, various reaction conditions have been tried to
perform the benzylic bromination of ethyl 2-methyl-1-naphtoate (8).
Treatment of compound 8 with benzoyl peroxide and bromotri-
chloromethane in refluxing benzene [20] or treatment with NBS and
benzoylperoxide in heated acetonitrile had failed [21]. Afterwards, a
method developed by Ishii and coworkers was used for benzylic bro-
mination reaction by using NaBrO3/NaHSO3 in EtOAc-water mixture
[22]. Although this procedure is quite sensitive to both temperature and
volume of the ethyl acetate used in the reaction, the yield of the
Table 1
Conversion of 2-methyl-1-naphthoic acid (7) into 2-alkoxymethyl-1-naphthaldehyde derivatives (13a-i).
Entry
R
Yield %
10a-i
11a-i
12a-i
13a-i
x
a
b
c
d
e
f
66a
-
83
85
77
78
74
76
86
23c
x
60a
-
x
-
52a
85
x
x
-
-
12b
81
x
44a
-
x
x
x
-
-
-
x
g
42a
-
97
84
h
i
80a, 28a
-x, 30a
90
65
86
68
x
14a
-
a,b,c
x
Yields for the last 2, 3 and 4 steps, respectively.
Compounds couldn’t be purified.
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H. Çetinkaya, et al.
BioorganicChemistry103(2020)104162
Table 2
Preparation of 2′-alkoxymethyl substituted klavuzon derivatives (16a-i) starting from 2-alkoxymethyl-1-naphthaldehydes (13a-i).
Entry
R
Yield %
15a-i
14a-i
16a-i
a
b
c
d
e
f
94
94
94
98
77
96
88
97
89
91
76
79
78
85
85
67
73
68
g
89
75
88
h
i
72
98
95
44
74
99
compound 9 was almost quantitative under optimized conditions. Re-
peatability of the quantitative yield was not so high and in some trials
the reaction gave an inseparable mixtures of compounds 8 and 9. Then,
this mixture of product (9) and starting material (8) was used in the
next step without purification (Table 1).
Formation of 6-(Boc-amino)-1-hexanoxy substituted ethyl ester
(10i) could not be achieved by this procedure, however it was suc-
cessfully produced by addition of alcohol in the presence of silver tri-
flate and DIPEA. Reduction of the esters (10 and 11) with lithium
aluminium hydride followed by hydrolysis gave the corresponding al-
cohols 12a-i. In the next step, oxidation of these primary alcohols (12a-
i) with PCC yielded 2-alkoxymethyl substituted 1-naphthaldehyde de-
rivatives (Table 1).
In the next step, various primary and secondary alcohols treated with
potassium tert-butoxide were reacted with ethyl 2-bromomethyl-1-
naphthoate (9) to yield corresponding ethyl 2-alkoxymethyl-1-naphtoate
derivatives (10a-h) besides their transesterification products (11a-i).
Because of their similar polarities, purification of the ethyl esters from
transesterification products was not always successful. Yields of the
purified esters were summarized in Table 1. Methoxy, ethoxy, 1-pentoxy,
isobutoxy and benzyloxy substituted ethyl esters (10a, 10b, 10e, and
10 g-h) were isolated successfully. Among the transesterification pro-
ducts, 1-propoxy and benzyloxy substituted ones (11c and 11h) were
isolated. On the other hand, the products for 1-butoxy and isopropoxy
substituted esters (10d, 10f, 11d and 11f) could not be purified. Hence,
mixtures of esters were used in the next step for these cases.
At this point, addition of allymagnesium bromide to 2-alkoxymethyl
substituted 1-naphthaldehydes (13a-i) yielded homoallylic alcohols
(14a-i) in 72–98% yields. Surprisingly, 1H NMR spectra of 1-butox-
ymethyl substituted homoallylic alcohol (14d) in CDCl3 implies the
formation of atropisomers. The absence of the atropisomerism in 1H
NMR spectra of compound 14d, acquired in CD3OD, strongly suggest
that atropisomers should be formed through an intramolecular hy-
drogen bonding between the hydroxyl group alcohol and oxygen of
ether. After conversion of homoallylic alcohols (14a-i) to acrylate esters
(15a-i) by addition of acryloyl chloride, ring-closing metathesis gave
the novel klavuzon derivatives in good yields (Table 2).
