CRM1 inhibitory and antiproliferative activities of novel 4′-alkyl substituted klavuzon derivatives

Article abstract of DOI:10.1016/j.bmc.2017.06.030

Klavuzons are 6-(naphthalen-1-yl) substituted 5,6-dihydro-2H-pyran-2-one derivatives showing promising antiproliferative activities in variety of cancer cell lines. In this work, racemic syntheses of nine novel 4′-alkyl substituted klavuzon derivatives were completed in eight steps and anticancer properties of these compounds were evaluated. It is found that size of the substituent has dramatic effect over the potency and selectivity of the cytotoxic activity in cancerous and healthy pancreatic cell lines. The size of the substituent can also effect the CRM1 inhibitory properties of klavuzon derivatives. Strong cytotoxic activity and CRM1 inhibition can be observed only when a small substituent present at 4′-position of naphthalen-1-yl group. However, these substituents makes the molecule more cytotoxic in healthy pancreatic cells rather than cancerous pancreatic cells. Among the tested compounds 1,2,3,4-tetrahydrophenanthren-9-yl substituted lactone was the most cytotoxic compound and its antiproliferative activity was also tested in 3D spheroids generated from HuH-7 cell lines.

Full text of DOI:10.1016/j.bmc.2017.06.030

Accepted Manuscript
CRM1 inhibitory and antiproliferative activities of novel 4′-alkyl substituted
klavuzon derivatives
Tuğ çe Kanbur, Murat Kara, Meltem Kutluer, Ayhan Şen, Murat Delman, Aylin
Alkan, Hasan Ozan Otaş, Ali Çağ ır
PII:
S0968-0896(17)31028-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.030
BMC 13813
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 May 2017
10 June 2017
16 June 2017
Please cite this article as: Kanbur, T., Kara, M., Kutluer, M., Şen, A., Delman, M., Alkan, A., Otaş, H.O., Çağ ır,
A., CRM1 inhibitory and antiproliferative activities of novel 4′-alkyl substituted klavuzon derivatives, Bioorganic
& Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.06.030
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CRM1 inhibitory and antiproliferative activities of novel 4′-alkyl
substituted klavuzon derivatives
Tuğçe Kanbur,a Murat Kara,a Meltem Kutluer,b Ayhan Şen,c Murat Delman,b Aylin Alkan,c
Hasan Ozan Otaş,c 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
*Corresponding Author: Assoc. Prof. Dr. Ali Çağır
Address:
İzmir Institute of Technology,
Faculty of Science, Department of Chemistry
35430 Urla-İzmir, Turkey
+090 232 7507572
Phone:
Fax:
+090 232 7507509
e-mail:
alicagir@iyte.edu.tr
1

Abstract
Klavuzons are 6-(naphthalen-1-yl) substituted 5,6-dihydro-2H-pyran-2-one derivatives
showing promising antiproliferative activities in variety of cancer cell lines. In this work,
racemic syntheses of nine novel 4′-alkyl substituted klavuzon derivatives were completed in
eight steps and anticancer properties of these compounds were evaluated. It is found that size
of the substituent has dramatic effect over the potency and selectivity of the cytotoxic activity
in cancerous and healthy pancreatic cell lines. The size of the substituent can also effect the
CRM1 inhibitory properties of klavuzon derivatives. Strong cytotoxic activity and CRM1
inhibition can be observed only when a small substituent present at 4′-position of naphthalen-
1-yl group. However, these substituents makes the molecule more cytotoxic in healthy
pancreatic cells rather than cancerous pancreatic cells. Among the tested compounds 1,2,3,4-
tetrahydrophenanthren-9-yl substituted lactone was the most cytotoxic compound and its
antiproliferative activity was also tested in 3D spheroids generated from HuH-7 cell lines.
Keywords: Klavuzon, Anti-proliferative activity, CRM1 inhibition, pancreatic cancer,
hepatocellular carcinoma, 3D cell culture
2

