New azafluorenones with cytotoxic and carbonic anhydrase inhibitory properties: 2-Aryl-4-(4-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-ones

Article abstract of DOI:10.1016/j.bioorg.2018.09.013

New azafluorenones, 2-aryl-4-(4-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-ones, were prepared to evaluate their cytotoxic/anticancer properties, also their inhibitory effects on hCA I and II isoenzymes. Aryl part was changed as [phenyl (H1), 4-methylphenyl

Full text of DOI:10.1016/j.bioorg.2018.09.013

Accepted Manuscript
New Azafluorenones with Cytotoxic and Carbonic Anhydrase Inhibitory Prop-
erties: 2-Aryl-4-(4-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-ones
Mehtap Tugrak, Halise Inci Gul, Hiroshi Sakagami, Ilhami Gulcin, Claudiu T
Supuran
PII:
S0045-2068(18)30626-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.013
YBIOO 2510
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 June 2018
31 August 2018
7 September 2018
Please cite this article as: M. Tugrak, H. Inci Gul, H. Sakagami, I. Gulcin, C.T. Supuran, New Azafluorenones with
Cytotoxic and Carbonic Anhydrase Inhibitory Properties: 2-Aryl-4-(4-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-
ones, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.09.013
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New Azafluorenones with Cytotoxic and Carbonic Anhydrase Inhibitory Properties:
2-Aryl-4-(4-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-ones
Mehtap Tugraka, Halise Inci Gula,*, Hiroshi Sakagamib, Ilhami Gulcinc, Claudiu T Supurand
b
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey; Division
c
of Pharmacology, Meikai University School of Dentistry, Sakado, Saitama, Japan; Department of Chemistry,
Faculty of Science, Ataturk University, Erzurum, Turkey; dNeurofarba Department, Sezione di Scienza
Farmaceutiche e Nutraceutiche, Universita egli Studi di Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino
(Florence), Italy
*Correspondence: Prof. Dr. Halise Inci Gul, Ataturk University, Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, 25240, Erzurum, Turkey.
Tel: +90 442 231 5219
Fax: +90 442 231 5201
E-mail: incigul1967@yahoo.com
1

Abstract
New azafluorenones, 2-aryl-4-(4-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-ones, were
prepared to evaluate their cytotoxic/anticancer properties, also their inhibitory effects on hCA I
and II isoenzymes. Aryl part was changed as [phenyl (H1), 4-methylphenyl (H2), 4-
methoxyphenyl (H3), 4-fluorophenyl (H4), 4-bromophenyl (H5), 4-chlorophenyl (H6), 3-
hydroxyphenyl (H7), and 4-hydroxyphenyl (H8)]. The structure of the synthesized compounds
was characterized by 1H-NMR, 13C-NMR and HRMS spectra.
Cytotoxicity results of the series pointed out that the compounds H6 (PSE: 28.0) and H5 (PSE:
27.3), with the highest potency selectivity expression (PSE) value, can be considered as leader
compounds of the study in designing novel anticancer agents. Additionaly, all azafluorenones
synthesized showed a good inhibition profile towards hCA I and II isoenzymes in the range of
54.14-73.72ꢀnM and 67.28-76.15ꢀnM, respectively.
The compounds H5 and H6 can be considered for further designs with their cytotoxic and CA
inhibitory profiles.
Keywords: Azafluorenone, Phenol, Cytotoxicity, Carbonic Anhydrase
2

1. Introduction
Cancer is a group of diseases characterized by uncontrolled growth and division of cells which
are able to invade other tissues and organs by spreading to other parts of the body through blood
and lymph vessels. Despite the advances in the field of anticancer drug discovery, 14.1 million
new cases of cancer were diagnosed worldwide in 2012 with 8.2 million deaths. Among the
limitations associated with the strategies currently available for cancer treatment, the
development of multidrug resistance stands as the major cause of failure as far as chemotherapy
is concerned [1].
Phenolic compounds have important biological and pharmacological properties such as
antioxidant, antimicrobial, cytotoxic/anticancer, carbonic anhydrase inhibitory, antiobesity, and
antidiabetic actions [2-14].
The carbonic anhydrases (CAs) are the metalloenzymes containing zinc ions (Zn2+) and they
−
catalyse the reversible hydration of CO2 in two-step reaction to yield bicarbonate (HCO3 ) ion
and proton (H+) [15, 16].
CA
HCO-3 + H+
H2CO3
CO2 + H2O
CAs are involved in various biochemical and metabolic processes such as gluconeogenesis,
lipogenesis, and ureagenesis [15]. Inhibition of CAs has pharmacologic applications in the field
of antiglaucoma, anticonvulsant, anticancer, and anti-infective agents [15].
The CA inhibitory effects of some natural products and their derivatives have been recently
investigated and reported that natural compounds carrying phenol or polyphenol moiety had
carbonic anhydrase inhibitory activity at micromolar concentrations [17]. The simple phenol
molecule was reported to act as an inhibitor of the zinc enzyme CA in a different mechanism of
action [18]. Indeed, sulfonamides which are the very well known inhibitors of CA coordinate to
3