As it was mentioned above, CRM1 and Topo I proteins are two re-
ported intracellular targets for both GTN and klavuzon derivatives
[9,16,23]. Before testing their effect over the activity of these proteins,
their antiproliferative activities on cancerous (MIA PaCa-2) and healthy
(HPDEC) pancreatic cell lines were determined by MTT cell prolifera-
tion assay (Table 3). Because, camptothecin (CPT) is one of the well-
known Topo I poison, and goniothalamin (GTN) inhibits the same in-
tramolecular targets with klavuzons both compounds were used as
positive control. Synthesis and biological activities of compound 3 has
been reported by our research group in a previous study [1] and it was
also used as a third control in order to draw a structure activity re-
lationship. As it can be seen from the table, CPT was the most cytotoxic
compounds on both cell lines without any selectivity. Contrarily, GTN
was found to be the least cytotoxic compound with the highest se-
lectivity index.
Table 3
Antiproliferative activity of 2′-alkoxymethyl substituted klavuzon derivatives
(16a-i) over cancerous (MIA PaCa-2) and immortalized healthy (HPDEC)
pancreatic cell lines after 48 h. of incubation.
Comp.
IC50 (in μM)
SI
MIA PaCa-2
HPDEC
0.21
CPT
GTN
3
0.18
8.53
1.86
0.61
0.72
2.46
3.63
3.25
2.87
3.02
3.21
0.70
0.01
0.88
0.17
0.09
0.11
0.42
0.14
0.39
0.51
0.02
0.19
0.05
0.01
0.68
0.05
0.10
0.05
0.03
0.23
0.16
0.08
0.22
0.02
0.02
1.17
2.08
0.28
0.89
0.97
0.80
0.88
1.11
0.43
1.83
0.50
0.60
17.75
0.53
0.54
0.70
1.98
3.21
3.61
1.23
5.53
1.60
0.42
16a
16b
16c
16d
16e
16f
16g
16h
16i
In terms of cytotoxic potencies, all klavuzon derivatives were more
cytotoxic in both cell lines compared to GTN. It seems that the activity
of the klavuzon derivatives inversely related with the size of the sub-
stituent at 2′- position of klavuzon with one exception compound 16i.
Selectivity of the klavuzon derivatives are sensitive to the size of the
substituent. A number of the klavuzon derivatives (3, 16f and 16 h-i)
SI: Selectivity Index = IC50 value calculated for HPDEC / IC50 value calculated
for MIA PaCa-2, where IC50 is the concentration that inhibit the cell growth by
50%. CPT: camptothecin.
3

H. Çetinkaya, et al.
BioorganicChemistry103(2020)104162
Fig. 2. Concentration dependent CRM1 inhibition in HeLa cells by using compounds 3, 6, 16a and 16g after 6 h. of incubation. Green channel shows RIOK2 protein which
is a Serine/threonine-protein kinase. Blue channel shows nucleus staining with DAPI. RIOK2 is observed as localized in nucleus with increasing concentrations. (Scale
bar = 20 µm, control group only 1% DMSO). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
showed selective toxicity over healthy (HPDEC) cell line. Another group
of klavuzon derivatives (16a-e) had comparable selectivity profile with
that of CPT. Interestingly, 2′-isobutoxymethyl substituted klavuzon
(16g) was significantly selective on pancreatic cancer (MIA PaCa-2) cell
line. Although the selective cytotoxic activity of GTN and compound
16g was quite similar, klavuzon derivative (16g) is 2.8 times more
cytotoxic in cancer (MIA PaCa-2) cell line and 3.2 fold more cytotoxic
in healthy (HPDEC) cell line (Table 3).