1. Introduction
Current the 5-year survival rates for lung and pancreatic cancers are 18% and 8%,
respectively. These low survival rates are partially because of the late diagnosis of one-half of
cases, in which 5-year survival decrease to 4% and 3%, respectively.1 Currently, FDA has
approved more than ten anticancer drugs for the treatment of pancreatic cancer but poor
survival rates indicates the urgency of the development of novel anticancer drug candidates.
In this work, development of novel klavuzon derivatives, as new anticancer drug candidates
for the treatment of pancreatic cancer, was studied.
In recent years, there is an increase in the number of studies for the discovery of drug
candidates, which act by covalent binding to the nucleophilic sites of the macromolecules
inside the cells. Almost, 30% of drugs in use today act as covalent inhibitor.2 One of the most
common motif in the structure of natural products is α,β-unsaturated-δ-lactone that can act as
an irreversible inhibitor through a covalent bond formation. It is believed that, nucleophilic
site of the enzymes can bind to the β-carbon of the lactone pharmacophore through by
Michael addition.3
(R)-Goniothalamin (1) is one of these natural products and was isolated first in 1967
by Hlubucek from Cryptocarya caloneura.4 Its selective cytotoxic activity against cancer cell
lines5-6 draw the attention of scientist and many research groups have put effort in
synthesizing novel derivatives of goniothalamin to understand the structure activity
relationship.7-16 On the other hand, a few number of studies were dedicated to explore the
mechanism of action of goniothalamin derivatives such as inhibition of CRM1
nucleocytoplasmic transport receptor protein17 and inhibition of topoisomerase I.18 In terms of
cell death, it was shown that (R)-enantiomer of goniothalamin induces apoptosis while (S)-
enantiomer induces autophagy.19 Efficiency of racemic and enantiomerically pure forms of
goniothalamin was also demonstrated in Ehrlich solid tumor in mice.20
Previously, synthesis and antiproliferative activity of conformationally constrained
(R)-goniothalamin ((R)-2) was reported by our group. Although it was found that
antiproliferative activity of compound (R)-2 was higher than that of (R)-goniothalamin (1),
naphthalen-1-yl substitution at position-6 of 5,6-dihydro-2H-pyran-2-one pharmacophore
further increases the activity. The inventors named this compound as “klavuzon”. In the same
study, (R)-4′-methylklavuzon ((R)-3) and (R)-2′-methylklavuzon ((R)-4) were found to be the
most cytotoxic compound.21 In a recent study, racemic 4′-methoxyklavuzon (5) and (R)-2′-
3

methoxyklavuzon (6) derivatives were also prepared and they were found to be less cytotoxic
compared to their methyl counterparts ((R)-3 and (R)-4). In the same study, topoisomerase I
inhibitory property of klavuzon derivatives was also represented (Figure 1).18
Figure 1. Structures of (R)-goniothalamin (1), conformationally constrained (R)-
goniothalamin (2), 4′- and 2′-substituted klavuzon derivatives (3-6).
Multi-target drug discovery gained significant attention in the last decade to overcome
the difficulty of curing diseases having complex etiology and drug-resistance issues.22 In this
respect, goniothalamin derivatives can be considered as a dual acting inhibitor because of its
capability to inhibit both CRM1 and topoisomerase I. Due to their structural similarity to
goniothalamin, klavuzons may also have similar dual acting inhibitor property. Previously,
topo I inhibitory property of klavuzons was shown. In this study, synthesis and
antiproliferative properties of novel 4′-alkyl substituted klavuzon derivatives in pancreatic
cancer cells (MIA PaCa-2) and Human pancreatic ductal epithelial cells (HPDEC) will be
presented. Their time and concentration dependent CRM1 inhibitory properties will also be
discussed.
2. Results and Discussion
Racemic syntheses of 4′-alkylklavuzon derivatives (16a-h) were completed in eight
steps. Strategically important ethyl 4-alkyl-1-naphthoates (10a-h) were prepared according to
the procedures reported in literature starting from 4-methyl-1-naphthoic acid (7) in three
steps. As can be seen in Scheme 1, benzylic bromination of ethyl 4-methyl-1-naphthoate (8)
was performed by a NBS-benzoyl peroxide mixture to produce compound 9 in 92% yield. In
the next step, the copper (I)-catalyzed addition of various freshly prepared Grignard reagents
4