the metal ion from the active site of the enzyme; phenols and derivatives which are less common
inhibitor of CA from SO2NH2 anchor to the water molecule/hydroxide ion coordinated to the
metal ion [19, 20]. In addition, some synthetic compounds having the phenol function showed
inhibitory potential on several hCA isoenzymes [3, 4, 13, 14, 21].
The 4-azafluorenone which is an alkaloid from Annonaceae species having the chemical
structure of 5H-indeno[1,2-b]pyridin-5-one comprises a small but biologically intriguing group
of alkaloids [22-24]. In general, they are rigid compounds, as compared to flexible structures,
have little conformational entropy and can be more efficiently fitted into the active site of several
enzymes [25]. They have the ability to intercalate into the enzyme–DNA complex [26].
Indenopyridines were reported with several biological activities such as anticancer, anti-
inflammatory and topoisomerase II inhibitory activities [27-29].
In this study, it was aimed to synthesize new azafluorenone derivatives, 4-(4-hydroxyphenyl)-2-
substitutedphenyl-5H-indeno[1,2-b]pyridin-5-one, to evaluate their cytotoxic/anticancer
properties. Substituent on phenyl ring was considered as (H1), (H2), (H3), (H4), (H5), (H6),
(H7), and (H8). It was also planned to investigate their inhibitory effects on hCA I and II
isoenzymes to find out new drug candidate/s for further studies.
2. Result and Discussion
2.1. Chemistry
Synthesis of H1-H8 were carried out successfully according to procedure described in detail in
the Material and Methods section. The compound synthesized were purified by crystallization
from ethanol and the compounds were obtained with the yield of 6.1%-41.1%. The chemical
1
13
structures of the final compounds were confirmed by H NMR, C NMR, and HRMS and the
data were provided in the Material and Methods section.
2.2. Cytotoxicity
4

The synthesis of eight new azafluorenone compounds, 4-(4-hydroxyphenyl)-2-substitutedphenyl-
5H-indeno[1,2-b]pyridin-5-one, H1-H8 were reported. Compound H, 2-(4-
Hydroxybenzylidene)-2H-indene-1,3-dione, was used as the starting compound for the synthesis
of H1-H8. Synthetic pathway for H1-H8 was shown in Scheme 1. The chemical structures were
1
elucidated by H NMR, 13C NMR, and HRMS spectra. Cytotoxic activities of the compounds
were tested towards human oral squamous cell carcinoma cell lines (Ca9-22, HSC-2, HSC-3,
HSC-4) and human oral normal mesenchymal cells [gingival fibroblast (HGF), pulp cell (HPC)
and periodontal ligament fibroblast (HPLF)]. The cytotoxicity results are presented at Table 1.
The first question to be addressed was whether the compounds synthesized are cytotoxic. The
CC50 values of the compounds towards cancer cell lines presented at Table 1. They displayed
cytotoxic activity at low μM concentrations suggesting that they have antineoplastic property.
The cytotoxicity of the compounds H1-H8 were compared with reference anticancer drug 5-
Fluorouracil (5-FU) and melphalan. Most of the compounds were more cytotoxic than the
reference compounds 5-FU [H1 (3.4 times), H2 (1.5 times), H3 (1.2 times), H5 (2.2 times), H6
(2.2 times), H7 (1.5 times), H8 (1.2 times) towards Ca9-22; the compunds H1 (4.6 times), H2 ve
H3 (1.2 times), H5 (2.5 times), H6 (1.6 times), H7 (1.7 times), H8 (1.4 times) towards HSC-2;
the compunds H1 (1.6 times), H4 (2.7 times), H5 (1.7 times), H6 (2.1 times), H7 (2 times), H8
(1.3 times) towards HSC-3; H1 (4.3 times), H5 (1.6 times), H6 (1.5 times), H7 (1.6 times), H8
(1.4 times) towards HSC-4] and/or melphalane [H1 (2.6 times), H2 (1.2 times), H5 and H6 (1.7
times), H7 (1.2 times) towards Ca9-22; the compunds H1 (3 times), H5 (1.6 times), H6 (1
times), H7 (1.1 times) towards HSC-2; the compunds H1 (3.9 times), H5 (1.5 times), H6 (1.4
times), H7 (1.5 times), H8 (1.2 times) towards HSC-4] .
The cytotoxicities of the azafluorenone type compounds (H1-H8) were always higher than
chalcone analogue compound H from which they were derived.
5