Fig. 3. Panel for time and concentration dependent relaxation of pUC19 (SC DNA: Supercoiled DNA and R is the relaxed form) by Topo I in the presence of klavuzon
derivatives. All reactions were carried out for 30 min. CPT (camptothecin) was used as positive control. A) Time dependent inhibition by 2′-methylklavuzon (3). B)
Concentration dependent inhibition of Topo I, preincubated for 10 min. with 2′-methoxymethylklavuzon (16a) and 2′-isobutoxymethylklavuzon (16 g). C)
Concentration dependent inhibition of Topo I, preincubated for 10 min. with 2′-ethoxymethyklavuzon (16b) and compound 16i. D) Concentration dependent
inhibition of Topo I, preincubated for 10 min. with 2′-propoxymethylklavuzon (16c) or 2′-isopropoxymethylklavuzon (16f). E) Concentration dependent inhibition of
Topo I, preincubated for 10 min. with 2′-butoxymethylklavuzon (16d) or 2′-pentyloxymethylklavuzon (16e).
4

H. Çetinkaya, et al.
BioorganicChemistry103(2020)104162
Fig. 4. Time and concentration dependent cytotoxic activity of 2′-methoxymethylklavuzon (16a) in comparison with 1% DMSO and 50 µM GTN (goniothalamin) in
3D spheroids of HuH-7 cells. Scale bar = 500 µm.
inhibitory properties. However the CRM1 inhibitory activities of com-
pounds 2, 3, and 6 are still comparable with the activity of selinexor.
Similar to 2′-methylklavuzon (3), two novel 2′-alkoxymethyl sub-
stituted klavuzon derivatives (16a and 16g) are not effective as much as
4′-methylklavuzon (2) in terms of CRM1 inhibition. Hence, the selective
toxicity of compound 16g may not be related with CRM1 inhibition
(Fig. 2). RIOK2 protein was used to demonstrate effects of CRM1 pro-
tein that facilitates the process of intracellular translocation of RIOK2
from nucleus to cytoplasm under normal conditions.
Previous studies showed that Topo I’s vicinal cysteine residues 504
and 505 should be the target for thiol reactive Topo I inhibitors such as
N-ethylmaleimide. In the same study, authors also mentioned that
Cys505 plays a critical role during the poisoning of the Topo I by de-
rivatives of CPT [31]. Afterwards, Topo I inhibitory properties of other
thiol reactive species such as derivatives of naphthoquinone, [32] cy-
clometalated gold III, [33] 1-ethylkuquinone, [34] CY13II, [35] and
oxindolimine-metal complexes [36] were reported. Since 2′-methylk-
lavuzon (3) is a thiol reactive compound it was preincubated with Topo
I enzyme for 0, 1, 5 and 10 min at two different concentrations before
the supercoiled DNA relaxation assay (Fig. 3A). Although compound 3
Fig. 5. Mean Fluorescence Intensity analysis for spheroids which are treated
inhibited Topo I at 50 and 100 µM concentrations in all preincubation
with PI after 24 h and 48 h incubation. Two-way ANOVA with Sidak’s multiple
times, 10 min of preincubation time was chosen for further experi-
ments.
comparison test: ****P value is smaller than 0.0001 (P < 0.0001).
Concentration dependent inhibitions of Topo I preincubated with
50, 10, 2 and 0.4 μM of novel klavuzon derivatives (16a-g and 16i)
were also investigated (Fig. 3B-3E). As a summary, most of the klavu-
zons showed Topo I inhibition at all tested concentrations except
compounds 16a, 16i and 16f. These three klavuzon derivatives showed
minimal or no Topo I inhibition at 0.4 µM concentrations which was the
lowest tested concentration (Fig. 3B, Lane 8; Fig. 3C, Lane 12; Fig. 3D,
Lane 12). More interestingly the same derivatives were the ones that
show selective toxicity in healthy (HPDEC) pancreatic cell line
(Table 3).
One of the well-known covalent inhibitor of CRM1 is leptomycin B
which reacts with Cys528 residue present at NES-binding groove of
CRM1 protein by Michael addition reaction [24]. Later, this residue was
targeted with many different thiol reactive compounds such as angui-
nomycin, [25] selinexor, [26] valtrate, [27] acetoxychavicol acetate,
[28] piperlongumine, [29] curcumin, [30] and goniothalamin [9].
Among these leptomycin, anguinomycin and selinexor inhibit CRM1 at
low nanomolar concentrations and only selinexor has been approved by
FDA for treatment of multiple myeloma so far.
