Synthesis, biological evaluation and virtual screening of some acridone derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bmc.2020.115426

Eleven novel acridone derivatives were synthesized and evaluated for their anticancer activity against 60 human cancer cell lines. Five compounds 8b, 8d, 8g, 8h, and 8k displayed very good in vitro antiproliferative activities well over 95% of the panels.

Full text of DOI:10.1016/j.bmc.2020.115426

Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and virtual screening of some acridone
derivatives as potential anticancer agents
⁎
Abiodun S. Oyedele, Deanna N. Bogan, Cosmas O. Okoro
Department of Chemistry, Tennessee State University, 3500 John A. Merritt Blvd., Nashville, TN 37209-1561, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Eleven novel acridone derivatives were synthesized and evaluated for their anticancer activity against 60 human
cancer cell lines. Five compounds 8b, 8d, 8g, 8h, and 8k displayed very good in vitro antiproliferative activities
well over 95% of the panels. The most active compound is 8k (5, 7-dibromo-3-phenyl-3,4-dihydroacridin-1 (2H)-
one). In addition, 8k was the most sensitive agent in all 9 panels starting with prostate (0.075 µm), leukemia
Acridones
Anticancer activity
Amsacrine
1
,3-cyclohexanedione, 2-aminobenzaldehyde
(
(
0.116 µm), non-small cell lung cancer (0.164 µm), colon cancer (0.193 µm), CNS cancer (0.264 µm), melanoma
0.317 µm), renal cancer (0.403 µm), ovarian cancer (0.410 µm), and breast cancer (0.608 µm). Virtual
Imidazoacridinone
screening studies also revealed that nine of the eleven compounds formed good binding interaction with the
active site ATPase domain of human topoisomerase IIα (PDB: 1zxm). All nine derivatives exhibited binding
affinities that ranged in values from −8.5 to −7.9 kcal/mol, indicating that they could be catalytic inhibitors of
the nuclear enzyme, topoisomerase.
1
4
1
. Introduction
those of m-AMSA and doxorubicin. Also, natural product glyfoline 4
11
possesses anticancer activities.
Cancer is the second leading cause of death worldwide, accounting
Previously, our group reported the synthesis of a novel series of
phenyl-substituted -3-(trifluoromethyl)-3,4-dihydroacridin-1-(2H)-one
derivatives using Friedlander one pot multicomponent reaction
for over 18 million new cases and 9.8 million deaths according to 2018
1
–3
data.
Despite the huge investment in research studies that runs into
1
5
millions of dollars, to elucidate the precise mechanisms of the different
types of cancer, the disease remains an unmet medical need. Several
small molecules have progressed to clinical use having found to be
successful in delaying the progression of the disease. Unfortunately, the
patient eventually dies. Some of the approved drugs are both expensive
and have several side effects and ultimately become resistance via
mutations.
(MCR). The preliminary cytotoxicity evaluation of an identified 5, 7-
dibromo-3-(trifluoromethyl)-3,4-dihydroacridin-1-(2H)-one 5 as an ac-
tive lead, with GI50 that ranged in values from 0.13 to 26 μM, covering
1
6
a wide range of cancer cell lines. When the 3-CF group was replaced
3
by a 3-phenyl group, another more active compound containing the
5,7-dibromo derivative was identified (included in this communica-
tion). We envisioned that a new series containing one substituent at the
7-position could be equipotent or more potent than the lead compound
described above.
