Synthesis and SAR studies of bis-chromenone derivatives for anti-proliferative activity against human cancer cells

Article abstract of DOI:10.1016/j.bmcl.2014.09.057

A novel family of 3-((4-oxo-4H-chromen-3-yl)methyl)-4H-chromen-4-one (bis-chromone) derivatives were designed, synthesized and studied for their anti-cancer activity using the XTT assay for the growth inhibition against various human cancer cells. Among them, 3-((5-(cyclohexylmethoxy)-4-oxo-4H-chromen-3-yl)methyl)-7-methoxy-4H-chromen-4-one and 3-((5-(cyclohexylmethoxy)-4-oxo-4H-chromen-3-yl)methyl)-7-hydroxy-4H-chromen-4-one showed micromolar level of in vitro anti-proliferative activity against human cancer cell lines. The SAR studies indicated bis-chromone as a basic scaffold to design anticancer agents. The 5-cyclohexylmethoxy on the first chromenone ring and electron donating group such as CH₃, OCH₃ or hydrogen bonding group (OH) on the other chromenone ring of bis-chromone increased the activity. However, saturation of one of chromenone to chromanone in bis-chromones decreased the activity.

Full text of DOI:10.1016/j.bmcl.2014.09.057

Accepted Manuscript
Synthesis and SAR Studies of Bis-Chromenone Derivatives for Anti-prolifera-
tive Activity against Human Cancer Cells
Eeda Venkateswararao, Vinay K. Sharma, Manoj Manickam, Jieun Yun, Sang-
Hun Jung
PII:
S0960-894X(14)01010-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.09.057
BMCL 22023
Reference:
Bioorganic & Medicinal Chemistry Letters
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2 September 2014
19 September 2014
Please cite this article as: Venkateswararao, E., Sharma, V.K., Manickam, M., Yun, J., Jung, S-H., Synthesis and
SAR Studies of Bis-Chromenone Derivatives for Anti-proliferative Activity against Human Cancer Cells,
Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.09.057
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Synthesis and SAR Studies of Bis-Chromenone Derivatives for Anti-proliferative
Activity against Human Cancer Cells
Eeda Venkateswararaoa, Vinay K. Sharmaa, Manoj Manickama, Jieun Yunb, Sang-Hun Junga*
aCollege of Pharmacy and Institute of Drug Research and Development, Chungnam
National University, Daejeon 305-764, Korea
bBio-evaluation Center, KRIBB, Cheongwon-gun chungcheongbuk-do 363-883, Korea
Abstract
A novel family of 3-((4-oxo-4H-chromen-3-yl)methyl)-4H-chromen-4-one (bis-chromone)
derivatives were designed, synthesized and studied for their anti-cancer activity using the
XTT assay for the growth inhibition against various human cancer cells. Among them, 3-((5-
(cyclohexylmethoxy)-4-oxo-4H-chromen-3-yl)methyl)-7-methoxy-4H-chromen-4-one and 3-
((5-(cyclohexylmethoxy)-4-oxo-4H-chromen-3-yl)methyl)-7-hydroxy-4H-chromen-4-one
showed micromolar level of in vitro anti-proliferative activity against human cancer cell
lines. The SAR studies indicated bis-chromone as a basic scaffold to design anticancer agents.
The 5-cyclohexylmethoxy on the first chromenone ring and electron donating group such as
CH3, OCH3 or hydrogen bonding group (OH) on the other chromenone ring of bis-chromone
increased the activity. However, saturation of one of chromenone to chromanone in bis-
chromones decreased the activity.
Key words: Bis-chromone, Anti-cancer, Cytotoxicity.
*Corresponding author: jungshh@cnu.ac.kr. Tel.; +82 42 821 5939; Fax +82 42 823 6566;
Present address: College of Pharmacy and Institute of Drug Research and development,
Chungnam National University, Daejeon 305-764, Korea