to 4-bromomethyl-1-naphthoate (9) was successfully performed.23 Interestingly, varying
amounts of dimerization product (11) of 4-bromomethyl-1-naphthoate (9) were also observed.
Scheme 1. Conversion of 4-methyl-1-naphthoic acid (7) into ethyl 4-alkyl-1-naphthoate
derivatives (10a-h).
Ethyl 1-naphthoates (10a-h) were successfully reduced to 1-naphthylmethanols (12a-
h) by using LiAlH4 with 8-87% yields. Then PCC was used to convert the alcohols into the
corresponding 4-alkyl-1-naphthaldehyde derivatives (13a-h) with relatively higher yields (58-
90%) as represented in Scheme 2. Then, a well-known three step synthesis was used to
complete the syntheses. Simply, addition of allylmagnesium bromide over 1-naphthaldehydes
(13a-h) and commercially available 4-methyl-1-naphthaldehyde (13i) yielded homoallylic
alcohols (14a-i) in 54-91% yields. Acrylation with acryloyl chloride in the presence of
triethylamine followed by ring closing metathesis reactions gave the final klavuzon
derivatives (3 and 16a-h, Scheme 2). Another important klavuzon derivative 20 was also
prepared by the same three step synthesis starting from commercially available 1,2,3,4-
tetrahydrophenanthrene-9-carbaldehyde (17) as represented in Scheme 3.
5

Scheme 2. Transformation of ethyl 4-alkyl-1-naphthoate derivatives (10a-h) to 4′-alkyl
substituted klavuzon derivatives (16a-h).
Scheme 3. Synthesis of 6-(1,2,3,4-tetrahydrophenanthren-9-yl)-5,6-dihydro-2H-pyran-2-one
(20).
Alternatively, 5,6-dihydro-2H-pyran-2-one pharmacophore can be prepared from the
isomerization of 3,6-dihydro-2H-pyran 2-one in the presence of an amine base. A similar
isomerization can also be observed for naphthalen-1-yl substituted 3,6-dihydro-2H-pyran 2-
one derivatives inside the cells to give the corresponding klavuzons, so derivatives of 3,6-
dihydro-2H-pyran 2-one may have therapeutic importance. For these reasons, a list of 4-
alkylnaphthalen-1-yl substituted 3,6-dihydro-2H-pyran 2-one derivatives (23a-e) were also
prepared starting from 4-methyl-1-naphthoic acid (Scheme 4).
Compounds 13a-h were synthesized by the same synthetic route reported above and
then reacted with vinylmagnesium bromide to give allylic alcohols 21a-e with 67-87% yields.
Steglich esterification of the formed alcohols with but-3-enoic acid gave esters 22a-e. Finally,
6

ring closing metathesis reactions of the esters 22a-e yielded 4-alkylnaphthalen-1-yl
substituted 3,6-dihydro-2H-pyran 2-one derivatives (23a-e).
Scheme 4. Synthesis of 4-alkylnaphthalen-1-yl substituted 3,6-dihydro-2H-pyran-2-one
derivatives (23a-e).
Although, compounds 23a-e were isolated and characterized successfully, it was noted
that these compounds totally decomposed even at +4 oC in three months. Decomposition of 6-
(4-methylnaphthalen-1-yl)-3,6-dihydro-2H-pyran 2-one (23a). was also shown by 1H-NMR
spectroscopy in Figure 2. Observed instability of the 4-alkylnaphthalen-1-yl substituted 3,6-
dihydro-2H-pyran 2-one derivatives led us to convert compound 23b-e to the corresponding
klavuzon derivatives 16b-e by isomerization with DBU, instead of testing them to determine
their antiproliferative properties (Figure 3).
7