Several compounds based on tricyclic template (Fig. 1) have been
studied in the search for better anticancer agents. In this regard, the use
of natural and synthetic acridine/acridone compounds as antitumor
It has also been shown that the incorporation of fluorine into bio-
logically active molecules as monofluoro, difluoromethyl, or tri-
4
agents have been extensively reviewed. Several acridine/acridone
compounds are now in clinical trial. The above includes acronycine
fluoromethyl groups frequently leads to enhanced pharmacological
5
,6
7
8–10
17–19
1
,
imidazoacridinone (C-1311) 2, amsacrine (m-AMSA) 3,
and
properties,
due to high membrane permeability, increased hydro-
1
1
glyfoline 4.
phobic binding, and, metabolic stability. In protein-ligand complexes,
fluorine, particularly trifluoromethyl, can have profound impacts on
the binding affinity. This impact may be direct through fluorine inter-
action with the protein, or it may be indirect through modulation of the
For example, acronycine 1, a natural alkaloid isolated in 1948 from
1
2,13
the bark of the Australian Rutaceous tree
is among the earliest
acridone-based anticancer compounds. Imidazoacridinone 2 was dis-
covered in 1990s and was found to be an inhibitor of the nuclear en-
zyme topoisomerase II.12 The compound displayed activities similar to
2
0
polarity of other ligand groups that interfere with the protein.
Another example that demonstrates the effect of fluorine
⁎
Corresponding author.
E-mail address: cokoro@tnstate.edu (C.O. Okoro).
https://doi.org/10.1016/j.bmc.2020.115426
Received 12 December 2019; Received in revised form 7 February 2020; Accepted 9 March 2020
0968-0896/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Abiodun S. Oyedele, Deanna N. Bogan and Cosmas O. Okoro, Bioorganic & Medicinal Chemistry,
https://doi.org/10.1016/j.bmc.2020.115426

A.S. Oyedele, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Fig. 1. Structures of known and investigative acridine/acridone derivatives and acridone numbering system.
introduction is the replacement of the phenyl group of the isoserine side
group as building block for attaching the trifluoromethyl group to or-
1
7
chain of docetaxel with a CF
3
as reported by Ojima et al. The CF
3
-
ganic frameworks. Parallel reactions were accomplished using com-
mercially available 5-phenylcyclohexane-1,3-dione 6. In addition, two
derivatives 8i and 8j were studied at the five positions of the acridone
derivatives. Compounds 8a-8k were synthesized with good yields as
shown in (Table 1).
docetaxel showed 14-fold activity compared with docetaxel. The above
study showed that fluorine substitution has a strong effect on the con-
formational equilibrium of the isoserine side chain in a hydrophobic
2
1
medium.
Previously our group reported that 3-trifluoromethyl-5,7-di-
bromoacridone derivatives showed anticancer activity in NCI panel of
2.2. In silico molecular descriptor studies of the analogs 8a-8k
6
0 cancer cell line over a wide range. The corresponding 3-phenyl de-
rivative exhibited better activity. The structure activity studies of the
above series showed that incorporation of alkyl or phenyl group at the
As shown in Table 2, all compounds showed zero violations in ac-
22
cordance with Lipinski’s rule. The LogP values ranged from 3.30 to
1
6
9
-position abolished anticancer activity.
4
.70. Thus, we can predict that all the compounds will be able to pe-
23
Herein, we report the synthesis and in-vitro antiproliferative activ-
netrate the membrane in significant concentration. The molar re-
fractivity (MR) of the synthesized derivatives were determined to be in
ities of a new series of acridone derivatives containing electron-with-
drawing groups, Cl, Br, F at position 7, respectively. Additionally,
electron-donating methoxy groups were introduced at the 5 and 7 po-
sitions of the acridone core (Fig. 1).
3
the range of 69.60–98.66 cm /Mol for the overall polarity of the mo-
lecules. All the compounds have less than 10 numbers of hydrogen bond
acceptors (n-HBA) and zero number of hydrogen bond donors (n-HBD),
which are consistent with Lipinski’s rule of five. Additionally, a com-
putational study for the prediction of absorption, distribution, meta-
bolism, excretion and toxicity (ADMET) properties of the acridone
compounds were carried out by evaluation of topological polar surface
area (TPSA) and percent absorption (%ABS). TPSA is the surface be-
longing to polar atoms and is a descriptor that has been shown to
correlate with passive molecular transport through membranes, and
thus enables estimation of transport properties in the intestine and
2
. Results and discussion
2.1. Chemistry
The synthesis of the acridone derivatives were done as shown in
Scheme 1, by reacting substituted 2-amino benzaldehyde 7 with cyclic
5
-trifluoromethylcyclohexane-1, 3-dione 6 previously developed by our
Scheme 1. Synthesis of some acridone derivatives (8a-8k).