Since tumour cells were surrounded by normal cells, it is important for a compound to be a
tumour specific cytotoxin. Selectivity index (SI) figures were generated which are the quotients
of the average CC50 values of the nonmalignant cells and the CC50 figure of a compound towards
a specific cell line. SI value is a marker of the selectivity of a compound for a spesific cell line.
The compound having a SI value over the value of 1 can be considered as tumour selective
cytotoxic agent. The SI values of the compounds were presented at Table 1. According to the
results at Table 1, many azafluorenone compounds seem tumour specific cytotoxins with the SI
value higher than the value of 1 [H5 (1.5 times), H6 (1.8 times), H7 (1.3 times), H8 (1.5 times)
towards Ca9-22; the compouds H (3.2 times), H1 (1.9 times), H2 (1.8 times), H3 (2.3 times), H5
(3.6 times), H6 (2.8 times), H7 (3.2 times), H8 (4.0 times) towards HSC-2; the compounds H3
(1.2 times), H2 ve H4 (1.1 times), H5 (2.1 times), H6 (2.9 times), H7 and H8 (3.0 times)
towards HSC-3; the compound H8 (3.8 times) towards HSC-4].
The leader compound should possess both remarkable cytotoxic potential and selective
cytotoxicity towards tumour cells. Potency selectivity expression (PSE) was devised which is
the product of the reciprocal of the average CC50 values towards Ca9-22, HSC-2, HSC-3 and
HSC-4 cells (a measure of potency) and the average SI figures towards these cell lines (a
determination of tumour-selectivity) expressed as a percentage. The PSE value of the compounds
were presented at Table1. The PSE values of the compounds H1-H8 were lower than the 5FU
and Melphalan’s and higher than the starting compound H which is a chalcone derivative from
which H1-H8 were derived. These data suggest that preparation of azafluorenones from
chalcone analogue H has been a useful chemical modification in terms of cytotoxicity and
tumour selectivity.
When PSE values were considered, halogen bearing compounds H6 (with chlorine) and H5
(with bromine) had PSE values around 27-28 while fluorine bearing compound H4 had the value
of 2.3 as PSE. It seems that the PSE values of H5 and H6 were 10 times higher than H4’s.
6

The position of hydroxy group on the phenyl ring whether it is at meta (H7) or para (H8)
position did not change so much the PSE values of the compounds. Similarly, the type of
substituents, whether it is electron donating or attracting, did not affect the PSE values of the
compounds in the compounds H2 (PSE: 7.6) and H3 (PSE:9.0). But, the replacement of a
methoxy (H3) group by a hydroxy (H8) group in which oxygen has less sterical hidrance led to
increase in PSE value about 2.5 times in H8. So, substition with chlorine (H6) or bromine (H5)
on phenyl ring, which are halogene, was a useful modification in increasing PSE values
comparing with H1. However, when another halogene fluorine was considered as a substituent
on phenyl ring (H4), its substitution was not useful in increasing PSE value. Contrary to the
expectation, it decreased PSE value comparing with H substition on phenyl (in H1) although
hydrogen and fluorine have similar size.
So, the cytotoxicity of the compounds may result from the physical properties of the compounds,
which direct their kinetics and metabolism in different tissues or differences in the cell lines
used, which may have different metabolic characteristics.
2.3. CA Inhibition
CA inhibitory effects of the compounds were tested and the results were presented at Table 2. As
shown in Table 2, IC50 values were in the range of 54.1–87.7 nM towards hCA I while they were
in the range of 10.7–76.153 nM towards hCA II. The IC50 values of the reference compound
AZA towards hCA I and hCA II were 197.3 nM and 115.5 nM, respectively. All compounds had
lower IC50 value than AZA toward hCA I and hCA II. When IC50 values of the compounds were
considered, all compounds had similar selectivity on both hCA I and hCA II isoenzymes. H3
was 1.3 times more selective towards hCA I than hCA II. According to IC50 values of the
compounds, methoxy-bearing compound H3 and hydroxy-bearing compound H7 were the most
effective compounds towards hCA I.
7