Figure 2. Decomposition of 6-(4-methylnaphthalen-1-yl)-3,6-dihydro-2H-pyran 2-one (23a).
(Spectra 1: freshly prepared; Spectra 2: after one month; Spectra 3: after three months).
Figure 3. Isomerization of 6-(4-alkylnaphthalen-1-yl)-3,6-dihydro-2H-pyran 2-one
derivatives.
Antiproliferative properties of all prepared klavuzon derivatives (3, 16a-h and 20)
were tested in healthy (HPDEC) and cancerous (MIA PaCa-2) pancreatic cell lines by MTT
cell viability assay. Camptothecin (CPT) was used as positive control. There were differences
between the cell culture conditions of both cell lines, and the most important difference was
that HPDEC should be cultured (and tested) over type I collagen coated surface. Because
there is a Michael acceptor in the structure of klavuzon, a small amount of the tested
compounds can bind to the collagen and actual level of cytotoxicity may not be observed. To
determine the effect of the collagen in MTT assay, three klavuzon derivatives (3, 16b and
16c) were also tested in MIA PaCa-2 cell lines which are cultured in type I collagen coated 96
well microplate. All IC50 values calculated for klavuzon derivatives were summarized in
Table 1.
According to these data, collagen coating did not have any influence in structure
activity relationship of klavuzon derivatives. It slightly increased the IC50 values for
compounds 3 and 16c. It might be possible that MIA PaCa-2 cell viability and resistance to
goniothalamin increases in the presence of type I collagen or a small amount of klavuzons
was bound to the collagen as expected. On the other hand, effect of collagen over the IC50
value of compound 16b was more dramatic. It is difficult to announce the reason for this
difference between the IC50 values of compounds 16b and 16c. In one thought, this difference
may not be the result of interactions between klavuzon and collagen because if that is the case
compounds 16b should have similar IC50 values for HPDEC and MIA PaCa-2 cell lines
cultured over collagen. Only explanation can be the development of a resistance in MIA
PaCa-2 cells when they grow over collagen. Overall, it can be concluded that SAR of
klavuzon derivatives was not affected by collagen coating (Table 1).
8

MTT results show that, both pancreatic cell lines were quite sensitive to camptothecin
(CPT), a well known topoisomerase I poison. Interestingly, CPT does not show any
selectivity over cancer cell lines probably due to the duplication times for cancerous (MIA
PaCa-2) and immortalized healthy (HPDEC) cell lines were quite close to each other (40 and
34 hours, respectively). As it can be seen in Table 1 compound 20 showed five and two times
stronger cytotoxic activity compared to compound 3 in MIA PaCa-2 and HPDEC cell lines,
respectively. Level of cytotoxic activity of 4′-ethylklavuzon (16a) was stronger compared to
4′-methylklavuzon (3) in both cell lines, but selectivity was getting worse. In general,
presence of a linear alkyl chain at position 4′- of klavuzon (3 and 16a-d) caused slightly
selective cytotoxic activity for healthy pancreatic cell line (HPDEC). The length of the alkyl
chain negatively affected potency of these compounds and shorter alkyl chains resulted more
cytotoxic klavuzon derivatives. On the other hand, presence of a more bulky group at 4′-
position gave klavuzon derivatives (16e-h) that have comparable potency with 4′-propyl and
4′-butyl substituted klavuzons (16b and 16c). Nevertheless, selectivity index enhanced to
0.91-1.31 interval (Table 1).
9

Table 1. Antiproliferative effect of 4′-alkyl substituted klavuzon derivatives (16a-i) over MIA
PaCa-2 pancreatic cancer cell line and HPDEC immortalized healthy pancreatic cell
line.
IC50 (in µM)
Comp. MIA PaCa-2
Collagen
SI
HPDEC
No collagen
CPT
3
0.18 0.01
0.21 0.01 1.17
0.34 0.09 0.35
0.19 0.05 0.23
0.47 0.15 0.31
0.49 0.17 0.08
2.83 0.38 0.42
4.50 0.48 1.10
2.88 0.49 1.31
3.97 0.50 1.24
1.28 0.11 0.91
0.15 0.04 0.94
0.96 0.17 1.23 0.34
0.83 0.12
16a
16b
16c
16d
16e
16f
16g
16h
20
1.50 0.61 2.62 1.09
6.30 0.73 7.37 1.21
6.70 0.20
4.09 0.20
2.20 0.11
3.21 0.39
1.40 0.69
0.16 0.03
SI: Selectivity Index = IC50 in HPDEC /IC50 in MIA PaCa-2, where IC50 is
the concentration required to decrease the cell population 50%. CPT:
camptothecin
Induction of apoptosis and cell cycle analysis of MIA PaCa-2 cells treated with
varying concentrations of 4′-methylklavuzon (3) were also studied by using Annexin V-FITC
apoptosis detection kit (Figure 4). At the end of 24 hours of incubation with compound 3 the
percentage of the early apoptotic MIA PaCa-2 cells slightly increased by concentration
dependent manner. A similar statistically significant amount of increase was observed for the
percentage of the late apoptotic cells, while the percentage of the necrotic cells did not
change.
Besides, cell cycle analysis gave the clues about the existence of more than one action
of mechanism for 4′-methylklavuzon (3). Incubation with low dose (1 µM) of compound 3
resulted a small increase at G1 phase while that causes a similar amount of decrease at S
phase. The number of cells at G1 phase decreased significantly in MIA PaCa-2 cell line
10