CRM1 inhibitory properties of two novel klavuzon derivatives (the
least and the most selective compounds: 16a and 16g) were studied
along with previously reported klavuzon derivatives (2, 3 and 6).
Compounds 2 and 6 are the most active klavuzon derivatives reported
in literature and as it can be seen Fig. 2, they completely inhibit CRM1
at 0.05 µM concentration. However, 2′-methylklavuzon (3) started to
inhibit CRM1 at 0.2 µM concentration but still it could not inhibit
CRM1 fully at this concentration. Somehow, displacement of methyl
substituent from position 4′- to 2′- made a dramatic effect in the CRM1
It is quite difficult to draw a clear conclusion, however it was ex-
pected to have a similar IC50 values in both cell lines. Although it is
expected that drug candidates should have limited cytotoxic activity in
healthy cell line, strong cytotoxic activity in HPDEC cell line can only
be explained by mechanism of actions of klavuzon derivatives.
Duplication times of MIA PaCa-2 and HPDEC cell lines are quite similar
(~40 h. and ~ 34 h. respectively) and Topo I plays an important role
during cell division process. Inhibition of Topo I by klavuzons may halt
the cell division in both cell lines.
5

H. Çetinkaya, et al.
BioorganicChemistry103(2020)104162
Fig. 6. Concentration dependent cytotoxic activity of 2′-methoxymethylklavuzon over HuH-7 derived 3D spheroids. Two-way ANOVA with Dunnett’s multiple
comparison test: *P value is smaller than 0.05 (P < 0.05).
Another important point is that, HPDEC cells are Human Pancreatic
Duct Epithelial Cells immortalized by HPV16 E6/E7 genes. Both E6 and
E7 proteins inhibits the tumor suppressor proteins p53 and Rb (re-
tinoblastoma) respectively. CRM1 is the cargo protein that transport
p53 from nuclei to cytoplasm. Hence stability of the p53 is also depends
on the activity of CRM1 cargo protein. Inhibition of CRM1 increases the
stability of p53 in healthy HPDEC cell lines and causes cell death by
evading immortality.
At last, compound 16a was incubated with 3D spheroids that are
assembled from HuH-7 (hepatocellular cancer) cell line in 96 well
hanging drop plates at five different concentrations for 24 and 48 h. GTN
(goniothalamin) was also used at 50 µM concentration as positive control
to evaluate the relative toxicity of klavuzon derivative 16a. At the end of
the incubation time propidium iodide (PI) staining was performed for 3D
spheroids in hanging drops. Visualization of the spheroids was performed
with an inverted phase contrast - fluorescence microscope by using 4X
objective just before and after the PI staining (Fig. 4). Image analysis of PI
stained spheroids resulted the mean fluorescence intensities as shown in
Fig. 5. In both control experiments (with or without 1% DMSO) the size of
the spheroids increased over time and PI staining indicated minimal cell
death. The sizes of the GTN treated spheroids were similar and the
number of PI stained death cells were increased compared to control at
the end of 24 and 48 h of incubation. On the other side, compound 16a
was much more effective and inhibits the growth of spheroids at the
lowest tested concentration (0.37 µM), although there was not significant
number of cell death. A clear dose and time dependent cell death in the
spheroids were seen and spheroids started to crumble at the end of 48 h of
incubation in the presence of 30 µM of compound 16a.
According to the literature time dependent Topo I inhibitory prop-
erties of N-ethylmaleimide can be seen clearly at 600 μM concentration
[31]. Naphthoquinone derivative causes Topo
I inhibition at
100–500 μM concentrations. Similar time dependent CRM1 inhibition
was also observed for cyclometalated gold III, 1-ethylkuquinone,
CY13II, and oxindolimine-metal complexes at 6.5, 5, 25, and 25 μM
concentrations respectively. It seems that most of the klavuzon deri-
vatives reported in this work show superior Topo I inhibition property
compared to other thiol reactive inhibitors present in the literature.