When Ki values of the compounds were considered, Ki values of the compounds were in the
range of 4.6577±1.022–8.4757±2.554 nM towards hCA I while Ki values were 3.5061±0.911–
10.533±3.197 nM towards hCA II. The Ki values of reference compound AZA were
183.390±19.71 nM and 104.60±27.60 nM towards hCA I and hCA II, respectively.
When Ki values of the compounds were considered, azafluorenone compounds had very low Ki
values. All these compounds can be considered as leader of the series towards hCA I. On the
other hand H1 (3.5280±1.135), H5 (3.8242±0.874) and H7 (3.5061±0.911) which had the
similar and lowest Ki values in series can be considered leader compounds of the series towards
hCA II.
When Ki values of the compounds were considered, H1, H3, H4, H5, and H7 were slightly more
selective towards hCA II isoenzyme than hCA I since Ki values of the compounds towards hCA
II were lower than Ki values of the compounds towards hCA I.
3. Conclusion
Azafluorenone compounds H1-H8 were reported for the first time with detailed spectral
analysis. Cytotoxicity experiments pointed out that compound H6 [4-(4-hydroxyphenyl)-2-(4-
chlorophenyl)-5H-indeno[1,2-b]pyridin-5-one]
and
H5
[4-(4-hydroxyphenyl)-2-(4-
bromophenyl)-5H-indeno[1,2-b]pyridin-5-one] were leader compounds of the series with the
highest PSE values. The compounds H5 and H6 can be considered for further designs with their
cytotoxic and CA inhibitory profiles.
4. Material and Methods
4.1. Chemistry
All reactions were routinely checked by thin-layer chromatography (TLC) on silica gel 60 F254
(Merck Art 5715) and visualized by UV light or iodine. Melting points (mp) were determined in
capillary tubes using an Electrothermal 9100 (IA9100, U.K.) and are uncorrected.
Reagents and solvents were purchased from common commercial suppliers [1,3-indandione
(Alfa Aesar), 4-chloroacetophenone, 4-methylacetophenone, 4-hydroxyacetophenone, 3-
8

hydroxyacetophenone (Fluka), acetophenone, 4-hydroxybenzaldehyde (Merck), 4-methoxy
acetophenone, 4-fluoro acetophenone, 4-bromo acetophenone (Aldrich), ammonnium acetate
1
13
(Riedel-de Haen)] and were used as such. H NMR and C NMR spectra were recorded at 400
MHz (Danbury, U.S.A.) in dimethyl sulfoxide (DMSO). Chemical shifts (δ) are given in ppm,
and the spectral data were consistent with the assigned structures. Reactions were carried out in a
CEM Discover Microwave Synthesis System, 908010 (Matthews, NC)
4.1.1. 2-(4-Hydroxybenzylidene)-2H-indene-1,3-dione (H), Scheme 1
An aqueous solution of sodium hydroxide (10% w/v, 10 mL) was added into the solution of the
4-hydroxy benzaldehyde (0.02 mol) and 1,3-indandione (0.02 mol) in ethanol (6 mL, Scheme 1).
The reaction mixture was stirred at room temperature overnight and poured into water (100 mL)
[3, 5, 7-9, 11, 30]. The content was neutralized with hydrochloric acid (10% w/v) to give a
yellow solid. It was crystallized from EtOH to yield H (43%): m.p: 238-239⁰C, lit m.p. 238-
1
239⁰C; H NMR (400 MHz, [D6]DMSO): δ 8.52 (d, 2H, Ar-H, J = 8.8 Hz), 7.91-7.90 (m, 4H,
Ar-H), 7.74 (s, 1H, vinylic-H), 6.92 ppm (d, 2H, Ar-H, J = 8.8 Hz); 13C NMR (100 MHz,
[D6]DMSO): δ 190.7, 189.7, 164.1, 146.9, 142.3, 139.9, 138.3, 136.3, 136.1, 125.8, 125.3, 123.5,
123.4, 116.7 ppm; HRMS (ESI-MS): m/z [M+H]+ calcd for C16H9O3: 249.0552, found:
249.0546.
4.1.2. General method for the preparation of H1-H8, 2-aryl-4-(4-hydroxyphenyl)-5H-
indeno[1,2-b]pyridin-5-ones, Scheme 1
A mixture of chalcone analogue H, a suitable acetophenone having a substituent at 4 or 3
position of phenyl ring 4-H (H1), 4-CH3 (H2), 4-OCH3 (H3), 4-F (H4), 4-Br (H5), 4-Cl (H6), 3-
OH (H7), 4-OH (H8) and anhydrous ammonium acetate in dimethylformamide was heated in a
microwave oven (13.8 barr, 120 0C, 200 W) for 30-60 min for the synthesis of H1-H8 (Scheme
1) (30 min (H1, H5), 40 min (H7, H8), 45 min (H3, H4, H6), 60 min (H2)). Reactions were
9