Cancer is a deadly disease which figures among the prominent causes of mortality
worldwide1 due to the lack of a breakthrough research in this field. Thus, discovery of anti-
cancer drugs has become a desperate objective to be solved. Even though some potential
candidates have been discovered, yet they lag behind due to the toxicity associated with them.
Therefore, there is a need for the development of new anti-cancer agents which can overcome
this problem.
Since the beginning of cancer research a large number of heterocyclic compounds have been
discovered for their role in the treatment of various kinds of cancers.2 Among them one of the
important class is naturally occurring flavonoid which plays a crucial role in anti-cancer drug
discovery.3 Structural elucidation of flavonoid revealed chromen-4-one (chromone)
connected phenyl ring at 2 or 3 position as a core structure. However, chromone motif seems
to be vital for anti-cancer activity.4-6 Therefore, a number of chromones derivatives have been
designed, synthesized and studied for their anti-cancer activity.7-9 Among them, some
examples include a series of chromone derivatives 110 bearing diverse dithiocarbamate
moieties, 2-styrylchromone analogs 211, 3-hydroxychromones 312, geiparvarin analogs 413
(chromone linked with 1,2,3-triazole residues), flavones 514 bearing a N1-(flavon-7-
yl)amidrazones incorporating N-piperazines and related congeners, 4H-chromen-1,2,3,4-
tetrahydropyrimidine-5-carboxylate derivatives 615 and chromone-based lavendustin analogs
716. Thus, the chromone scaffold has emerged as one of the most important synthetic analogs
for their anti-cancer activity.
Although a large number of chromone analogs have been studied for their anti-cancer activity,
bis-chromone analogs have never been investigated as anti-cancer agents. Accordingly, we
have designed and synthesized bis-chromone derivatives 8 - 11 as shown in Figure 2 and
their anti-cancer activities were measured against human prostate (PC-3), lung (NCI-H23),

breast (MDA-MB-231), colon (HCT-15), stomach (NUGC-3) and renal ( ACHN) cancer cell
lines.
Preparation of bis-chromone analogs 8 and 9 were outlined in Scheme 1. The ,-unsaturated
intermediates 14 were synthesized by the reaction of the substituted chromone aldehydes 12
with appropriate triphenyl phosphine salts 13.17,18 In order to obtain 16, the reduction of
double bond and additional debenzylation of 14 were initially attempted with 40 psi hydrogen
gas in the presence of 20 % Pd/C in THF/methanol (1:1) at room temperature. However this
reaction gave mixture of inseparable multiple products containing saturated chromone 15.
Thus the reaction conditions were changed to refluxing with cyclohexene19 in the presence of
20 % Pd/C in THF/methanol (1:1). Five hour reflux of this reaction mixture partially reduced
14 to 16, which was isolated by column chromatography with moderate yield. TLC
monitoring of the reaction under transfer hydrogenation conditions indicated formation of
chromanone 15 around seventh hour of reaction. The prolongation of reaction time to ten
hours entirely gave chromanone 15. Compound 16 was efficiently cyclized to bis-chromone 8
by the treatment with boron trifluoride diethyl etherate (6.5 equivalents), methanesulfonyl
chloride (4.2 equivalent) and N,N-dimethylformamide at 50-60 oC. The same conditions were
applied for the preparation of 9 from 15. Compound 8b was obtained from 16b and
subsequent methylation and benzylation reactions afforded 8c and 8f, respectively (Scheme
2).
The phosphorous ylide derivative 2020 was prepared from the chromone aldehyde 17 by
reducing the aldehyde to the alcohol 18 in moderate yield using 9-BBN. The chromone
alcohol was treated with tetrabromomethane in the presence of PPh3 to afford the bromo
methyl analog 19. The bromo analog was reacted with PPh3 to provide the corresponding
phosphorous ylide 20 in good yield. Ylides thus formed was reacted with chromone aldehyde
12 as shown in Scheme 3 to get vinyl bridged bis-chromone 10 with an excellent yield. Using