2

A.S. Oyedele, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Table 1
The panel consisted of nine subpanels of cell lines for leukemia, non-
small cell lung, colon, central nervous system, melanoma, ovarian,
renal, prostate and breast cancer. Growth inhibition was determined
from sulforhodamine B-derived color optical density measurements to
test compound or control vehicle before and after 48 h of exposure. The
single-dose data shows the percentage growth of the drug-treated cells
in relation to vehicle control (non-drug-treated cells) and the number of
cells “time zero.” The resulting growth percentage is then used to
measure both growth inhibition (ranging from 0 to 100) and lethality
Physical and Chemical Properties of 8a-8k.
Entry Compounds
R
1
R
2
Molecular
Weight
Melting
%Yield*
Point (°C)
(
value less than 0). Compounds indicating 60 percent or more growth
−5
1
2
3
4
5
6
7
8
9
1
1
8a
8b
8c
8d
8e
8f
CF
Ph
CF
Ph
CF
Ph
CF
Ph
CF
Ph
Ph
3
3
3
3
3
7-Cl
298.64
306.79
343.14
351.24
282.23
290.33
295.26
303.36
295.26
303.36
196–197
272–274
227–229
302–305
168–170
249–250
262–263
340–342
262–263
341–343
440–441
70
97
75
60
86
80
70
65
70
80
85
inhibition (Table 3) in at least eight cell lines at a concentration of 10
−4
−5
−6
7-Cl
M were further screened at five doses (i.e. 10
M, 10
M, 10
M,
7-Br
−7
−8
1
0
M, and 10
M) and growth inhibition were calculated as GI50
7-Br
values reflecting the concentration of drugs required to cause 50 per-
cent cell growth inhibition. Single dose testing revealed that five out of
the eleven acridones demonstrated growth inhibition in at least eight
cell lines from the 60 cell lines panel (Table 3). These five compounds
out of eleven (8b, 8d, 8g, 8h and 8k) display more than 60% growth
inhibition or active lethality (cancer cell killing) against about 40 cell
lines at 10 μM concentration (Table 3). As a result, (8b, 8d, 8g, 8h and
8k) were selected for a 5-dose repeat screening at five different con-
7-F
7-F
8g
8h
8i
7-OMe
7-OMe
5-OMe
5-OMe
0
1
8j
8k
5,7-dibromo 431.12
*
Yields refer to pure products.
−
4
−5
−6
−7
−8
centrations (10 M, 10 M, 10 M, 10 M and 10 M) after 48 h
of incubation. The result for the 5-dose testing is shown in Table 4.
Clearly, these analogs show good anticancer potency in a wide spec-
trum of cell lines causing 50% cell growth inhibition (GI50), and the
values range from 0.03 μM for melanoma (MDA-MB-435) to 6.50 μM
for renal cancer (786–0). (Table 4).
blood brain barrier. The topological polar surface area (TPSA) of all
analogs were in the range of 29.96–39.19 Ã (Table 2). The absorption
(
%ABS) was determine using the equation %ABS = 109–0.345 × TPSA
24
following the method of Zhao et al.
All the synthesized compounds
showed %ABS in the range 95–99%, indicating potential for good oral
bioavailability. Since biological activity depends on interaction be-
tween ligand and a putative target, we carried out virtual screening of
all 11 compounds using Topoisomerase IIα ATPase domain as a model.