monitored by TLC (chloroform/methanol 4.8:0.2). Whenever the reaction completed, 50 mL of
water was added. The solid obtained was filtered, dried and crystallized from ethanol.
4.1.2.1. 4-(4-Hydroxyphenyl)-2-phenyl-5H-indeno[1,2-b]pyridin-5-one (H1), Scheme 1
1
Yield (6.1%): m.p: 298-299⁰C; H NMR (400 MHz, [D6]DMSO): δ 10.02 (s, 1H, 4-phenyl 4-
OH), 8.25 (dd, 2H, Ar-H, J = 8.1, 2.2 Hz), 7.91 (d, 1H, Ar-H, J = 7.3 Hz), 7.74 (s, 1H,
13
indenopiridine H-3), 7.69-7.49 (m, 8H, Ar-H), 6.87 ppm (d, 2H, Ar-H, J = 8.4 Hz); C NMR
(100 MHz, [D6]DMSO): δ 191.1, 166.4, 160.4, 159.8, 149.9, 142.7, 138.2, 135.9, 135.6, 132.1,
131.9, 130.9, 129.5, 128.0, 126.0, 124.1, 122.4, 121.2, 121.1, 115.6 ppm; HRMS (ESI-MS): m/z
[M+H]+ calcd for C24H16NO2: 350.1181, found: 350.1172.
4.1.2.2. 4-(4-Hydroxyphenyl)-2-p-tolyl-5H-indeno[1,2-b]pyridin-5-one (H2), Scheme 1
1
Yield (8.7%): m.p: 273-276⁰C; H NMR (400 MHz, [D6]DMSO): δ 9.95 (s, 1H, 4-phenyl 4-
OH), 8.18 (d, 2H, Ar-H, J = 8.1 Hz), 7.91 (d, 1H, Ar-H, J = 7.3 Hz), 7.72 (s, 1H, indenopiridine
H-3), 7.69-7.62 (m, 5H, Ar-H), 7.33 (d, 2H, Ar-H, J = 8.4 Hz), 6.86 (d, 2H, Ar-H, J = 8.4 Hz),
2.36 ppm (s, 3H, 2-phenyl-CH3-); 13C NMR (100 MHz, [D6]DMSO): δ 191.1, 166.4, 160.3,
159.8, 149.8, 142.8, 140.8, 135.9, 135.6, 135.5, 132.0, 131.9, 130.2, 127.9, 126.1, 124.1, 122.2,
121.1, 120.7, 115.5, 21.7 ppm; HRMS (ESI-MS): m/z [M+H]+ calcd for C25H16NO2: 362.1181,
found: 362.1176.
4.1.2.3. 4-(4-Hydroxyphenyl)-2-(4-methoxyphenyl)-5H-indeno[1,2-b]pyridin-5-one (H3),
Scheme 1
1
Yield (19.7%): m.p: 282-284⁰C; H NMR (400 MHz, [D6]DMSO): δ 9.90 (s, 1H, 4-phenyl 4-
OH), 8.27 (d, 2H, Ar-H, J = 8.8 Hz), 7.92 (d, 1H, Ar-H, J = 7.3 Hz), 7.69 (s, 1H, indenopiridine
H-3), 7.65-7.62 (m, 5H, Ar-H), 7.07 (d, 2H, Ar-H, J = 8.8 Hz), 6.86 (d, 2H, Ar-H, J = 8.8 Hz),
3.83 ppm (s, 3H, 2-phenyl-OCH3); 13C NMR (100 MHz, [D6]DMSO): δ 191.1, 166.5, 161.8,
160.2, 159.7, 149.8, 142.8, 135.8, 135.7, 132.0, 131.8, 130.6, 129.7, 126.2, 124.0, 121.7, 121.2,
10