incubated with 5 and 10 µM of 4′-methylklavuzon for 24 hours. Contrarily, number of cells at
G2 phase increased significantly at the same concentrations. There is a small rise in the
number of cells at S phase in the presence of 10 µM of 4′-methylklavuzon but this increment
was not significant (Figure 4). Cell cycle arrest at S phase may be the indication of Topo I
inhibitory property of 4′-methylklavuzon (3) and it is required high doses of compound 3. In
addition, cell cycle arrest at G2 phase could mean that a number of cells complete the DNA
duplication and another mechanism blocks the cell division at this stage.
15
DMSO (Cont.)
****
1 µM
10
5
5 µM
10 µM
**
0
Early Apop. Late Apop.
Necrosis
According to Two-way ANOVA Bonferroni's multiple comparisons test P Value for; ** is smaller than
0.01 (P<0.01) and for **** is smaller than 0.0001 (P<0.0001)
100
DMSO (Cont.)
1 µΜ
80
60
40
20
0
***
5 µΜ
****
10 µΜ
****
***
G1
S
G2
Two-way ANOVA Bonferroni's multiple comparisons test: *** P Values are equal to 0.0008 for G1 and
0.0002 for G2; **** P Value is smaller than 0.0001 (P<0.0001)
Figure 4. Concentration dependent apoptosis and cell cycle analysis for 4′-methylklavuzon
(3) treated MIA PaCa-2 cells.
According to apoptosis and cell cycle analysis, compound 3 causes apoptosis or cell
cycle arrest only in a small fraction of cell population even at 10 µM concentration at the end
of 24 h incubation period. However, IC50 value of compound 3 in MIA PaCa-2 cells was
calculated as 0.96 µM for 48 h treatment. There should be a sign for this cytotoxic effect at 24
h incubation. To control DNA integrity comet assay was performed for MIA PaCa-2 cells
treated with varying concentrations of compound 3 (Figure 5). An alkaline lysis solution was
11

used to detect the possible double and single strand DNA breaks.24 Hydrogen peroxide was
used as positive control. Formation of DNA breaks could easily be seen for 53% MIA PaCa-2
cells treated with 5 µM of 4′-methylklavuzon (3), although total number of the apoptotic cells
was around 14% of the total cell population. A similar inconsistency was also found for MIA
PaCa-2 cells treated with 10 µM of compound 3. While only 20% of the cells were apoptotic
at this concentration, 42% of the cell population gave comet as an indication of DNA breaks.
Mismatch between the percentages of cells at apoptosis and percentages of cells giving comet
can be explained by Topo I inhibition property of klavuzons. Topoisomerases, including
topoisomerase I, play crucial role in the cellular response to DNA damage, DNA repair and
apoptosis.26 Any damage in DNA can not be repaired when all Topo I enzymes are inhibited
by compound 3.
Figure 5. Comet assay for MIA PaCa-2 cells treated with 4′-methylklavuzon (3).
Nucleocytoplasmic transport systems play a critical role in intracellular localization
and regulation of proteins in each cell. This balance is quite important and is typically
uncontrolled in cancer cells. Specific localization of tumor suppressor and oncoproteins is
quite important because any abnormal localization of these proteins can cause inactivation or
over-activation. Hence it was believed that inhibitors of nucleocytoplasmic transport systems
could be an anticancer agent for a long time.27 In this respect, concentration dependent CRM1
inhibitory properties of klavuzons 3, 16b, 16c, 16d and 20 were studied by visualization of
CRM1 mediated nuclear export of Riok2 (Serine/threonine-protein kinase Rio2) in HeLa
cells. CRM1 inhibitory property of goniothalamin was shown previously and it was used as
positive control.17 DAPI was used to stain the nucleus of the cells for comparison purpose.
For each molecule, number of cells in three separate pictures were counted and percent
inhibition were calculated by dividing the number of CRM1 inhibited cells to the number of
total cells.
12