The binding affinity of the compounds ranged in values from −8.5 to
The most active derivative 8k showed an average activity of
0.283 μM in the nine cell line panels (Table 4). On the other hand, other
derivatives exhibited the following average activities, 8b (0.527 μM),
8d (0.581 μM), 8h (1.376 μM) and 8g (1.588 μM). These data indicate
that the 8k represents a good lead molecule for further development as
therapeutic agent for cancer treatment.
−
7.9 indicating favorable binding to the human topoisomerase IIα
ATpase domain active site.
2
.3. Screening of anticancer activity
2.4. NCI in vitro testing results of compounds 8b, 8d, 8 g, 8 h & 8 k at five
different dose levels in μM
2
.3.1. In vitro cell growth inhibition assay: anticancer activity of acridone
derivatives 8b, 8d, 8g, 8h, and 8k against a panel of sixty human cancer
cell lines (NCI-60 panel)
Initially, monosubstituted acridone derivatives were tested against a
panel of 60 human cancer cell lines at a single dose of 10 μM using the
Rubinstein et al technique.26
3. Conclusion
All the acridone compounds were synthesized in moderate to good
Table 2
In silico drug-like property predictions of acridone derivatives.a
3
Entry
Compound
R
1
R
2
Log P
TPSA (Å)
n-HBA
n-HBD
n- violations
%ABS
Molar Refractivity (cm /Mol)
Binding Affinity (kcal/Mol)
1
2
3
4
5
6
7
8
9
8a
8b
8c
8d
8e
8f
CF
3
Ph
CF
3
Ph
CF
3
Ph
CF
3
Ph
CF
3
Ph
Ph
7-Cl
3.83
4.21
4.02
4.30
3.71
3.99
3.39
3.67
3.37
3.64
4.62
29.96
29.96
29.96
29.96
29.96
29.96
39.19
39.19
39.19
39.19
29.96
5
2
5
2
6
3
6
3
6
3
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
98.66
98.66
98.66
98.66
98.66
98.66
95.48
95.48
95.48
95.48
98.66
69.60
89.09
72.29
91.78
64.55
84.04
71.09
90.58
71.09
90.58
99.48
−8.1
−7.9
−8.1
−8.3
−8.3
−8.5
−8.4
−8.5
−8.1
NA
7-Cl
7-Br
7-Br
7-F
7-F
8g
8h
8i
7-OMe
7-OMe
5-OMe
5-OMe
5,7-dibromo
1
1
0
1
8j
8k
NA
2
4
%
ABS = 109–0.345× TPSAn
a
%
ABS: Percentage of absorption, TPSA: topological polar surface area, n-HBA: number of hydrogen bond acceptors, n-HBD: number of hydrogen bond donors,
MR: Molar Refractivity. Calculations were done using SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness
2
5
of small molecules (http://www.swissadme.ch/index.php).
3

A.S. Oyedele, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Table 3
4. Experimental section
One-Dose percent growth inhibition.
4.1. Synthetic procedure and analytical data
Percent Inhibition of growth at 10 μM of compounds 8b, 8d, 8g, 8h, 8k
Cell line
Percentage growth inhibition
Melting points (mp) were determined using capillary melting ap-
paratus, MELT-TEMP and were uncorrected. Infrared (IR) spectra were
recorded on 1600 Model FTIR spectrophotometer. All IR spectra were
8
b
8d
8g
8h
8k
1
13
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
obtained as neat samples. Proton ( H NMR) and carbon ( C NMR)
nuclear magnetic resonance spectra were taken on Bruker ARX 400
NMR instrument with tetramethylsilane as an internal standard, using
solvent as indicator (DMSO). Chemical shifts are reported in parts per
million (δ), and signals were expressed as s (singlet), d (doublet), dd
89.6
91.49
128.41
93.86
89.45
86.89
90.12
115.07
83.25
84.72
83.58
89.27
101.74
86.81
82.2
–
120.68
79.32
93.17
87
95.36
86.8
79.15
80.49
MOLT-4
SR
82.32
(
doublet of doublet), t (triplet), q (quartet), m (multiplet). Coupling
Non-Small Cell Lung Cancer
A549/ATCC
constant are in hertz (HZ). Mass spectra of the compounds were re-
corded on Varian 3900 GC/MS. Each reaction was monitored and
judged complete by removing aliquots at intervals and analyzed by thin
layer chromatography (TLC) (Whatman 250 μm LAYER, 25 PLATES,
20 × 20 cm.