120.0, 115.5, 114.9, 56.0 ppm; HRMS (ESI-MS): m/z [M+H]+ calcd for C25H18NO3: 380.1287,
found: 380.1272.
4.1.2.4.
4-(4-Hydroxyphenyl)-2-(4-fluorophenyl)-5H-indeno[1,2-b]pyridin-5-one
(H4),
Scheme 1
1
Yield (28.8%): m.p: 314-317⁰C; H NMR (400 MHz, [D6]DMSO): δ 9.97 (s, 1H, 4-phenyl 4-
OH), 8.37-8.33 (m, 2H, Ar-H), 7.92 (d, 1H, Ar-H, J = 7.3 Hz), 7.76 (s, 1H, indenopiridine H-3),
7.73-7.50 (m, 5H, Ar-H), 7.35 (t, 2H, Ar-H, J = 8.8 Hz), 6.86 ppm (d, 2H, Ar-H, J = 8.8 Hz); 13C
NMR (100 MHz, [D6]DMSO): δ 191.0, 166.4, 159.8, 159.2, 149.9, 142.7, 135.9, 135.6, 132.1,
131.9, 130.5, 130.4, 125.9, 124.1, 122.3, 121.3, 121.0, 116.5, 116.3, 115.5 ppm; HRMS (ESI-
MS): m/z [M+H]+ calcd for C24H15NO2F: 368.1087, found: 368.1074.
4.1.2.5. 4-(4-Hydroxyphenyl)-2-(4-bromophenyl)-5H-indeno[1,2-b]pyridin-5-one (H5),
Scheme 1
1
Yield (9.7%): m.p: 303-305⁰C; H NMR (400 MHz, [D6]DMSO): δ 9.97 (s, 1H, 4-phenyl 4-
OH), 8.24 (d, 2H, Ar-H, J = 8.4 Hz), 7.92 (d, 1H, Ar-H, J = 7.3 Hz), 7.79 (s, 1H, indenopiridine
H-3), 7.73-7.53 (m, 7H, Ar-H), 6.87 ppm (d, 2H, Ar-H, J = 8.4 Hz); 13C NMR (100 MHz,
[D6]DMSO): δ 190.9, 166.4, 159.8, 159.0, 149.9, 142.7, 137.4, 135.9, 135.6, 132.5, 132.2, 131.9,
131.6, 130.0, 125.9, 124.7, 124.2, 122.7, 121.3, 115.5 ppm; HRMS (ESI-MS): m/z [M+H]+ calcd
for C24H15NO2Br: 428.0286, found: 428.0270.
4.1.2.6.
4-(4-Hydroxyphenyl)-2-(4-chlorophenyl)-5H-indeno[1,2-b]pyridin-5-one
(H6),
Scheme 1
1
Yield (20.1%): m.p: 308-310⁰C; H NMR (400 MHz, [D6]DMSO): δ 9.97 (s, 1H, 4-phenyl 4-
OH), 8.31 (d, 2H, Ar-H, J = 8.1 Hz), 7.92 (d, 1H, Ar-H, J = 7.3 Hz), 7.78 (s, 1H, indenopiridine
H-3), 7.72-7.51 (m, 7H, Ar-H), 6.86 ppm (d, 2H, Ar-H, J = 8.4 Hz); 13C NMR (100 MHz,
[D6]DMSO): δ 190.9, 166.4, 159.9, 158.9, 149.9, 142.7, 137.0, 135.9, 135.8, 135.6, 132.2, 131.6,
11

131.9, 129.8, 129.5, 125.9, 124.2, 122.6, 121.3, 115.5 ppm; HRMS (ESI-MS): m/z [M+H]+ calcd
for C24H15NO2Cl: 384.0791, found: 384.0773.
4.1.2.7. 4-(4-Hydroxyphenyl)-2-(3-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-one (H7),
Scheme 1
1
Yield (41.1%): m.p: 315-316⁰C; H NMR (400 MHz, [D6]DMSO): δ 9.90 (s, 1H, 4-phenyl 4-
OH), 9.64 (s, 1H, 2-phenyl 3-OH), 7.88 (d, 1H, Ar-H, J = 7.3 Hz), 7.17-7.50 (m, 8H, Ar-H,
indenopiridine H-3), 7.31 (t, 1H, Ar-H, J = 7.7Hz), 6.90 (d, 2H, Ar-H, J = 8.8 Hz), 6.86 ppm (d,
13
1H, Ar-H, J = 8.4 Hz); C NMR (100 MHz, [D6]DMSO): δ 191.1, 166.4, 160.5, 159.7, 158.4,
149.8, 142.7, 139.6, 135.9, 135.6, 133.1, 132.1, 131.8, 130.6, 126.1, 124.1, 122.4, 121.2, 118.9,
117.9, 115.6, 114.6 ppm; HRMS (ESI-MS): m/z [M+H]+ calcd for C24H16NO3: 366.1130, found:
366.1122.
4.1.2.8. 2,4-Bis(4-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-one (H8), Scheme 1
1
Yield (16.4%): m.p: 336-338⁰C: H NMR (400 MHz, [D6]DMSO): δ 10.03 (s, 1H, 4-phenyl 4-
OH), 9.93 (s, 1H, 2-phenyl 4-OH), 8.16 (d, 2H, Ar-H, J = 8.8 Hz), 7.89 (d, 1H, Ar-H, J = 7.3
Hz), 7.70-7.49 (m, 6H, Ar-H, indenopiridine H-3), 6.91-6.85 ppm (m, 4H, Ar-H); 13C NMR (100
MHz, [D6]DMSO): δ 191.1, 166.5, 160.5, 160.4, 159.7, 149.7, 142.8,135.9, 135.7, 131.9, 131.8,
129.8, 129.1, 126.3, 123.9, 121.4, 121.1, 119.6, 116.3, 115.5 ppm; HRMS (ESI-MS): m/z
[M+H]+ calcd for C24H16NO3: 366.1130, found: 366.1122.
4.2. Biological activity
4.2.1. Cytotoxicity evaluation
The cytotoxicities of the compounds H and H1-H8 were assayed towards human tumour cell
lines [gingival carcinoma (Ca9–22), oral squamous cell carcinoma (HSC-2, HSC-3, HSC- 4)]
and human normal oral cells [gingival fibroblasts (HGF), periodontal ligament fibroblasts
(HPLF) and pulp cells (HPC)] as described [3, 5-8, 31,32]. In brief, all cells were cultured in
DMEM supplemented with 10% fetal bovine serum (FBS). The following concentrations of the
12