According to the results, goniothalamin started to inhibit CRM1 (50%) at 1.0 µM
concentration at the end of the 90 minutes of incubation and CRM1 was inhibited completely
in 100% of the cell population at 5 µM concentration. A longer incubation period (6 hours)
gave similar trend and at 5 µM concentration, goniothalamin completely inhibited the
nucleocytoplasmic transport system in all cells (Figure 6). On the other hand, compound 3
started to inhibit CRM1 at 0.2 µM concentration even at the end of the 90 minutes of
incubation (87%). A similar CRM1 inhibition potency was also found for compounds 16b and
16c. Additionally, compound 16d could not inhibit CRM1 at 0.2 µM concentration. However,
it inhibited CRM1 completely at 1.0 µM concentration at the end of 6 hours of incubation.
Interestingly, compounds 16c and 16d had quite similar IC50 values in MIA PaCa-2 cells
(6.30 and 6.70 µM, respectively) and it is quite difficult to correlate their CRM1 inhibitory
potency with these IC50 values. Lastly compound 20 showed strong CRM1 inhibition activity
at 0.2 µM concentration at the end of 6 hours of incubation (Figure 6). Since compounds 3
and 20 were the most cytotoxic klavuzon derivatives, their CRM1 inhibitory properties were
also tested at lower doses (Figure SM1, Supplementary materials). It was found that both
compounds started to inhibit CRM1 at 20 nM concentrations, and they almost inhibited
CRM1 completely in all cells at 50 nM concentrations.
13

0.2 M
1.0 M
5.0 M
Figure 6. Time and concentration dependent inhibition of CRM1 in HeLa cells treated with
goniothalamin (1), 4′-methylklavuzon (3), 4′-propylklavuzon (16b), 4′-butylklavuzon (16c),
4′-pentylklavuzon (16d) derivatives and compound 20.
Lastly, antiproliferative activity of compound 20 was tested over 3D spheroids
generated from HuH-7 hepatocellular cancer cell line in 96 well hanging drop plates in 48
14

hours. Then varying concentrations of compound 20 were applied to the spheroids.
Goniothalamin was also applied in a single dose just for comparison purpose (Figure 7).
Spheroids were monitored by microscope at the end of 24, 48, and 72 hours of incubation by
using 4X objective, then the same spheroids were treated with propidium iodide (PI) staining
and monitored by fluorescence microscope by using 4X objective to visualize the dead cells.
As it can be seen in Figure 7, 50 µM concentration of goniothalamin caused significant cell
death just after 24 hours of incubation compared to untreated or DMSO control HuH-7
spheroids. Although its cytotoxic activity did not increase in 48 hours of incubation, the size
of the spheroid was smaller than the sizes of the untreated or DMSO control spheroids. At the
end of the 72 hours of incubation, all of the cells in goniothalamin treated spheroids were
dead.
On the other hand, compound 20 inhibited the growth of 3D spheroids almost in all
tested concentrations and it caused significant cell death at 10 and 30 µM concentrations in 48
hours. Spheroid even started to become disintegrated at 30 µM concentration. Similar dose
dependent response was observed at the end of 72 hours of incubation with compound 20.
15

Cont +
DMSO
Comp. 20
3.33 µM
GTN
50 µM
Cont.
0.37 µM
1.11 µM
10 µM
30 µM
Figure 7. Time and concentration dependent cytotoxic activity of compound 20 in 3D
spheroids of HuH-7 cells.
3. Conclusion
Synthesis of novel 4′-alkyl substituted klavuzon derivatives were successfully
completed in eight steps starting from 4-methyl-1-naphthoic acid. MTT cell viability assay
over cancerous (MIA PaCa-2) and immortalized healthy (HPDEC) pancreatic cell lines
revealed that compound 20 has a remarkable cytotoxic effect in both cell lines without having
any selectivity. Two other novel klavuzon derivatives, 4′-methylklavuzon (3) and 4′-
ethylklavuzon (12a), are quite cytotoxic and selective to HPDEC cell line with IC50 values at
nanomolar concentrations. Among these, 4′-methylklavuzon (3) and compound 20 were
chosen for further biological activity studies and it is found that 4′-methylklavuzon (3)
induces apoptosis in MIA PaCa-2 cells at 5 and 10 µM concentrations. Comet assay also
indicates the formation of single and double strand DNA breaks in MIA PaCa-2 cells treated
with 4′-methylklavuzon (3) for 24 h. Cell cycle analysis of MIA PaCa-2 cells treated with
varying concentrations of compound 3 surprisingly implies the presence of more than one
16