76.72
79.24
96.67
96.98
76.15
75.86
95.37
103.56
60.68
60.21
89.47
68.13
65.22
73.53
91.21
84.64
–
NCI-H226
85.91
97.68
91.55
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
131.66
94.72
94.55
86.07
95.61
98.27
76.95
136.33
72.99
94.44
83.96
97.8
90.86
15.99
88.06
62.2
98.07
8.74
148.3
93.49
93.8
88.91
75.21
90.03
89.16
82.24
4.2. General procedure for the synthesis of acridone derivatives
89.74
91.67
97.98
77.57
HT29
89.11
88.44
58.53
To a mixture of substituted 2-aminebenzaldehyde (1.0 mmol) and 5-
substituted-1,3-cyclohexanedione (1.2 mmol), 1 mL of 1 N HCl aqueous
solution was added. The resulting mixture was stirred at 75–80 °C. After
completion of the reaction (monitored by TLC), the resulting suspension
was neutralized with 1 mL of 1 N NaOH. The solid was filtered, washed
with water (3 × 6 mL), and air-dried to give the product as white or
slightly yellow powder. The solid product was further purified by re-
crystallization with ethanol when necessary.
KM12
91.06
74.9
SW-620
CNS Cancer
SF-295
103.44
111.96
75.05
70.3
82.82
98.4
54.66
77.43
49.66
92.13
15.34
69.02
92.08
65.7
132.15
93.09
65.2
SF-539
SNB-19
SNB-75
U251
72.62
63.37
81.86
109.09
54.97
64.8
81.19
91.38
Melanoma
M14
91.02
89.85
139.84
97.9
91.35
147.68
68.57
60.24
90.72
167.71
69.85
63.08
94.7
.3. Synthesis of 2-amino-5-bromobenzaldehyde32
MDA-MB-435
SK-MEL-5
UACC-62
138.55
114.31
70.73
124.41
118.01
61.73
4
63.78
To a hot, stirred solution of Na
12.8 g, 0.12 mol) in H O (500 mL) was added slowly a solution of 5-
bromo-2-nitrobenzaldehyde (6.20 g, 27 mmol) in MeOH (100 mL) over
5 min. The solution was then refluxed for 2 h and cooled to 0 °C. The
precipitate was collected by filtration, washed with H O and MeOH to
afford 2-amino-5-bromobenzaldehyde (0.99 g). The filtrate was ex-
tracted with methylene chloride solution washed in H O, dried over
2
S
2
O
4
(26.0 g, 0.15 mol) and Na
2
CO
3
Ovarian Cancer
(
2
IGROV1
83.45
129.11
73.58
77.6
78
63.39
94.7
42.32
60
64.17
107.18
38.8
84.85
140.12
52.93
95.99
113.49
OVCAR-3
128.36
72.84
75.93
82.89
2
OVCAR-5
OVCAR-8
60.97
86.77
2
NCI/ADR-RES
92.04
81.07
Renal Cancer
2
A498
117.41
69.19
70.84
78.2
112.39
65.67
69.28
71.58
67.82
107.7
50.79
62.95
101.91
57.87
113.46
56.08
68.74
102.76
59.31
–
Na
SO , and concentrated under reduced pressure to afford additional
2
4
ACHN
70.29
98.78
69.98
60.51
2
7
-amino-5-bromobenzaldehyde for a total yield of 1.70 g (32%): mp
CAKI-1
1
4–76 °C; H NMR (CDCl
3
) δ 9.79 (s, 1H), 7.58 (d, 1H, J = 2.4 Hz),
RXF 393
SN12C
71.75
7.37 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 6.57 (d, 1H, J = 8.7 Hz), 6.12 (b,
2
H).