same reaction condition for reduction of 14 (Pd/C, cyclohexene) gave only ethylene bridged
compound 11.
The in vitro anti-proliferative activities of compounds 8a - k, 9 - 11 were measured against
human cancer cell lines [prostate (PC-3), lung (NCI-H23), breast (MDA-MB-231), colon
(HCT-15), stomach (NUGC-3) and renal (ACHN)] using XTT assay21 as summarized with
IC50 values in Table 1.
In the preliminary investigation, simple bis-chromone derivative 8a (ACHN IC50 = 0.51),
(HCT-15 IC50 = >10), (MDA-MB-231 IC50 = >10), (NCI-H23 IC50 = >1.40), (NUGC-3 IC50 =
7.77) and (PC-3 IC50 = 7.40) without any substitution interestingly showed sub micro-molar
range activity. Thus the structure activity relationship of anticancer activity of bis-chromone
analogs was further studied. We initially planned to study the effect of substituents’ at
position 5 of ring A of bis-chromone scaffold (Table 1). Hydroxyl analog 8b showed similar
potency to those of 8a. Compound 8c (ACHN IC50 = 0.34), (HCT-15 IC50 = 3.43), (MDA-
MB-231 IC50 = >10), (NCI-H23 IC50 = >1.93), (NUGC-3 IC50 = 3.19) and (PC-3 IC50 = 3.49)
substituted with electron donating methoxy group showed nearly two fold better activities.
Interestingly, compounds 8a - c showed potent anti-cancer activity against ACHN, NCl-H23,
NUGC-3 and PC-3 cell lines. Increasing size of alkoxy group with isobutoxy (8d) and
isopentyloxy (8e) reduced the activity. Both analogs only exhibited good activity against
ACHN and PC-3 cell lines. However, the flat benzyloxy analog 8f showed good activity not
only against ACHN, PC-3 but also against NUGC-3 cell lines. These results gave the
impression that bulkiness of substituent at position 5 of ring A of bis-chromone scaffold can
be one of the factors for good activity. Therefore, the bulky cyclohexylmethoxy group (8g) at
the same position was further introduced and this analog displayed the best activity almost
against all cell lines. The above results confirmed our view point that increasing

hydrophobicity or bulkiness at this position is critical for the potent growth inhibitory activity
against cancer cell lines.
In the next set of experiments, hydrophobic electron donating groups (CH3, OCH3), electron
withdrawing group (Cl) and hydrogen bonding group (OH) at ring D of bis-chromones were
introduced to study their effect on anti-cancer activity (Table 1). Interestingly, Substitution
with electron donating groups as shown in 8h (CH3; CLogP: 6.2) and 8i (OCH3; CLogP: 5.8)
exhibited potent anti-proliferative activity whereas the introduction of electron withdrawing
group on 8j (Cl; CLogP: 6.4) led to suppress activity. The presence of hydrogen bonding
group on 8k (OH; CLogP: 5.4) resulted (ACHN IC50 = 2.10), (HCT-15 IC50 = 2.11), (MDA-
MB-231 IC50 = 3.22), (NCI-H23 IC50 = >2.15), (NUGC-3 IC50 = 3.22) and (PC-3 IC50 = 4.25)
in good anti-cancer activity against all cancer cell lines. Saturation of the double bond of one
of the chromone ring as shown in compound 9 reduced its activity compared to 8g. To get
more insight in the SAR of bis-chromones, methylene linker between chromenones in bis-
chromone analog were increased to vinyl (10) or ethylene group (11). These analogs did not
show any anti-cancer activity. The overall SAR study is represented in Fig.2.
To conclude, a novel family of bis-chromone derivatives were designed, synthesized and
evaluated using XTT assay for growth inhibition against various human cancer cells. Among
them compounds 3-((5-(cyclohexylmethoxy)-4-oxo-4H-chromen-3-yl)methyl)-7-methoxy-
4H-chromen-4-one (8i) and 3-((5-(cyclohexylmethoxy)-4-oxo-4H-chromen-3-yl)methyl)-7-
hydroxy-4H-chromen-4-one (8k) displayed the most potent anti-cancer activity. According to
the SAR studies, the activity increases by increasing the bulkiness at position 5 of ring A and
also by the introduction of electron donating group (CH3 and OCH3) and hydrogen bonding
group (OH) at ring D. However, upon saturation of bis-chromones of 8 to 9 the activity
decreased which indicates the critical importance of planar two chromenones of these bis-
chromones for their the anti-proliferative activity. Moreover, increasing the chain length in-