compounds in dimethylsulfoxide (DMSO) were added to the medium and incubated at 37⁰ C for
48 h: H and H1-H8 (0.32, 1, 3.2, 10, 31.6, 100, 316 and 1000 mmol/L), melphalan (3.12, 6.25,
12.5, 25. 50, 100, 200 and 400 mmol/L) and 5-FU (7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000
mmol/L). The media that contained the same concentration of DMSO (0.0078, 0.156, 0.03125,
0.0625, 0.125, 0.25, 0.5 or 1%) were used as controls since DMSO above 0.25% is cytotoxic.
The viable cell numbers were determined by the MTT method. The CC50 values were determined
from dose-response curves.
4.2.2. Carbonic anhydrase enzyme assay
The Carbonic Anhydrase (CA) I and II isoenzymes were purified from fresh human blood
erythrocytes using by Sepharose-4B-LTyrosine-sulfanilamide affinity chromatography [9, 13,
14, 33-39]. This method contains the purification of CA isoenzymes via a single step described
previously. CA isoenzyme activity was determined spectrophotometricaly at 348 nm as
described by Verpoorte et al. [40]. According to this method the absorbance changes were
measured during the time of 3 min at 25°C as p-nitrophenylacetate (PNA) converted to 4-
nitrophenylate ion. Bradford method was used to quantify the amount of protein during the
purification steps [41]. Bovine serum albumin was used as standard protein [42]. After the
purification process of the CA isoenzymes, SDSpolyacrylamide gel electrophoresis (SDS–
PAGE) has been carried out. According to literature [9, 33-39] stacking and resolving gel
containing 3% and 10% acrylamide, and 0.1% SDS was used for running the process using a
Minigel system (Mini-PROTEAN system Casting stand, Catalog 1658050, Bio-Rad
Laboratories, Inc. China). The method used for visualization of protein has been explained in
detail in our previous studies [43]. According to this method, the gel was fixed then stained with
Coomassie Brilliant Blues R-250 later on the gel stained by using standard methods for detecting
protein bands that are belong to purified CA isoenzymes.
13

Declaration of Interest
The authors report no conflict of interest and are responsible for the contents and writing of the
paper.
Acknowledgements
This work was supported by Ataturk University Research Found, Turkey (Grand Number
2015/078) .The authors are thankful to Dr. Murat ŞÜKÜROĞLU (Gazi University, Turkey) for
HRMS spectra and Ataturk University, Faculty of Science, Department of Organic Chemistry,
Turkey for NMRs spectra.
Captions
Scheme 1. Synthesis of chalcone analogue H and azafluorenone derivatives H1-H8.
Table 1. Cytotoxicity of the compunds H, H1-H8.
Table 2. Human CA isoenzymes (hCA I and II) inhibition values by the compounds H-H8.
14