action of mechanism for this compound. At 1 µM concentration, compound 3 causes an arrest
at G1 phase while at higher concentrations that results in G2 arrest.
Beside the previously reported Topo I inhibition, time and concentration dependent
CRM1 inhibitory properties of klavuzon derivatives have been shown as a second mechanism
of action in this work. Especially, compounds 3 and 20 are the most effective derivatives and
they completely inhibit CRM1 activity at 50 nM concentrations in 6 hours of incubation.
Compared to traditional two-dimensional cell cultures, 3D cell cultures are considered
as a much better way to represent in vivo environment. Hence, the most active derivative,
compound 20, has been tested in HuH-7 derived 3D spheroids and it has been found that
compound 20 inhibited the growth of 3D spheroids and it caused significant cell death at 10
and 30 µM concentrations in 48 hours. Therefore, compound 20 can be a good lead compound
for further studies.
Author contributions
All authors have approved the final version of the manuscript.
Acknowledgements
This work was supported by the Scientific and Technological Research Council of
Turkey (TÜBİTAK, 113Z146). The authors would like to acknowledge networking support
by the COST Action BM1204. We also would like to thank Mehmet S. Yıldız due to his help
in MTT assays. We thank to Biotechnology and Bioengineering Research Central Research
Laboratories at Izmir Institute of Technology for their help in bioactivity studies.
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18

Captions of Figures and Schemes
Figure 1. Structures of (R)-goniothalamin (1), conformationally constrained (R)-
goniothalamin (2), 4′- and 2′-substituted klavuzon derivatives (3-6).
Scheme 1. Conversion of 4-methyl-1-naphthoic acid (7) into ethyl 4-alkyl-1-naphthoate
derivatives (10a-h).
Scheme 2. Transformation of ethyl 4-alkyl-1-naphthoate derivatives (10a-h) to 4′-alkyl
substituted klavuzon derivatives (16a-h).
Scheme 3. Synthesis of 6-(1,2,3,4-tetrahydrophenanthren-9-yl)-5,6-dihydro-2H-pyran-2-one
(20).
Scheme 4. Synthesis of 4-alkylnaphthalen-1-yl substituted 3,6-dihydro-2H-pyran-2-one
derivatives (23a-e).
Figure 2. Decomposition of 6-(4-methylnaphthalen-1-yl)-3,6-dihydro-2H-pyran 2-one (23a).
(Spectra 1: freshly prepared; Spectra 2: after one month; Spectra 3: after three
months)
Figure 3. Isomerization of 6-(4-alkylnaphthalen-1-yl)-3,6-dihydro-2H-pyran 2-one
derivatives.
Figure 4. Concentration dependent apoptosis and cell cycle analysis for 4′-methylklavuzon
(3) treated MIA PaCa-2 cells.
Figure 5. Comet assay for MIA PaCa-2 cells treated with 4′-methylklavuzon (3).
Figure 6. Time and concentration dependent inhibition of CRM1 in HeLa cells treated with
goniothalamin (1), 4′-methylklavuzon (3), 4′-propylklavuzon (16b), 4′-
butylklavuzon (16c), 4′-pentylklavuzon (16d) derivatives and compound 20.
Figure 7. Time and concentration dependent cytotoxic activity of compound 20 in 3D
spheroids of HuH-7 cells.
19

Graphical Abstract:
20

CRM1 inhibitory and antiproliferative activities of novel 4'-alkyl substituted klavuzon
derivatives
Highlights
1. Syntheses of nine novel 4’-alkyl substituted klavuzon derivatives were performed.
2. Roles of substituents over potency and selectivity cytotoxicity were shown.
3. Effects of the substituents over CRM1 inhibition property were presented.
4. Induction of apoptosis, cell cycle arrests in MIA PaCa-2 cells were shown.
5. Time and concentration dependent disruptions of 3D spheroids were also presented.
21