Recently we have shown that klavuzon derivatives also inhibit
SIRT1 protein and that causes the accumulation of p53 protein inside
the cell which may be the cause of sensitivity of the immortalized
healthy cells toward klavuzon derivatives [37]. Additionally, inhibition
of p53 activity in healthy cell lines was maintained by E6 protein which
can bind directly to p53 and trigger proteasomal degradation via the
ubiquitin pathway [38]. It is reported that acetylation of p53 can
contribute the stability and activation of p53 protein. Acetylation of
lysines in p53 inhibits proteasomal degradation by inhibiting ubiqui-
tination, and acetylation of C-terminal region causes sequence-specific
DNA binding activity by activating p53 [39]. Interestingly, acetylated
p53 is one of the substrates of SIRT1 enzyme [40]. In the absence of
SIRT1 activity cells became more sensitive to stress conditions apop-
tosis can be triggered by transcriptional activities of increased expres-
sion levels of acetylated p53 and its targets p21 and Bax proteins [39].
In this sense, SIRT1 inhibitory properties of klavuzons are also con-
tributes to the stabilization p53 in the HPDEC cell lines. Hence cyto-
toxic activity is not unusual in immortalized healthy cell line (HPDEC)
when CRM1 and SIRT1 proteins are inhibited by klavuzon. Inhibition of
Topo I, CRM1 and SIRT1 may be the cause of lack of selective cytotoxic
activity.
Alternatively, WST-1 assay was also performed as a measure of cell
viability in spheroids. As it shown in Fig. 6, 2′-methoxymethylklavuzon
showed a clear dose dependent cytotoxic activity over the spheroids.
One interesting point is the presence of significant amount of cell via-
bility difference between the control and DMSO treated control spher-
oids. Such difference could not be seen in PI stained spheroids. Al-
though WST-1 assay is mostly depends on the enzymatic activities of
mitochondrial dehydrogenases, PI staining can only show late apoptotic
or necrotic cells. Thus normalized dose dependent percent inhibition
graph for compound 16a was drawn and IC50 value for the cytotoxic
activity of compound 16a in spheroids was calculated as 0.46 µM.
3. Conclusions
A number of 2′-alkoxymethyl substituted klavuzon derivatives have
been reported as an effort to contribute multi-target drug discovery
studies. Syntheses of these novel compounds were completed in eight
steps. Previously 4′-alkyl substituted klavuzon derivatives were also
6

H. Çetinkaya, et al.
BioorganicChemistry103(2020)104162
reported by our group for the same purpose. In terms of biological
activities there were some similarities and differences between these
two groups of klavuzon derivatives. One of the similarities is the po-
tency and selectivity of the klavuzon derivatives in healthy and can-
cerous pancreatic cell lines. Substituents, that are larger in size, resulted
a decrease in the cytotoxic activity of klavuzon, while larger sub-
stituents at 4′- and 2′-positions gave better selectivity index compared
to that of 4′- and 2′-methyl substituted klavuzons. It is important to
point out that none of the 4′-alkylklavuzons showed significant selec-
tive cytotoxic activity against cancer cells. On the other hand novel 2′-
isobutoxymethylklavuzon (16g) was reported as the most selective
klavuzon derivative among the reported klavuzons in the literature.
Both 2′- and 4′- substituted klavuzon derivatives shown to be the
inhibitors of CRM1 and Topo I proteins. One interesting point in here
was the potency of the CRM1 inhibition property was altered by the
positions and the sizes of the substituents. It seems that a smaller
substituents at 4′-position gave stronger CRM1 inhibition compared to
those of 2′-substituted ones. This finding was especially important for
us because it might be possible to tune klavuzons to inhibit a specific
target by playing the sizes and the positions of the substituents.
3D cell cultures are one of the convenient ways of showing newly
synthesized drugs on normal and cancerous cells. Because of the unique
properties of 3D cell cultures, cytotoxic effects of the drugs can be
evaluated as closer to in vivo conditions than in vitro conditions. In our
case the compound 16a, the most cytotoxic compound among the
tested 2′-alkoxymethyl klavuzon derivatives, inhibited the increment in
the size of HuH-7 derived 3D spheroids. Cytotoxic activity of compound
16a in spheroids was visualized by PI staining and IC50 value was
calculated as 0.46 µM by WST-1 assay.
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Declaration of Competing Interest
The authors declared that there is no conflict of interest.
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Acknowledgements
This work was supported by the Scientific and Technological
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