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17

Ar: C6H5 (H1), 4-CH3C6H4 (H2), 4-CH3OC6H4 (H3), 4-FC6H4 (H4), 4-BrC6H4 (H5), 4-ClC6H4 (H6), 3-HOC6H4 (H7), 4-HOC6H4 (H8)
Table 1. Cytotoxicity of the compunds H, and H1-H8
Tumour cell lines
Non-tumour cell lines
CC50 (μM)
CC50 (μM)
PSEx100
J/E
Compound Ca9-22
(A)
SI
HSC-2
(B)
182
2.8
SI
HSC-3
(C)
219
9.9
18
SI
HSC-4
(D)
182
3
19
15
21
7.9
8.4
7.9
SI
E
J
HGF HPLF HPC
I
(mean)
213±41
6.1±3.7
17±3.9
18±5.9
22±17
8.8±3.2
9.3±2.5
11±5.6
14±7.0
35.47
(F)
968
6.6
21
25
5.7
13
22
9.7
25
(G)
448
7.1
20
(H)
320
2.8
18
(mean)
579±343
5.5±2.4
20±1.5
25±3.0
6.4±0.7
19±5.3
23±15
269
8.5
19
2.1
0.6
1.1
3.2
1.9
1.8
2.6
0.6
1.1
1.2
1.1
2.1
2.9
3.0
3.0
3.2
1.8
1.1
1.7
0.3
2.4
2.7
3.0
3.8
2.22
2.8
1.2
1.3
1.6
0.5
2.4
2.6
2.6
3.1
1.3
19.7
7.6
9.0
2.3
27.3
28.0
23.6
22.1
15.7
127.7
348.8
H
H1
H2
11
24
1.0
11
2.3
21
28
22
H3
46
0.1
17
0.4
5.9
9.1
7.7
8
7.1
21
6.3
23
H4
13
1.5
5.2
3.6
H5
13
1.8
8.1
2.8
38
32
8.5
30
H6
19
1.3
7.5
3.2
24±12
36±14
81.99
H7
24
1.5
9.1
4.0
12
9.6
52
32
H8
43.39
22.6
29
1.22
6.5
˃34.5
28.19
8.5
13
2.58
17.3
˃77.0
34.51
5.6
16
1.96
26.2
˃62.5
30.42
11.9
13
2.01 121.78 72.58
51.4
Average
Melphalan
5-FU*
12.3 12.2±7.4 15.6 140.0 179.0 123.0 147.0±29.0
˃77.0 18±7.6
62.8 >1000 >1000 >1000 >1000
CC50 values refer to the concentrations of the compounds in micromoles which reduce the viable cell number by 50%. Selectivity index (SI) figures were generated which are quotients of the
average CC50 values of non-malignant cells and CC50 figure of a compound towards a specific cell line. A potency selectivity expression (PSE) was devised which is reciprical of the average CC50
value (a measure of potency) and the average SI figure (a determination of tumour selectivity) (Column J/ Column E x 100).
*5-FU (5-Fluorouracil) was also used as a reference drug. μM : micromolar
18

Ar: C6H5 (H1), 4-CH3C6H4 (H2), 4-CH3OC6H4 (H3), 4-FC6H4 (H4), 4-BrC6H4 (H5), 4-ClC6H4 (H6), 3-HOC6H4 (H7), 4-HOC6H4 (H8)
Table 2. Human CA isoenzymes (hCA I and II) inhibition values by the compounds H-H8
IC50 (nM)
KI (nM)
Compound
H
hCA I
87.721
61.875
70.020
58.728
68.613
59.230
61.327
54.140
73.723
197.304
r2
hCA II
10.661
67.281
71.443
74.516
73.723
59.741
67.281
68.613
76.153
115.50
r2
hCA I
hCA II
0.9738
0.9688
0.9292
0.9495
0.9646
0.9689
0.9791
0.9103
0.9399
0.9889
0.9277
0.9450
0.9792
0.9455
0.9626
0.9705
0.9725
0.9832
0.9880
0.9719
4.7987±1.282
6.8316±2.022
4.6577±1.022
7.2645±3.140
8.4757±2.554
5.1777±1.463
5.0125±1.258
7.5103±3.442
6.6933±1.386
183.390±19.71
10.533±3.197
3.5280±1.135
6.7563±1.535
5.6070±1.671
7.9153±2.016
3.8242±0.874
7.2065±1.740
3.5061±0.911
6.1730±1.155
104.60±27.60
H1
H2
H3
H4
H5
H6
H7
H8
AZA
AZA: Acetazolamide, reference compound used.
H: Starting compound for the synthesis of H1-H8, H1-H8: New azaflorenones synthesized
19

Ar: C6H5 (H1), 4-CH3C6H4 (H2), 4-CH3OC6H4 (H3), 4-FC6H4 (H4), 4-BrC6H4 (H5), 4-ClC6H4 (H6), 3-HOC6H4 (H7), 4-
HOC6H4 (H8)
Reagent and conditions: i) EtOH, aqueous solution of NaOH 10%, ii) CH3COONH4, 120°C, 200 Watt, DMF
Scheme 1. Synthesis of chalcone analogue H and azafluorenone derivatives H1-H8.
H: Starting compound for the synthesis of the H1-H8
20

21