Prostate Cancer
PC-3
72.27
99.82
68.01
66.95
95.69
71.19
98.88
77.36
99.05
DU-145
103.21
4
.4. Synthesis of 2-amino-3-methoxybenzaldehyde33
Breast Cancer
MDA-MB-231/ATCC
BT-549
80.53
95.38
89.79
86.82
93.52
99.65
40.00
39.07
64.43
45.36
48.29
31.74
84.89
94.71
115.64
A
mixture of 3-methoxy-2-nitrobenzaldehyde and ammonium
chloride in methanol-water (30 mL, 1:1) was heated to 80 °C. Iron
powder was added slowly, and the resulting mixture was stirred at this
temperature for 6 h. After completion of reaction, as judged by TLC,
resulting suspension was filtered and the filtrate was poured into water
(100 mL), wherein a precipitate formed. The precipitate was filtered
and was washed with water (3 × 20 mL). The solid was dried to get
crude product without further purification.
MDA-MB-468
yields (67–97%). In summary, the 3-phenyl-substituted acridone deri-
vatives (8b, 8d, 8h, 8k) have been identified as potential anti-
proliferative agents.in several cancer cell lines. Additionally, the 3-tri-
fluoromethylated acridone derivative 8g was also active in the in vitro
anticancer test. Compound 8k was the most active compound in the
series indicating that electron-withdrawing halogens are important for
antiproliferative activity of this class of tricyclic nitrogen heterocylces.
Thus, all the analogs represent new lead compounds for further de-
velopment into potential agents for cancer treatment. The in silico
studies provided data to support the results of the biological activity
determination, conducted by the Developmental Therapeutic Program
of the National Cancer Institute (NCI).
4
.4.1. 7-chloro-3-(trifluoromethyl)-3,4-dihydroacridin-1(2H)-one (8a)
Light Yellow solid, mp = 196–197 °C. IR (neat) 3051, 2435, 1589,
−
1
1
1
2
1
439, 977, 714 cm
;
H NMR (DMSO) δ 2.20–2.49 (dd, 2H),
.88–3.31 (dd, 2H), 3.61 (m, 1H), 8.20 (d, 1H), 8.59 (d, 1H), 8.91 (s,
H). GCMS m/z; 165 (12%), 196 (12.5%), 283 (15%), 288 (40%) 296
1
3
(
100%), (M++)298 (95%). C (400 MHz, DMSO) δ 194.21, 159.3,
1
1
48.0, 136.02, 135.99, 132.25, 131.83, 130.68, 128.11, 127.34,
26.33, 120.20, 40.11, 37.16.
4

A.S. Oyedele, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Table 4
Five dose testing for 8b, 8d, 8g, 8h and 8k.