between the chromone rings (n = 2) abolished the activity completely. It concludes that bis-
chromone scaffold might serve as a lead molecule for finding a novel family of anti-cancer
agents.
Acknowledgment
This research was supported by Basic Science Research Program through the National Resear
ch Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology
(Grant number 2011-0014889) and by a Grant from KRIBB Research Initiative Program.
Supplementary data
The detailed experimental part of this article can be found in the Supplementary data.
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Figures captions
Fig. 1. Synthetic anti-cancer chromones
Fig.2. Graphical representation of SAR design.
Table 1. In vitro anti-proliferative activity of bis-chromones 8a-k, 9, 10 and 11.
Scheme 1: Synthesis of bis-chromones 8a-b, 8d-f, 8h-k, 9
Reagents and conditions;
a) Methylene chloride, rt.; b) Pd/C, Cyclohexene, THF/ MeOH, reflux; c) BF3/Et2O, MsCl,
DMF, 50 oC.
Scheme 2: Synthesis of Bis-chromones 8c and 8f
Reagents and conditions;
a) BF3/Et2O, MsCl, DMF, 50 oC; b) NaH, CH3I, THF, rt. ; c) BnBr, K2CO3,Acetone, reflux.
Scheme 3: Synthesis of Bis-chromones 10 and 11
Reagents;
a) Aluminum oxide, 2-Propanol, reflux; b) CBr4/PPh3 c) PPh3, Methylene Chloride, reflux;
d) Methylene chloride, rt.; e) Pd/C, Cyclohexene, THF, reflux.

Fig. 1

Spacer n=1 is ideal
H bonding
Acceptor required
Hydrophobic alkoxy;
Improved activity
Double bond;
Essential for activity
Fig. 2

Table 1
IC50(µM)a)
MDA-
Comp.
R
R1
ACHN
HCT15
NCI-H23 NUGC-3
PC-3
No.
MB231
H
H
H
H
H
H
H
H
0.51  0.05
0.74  0.07
>10
>10
>10
1.40  0.09 7.77  0.10 7.40  0.57
7.55  0.35 3.27  0.55 5.95  0.24
1.93  0.07 3.19  0.14 3.49  0.07
8a
8b
8c
8d
8e
8f
OH
>10
>10
>10
>10
>10
OMe
IBO
0.34  0.04 3.43  0.23
1.46  0.05
3.96  0.24
3.99  0.14
>10
>10
>10
>10
>10
>10
>10
>10
1.72  0.01
IPO
>10
BZO
CHM
CHM
CHM
CHM
CHM
7.22  0.18 4.83  0.34
4.23  0.09 4.24  0.17 4.11  .22 4.13  0.31 3.53  0.10 4.51  0.14
8g
8h
8i
CH3 2.25  0.11 2.22  0.07 3.320.19 3.22  0.20 2.15  0.17 2.44  0.21
OMe 1.35  0.05 1.45  0.08 1.2  0.10 1.32  0.06 1.44  0.07 1.12  0.05
Cl
>10
>10
>10
>10
>10
>10
8j
OH 2.10  0.15 2.11  0.11 3.220.18 2.150.07
3.220.10
4.250.13
8k
CHM
H
8.20  0.74 8.360.94
9.110.88 9.490.54
8.420.23
>10
9.380.77
>10
9
H
H
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
10
11
>10
>10
Adriamycin
0.260.02
0.430.02
0.600.03 0.490.01
0.380.03
0.50.02
a) IC50 values are taken as a mean from 3 experiments. Mean  SE
IBO = Isobutoxy , IPO = Isopentyloxy, BZO = Benzyloxy, CHM = Cyclohexyl methoxy,

Scheme 1

Scheme 2

Scheme 3

Synthesis and SAR Studies of Bis-Chromenone Derivatives for Anti-proliferative
Activity against Human Cancer Cells
Eeda Venkateswararaoa, Vinay K. Sharmaa, Manoj Manickama, Jieun Yunb, Sang-Hun Junga*
aCollege of Pharmacy and Institute of Drug Research and Development, Chungnam
National University, Daejeon 305-764, Korea
bBio-evaluation Center, KRIBB, Cheongwon-gun chungcheongbuk-do 363-883, Korea
Graphical abstract
Spacer n=1 is ideal
H bonding
Acceptor required
Hydrophobic alkoxy;
Improved activity
Double bond;
Essential for activity
8i ( R= Cyclohexylmethoxy, R1= OMe ): IC50 values (M, cancer cell line) = 1.35(ACHN),
1.45 (HCT-15), 1.21 (MDA-MB-231), 1.32 (NCI-H23), 1.44 (NUGC-3), 1.12 (PC-3).