In vitro antitumor activity (GI50/μM) data for compounds 8b, 8d, 8g, 8h, 8k from the five dose 60 human cancer cell lines
Panel/Cell Line
8b
8d
8g
8h
8k
GI50 (μM)
MG-MID*
GI50 (μM)
MG-MID*
GI50 (μM)
MG-MID*
GI50 (μM)
MG-MID*
GI50 (μM)
MG-MID*
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
0.04
0.0981
0.0498
0.214
0.110
0.238
0.325
0.437
0.0882
0.261
0.322
0.274
0.344
0.431
0.339
0.0889
0.226
0.116
0.211
0.119
0.0675
0.175
0.0676
0.104
MOLT-4
RPMI-8226
SR
0.0537
*
*
**
**
**
**
DNS
DNS
DNS
DNS
DNS
0.0532
0.0567
0.217
0.322
0.0913
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
0.42
0.620
0.694
0.419
0.756
0.354
0.700
0.618
3.03
4.19
3.61
2.923
1.136
2.07
5.92
1.96
3.441
0.891
0.0792
0.391
0.18
0.164
0.193
0.607
0.428
HOP-62
*
*
**
**
*
**
HOP-92
DNS
DNS
DNS
DNS
0.0668
0.241
0.26
*
**
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
1.61
2.25
DNS
DNS
0.517
0.72
0.616
0.623
0.344
0.236
5.15
1.32
2.61
6.18
5.78
1.71
0.471
0.0748
0.0423
0.137
0.345
0.314
0.582
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
0.346
2.89
0.261
2.58
1.16
0.608
0.152
0.282
0.0478
0.312
0.233
0.242
0.0839
*
*
**
DNS
DNS
0.0468
0.588
0.271
0.349
0.37
0.0515
0.528
0.205
0.341
0.36
0.133
1.37
0.432
2.68
HT29
0.387
0.316
3.45
0.428
0.45
KM12
SW-620
0.747
CNS Cancer
**
SF-268
DNS**
0.196
0.219
0.425
0.215
0.729
0.357
0.453
3.45
0.849
0.350
DNS**
0.853
1.89
2.128
0.895
DNS
1.244
0.555
0.167
0.196
0.192
0.406
0.199
0.421
0.264
0.317
SF-295
0.206
0.241
0.434
0.21
0.634
1.15
SF-539
SNB-19
SNB-75
U251
3.61
2.62
2.16
0.572
**
0.553
DNS**
DNS
Melanoma
LOX IMVI
MALME-3M DNS
M14
0.558
0.707
0.0949
0.0505
0.0271
0.238
0.954
0.288
3.08
0.887
0.758
0.119
0.284
0.211
0.0492
0.478
0.298
*
*
**
**
DNS
DNS
0.038
0.0266
0.32
0.238
0.143
0.802
0.259
0.137
0.777
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
*
*
**
1.69
DNS
DNS
0.203
0.411
0.54
*
*
**
**
**
**
DNS
DNS
DNS
DNS
DNS
0.333
0.444
0.697
0.728
0.338
Ovarian Cancer
IGROV1
0.375
0.302
0.523
0.400
0.423
0.289
0.446
0.432
0.769
1.83
1.244
1.681
2.31
1.23
1.420
1.088
0.125
0.0302
1.41
0.410
0.403
OVCAR-3
*
*
**
**
**
OVCAR-4
DNS
DNS
DNS
DNS
*
*
**
OVCAR-5
1.02
0.825
0.461
0.0669
0.611
DNS
DNS
0.461
0.507
0.236
0.102
OVCAR-8
0.534
0.0644
0.845
2.04
1.06
0.408
2.09
NCI/ADR-RES
SK-OV-3
0.336
DNS**
Renal Cancer
7
86–0
0.838
0.087
0.508
0.151
0.195
0.533
0.784
0.0745
0.583
0.207
0.208
0.526
6.57
4.37
0.813
0.14
A498
0.355
0.768
0.69
0.346
0.756
0.525
0.44
ACHN
CAKI-1
RXF 393
SN12C
TK-10
0.595
0.145
0.0916
0.661
0.7
0.562
0.823
0.639
*
*
**
**
**
DNS
DNS
DNS
DNS
UO-31
0.489
0.64
2.00
0.543
0.0795
Prostate Cancer
PC-3
0.28
0.307
1.289
0.254
0.274
0.264
1.459
0.835
0.849
0.842
3.182
0.715
0.593
0.654
2.754
0.0666
0.0826
0.075
0.608
DU-145
0.334
Breast Cancer
MCF7
2.55
0.617
0.306
0.5
2.76
3.42
3.98
3.35
DNS**
3.49
3.42
1
0.382
0.402
0.191
0.541
MDA-MB-231/ATCC
HS 578T
0.598
0.411
0.685
BT-549
2.21
(
continued on next page)
5

A.S. Oyedele, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Table 4 (continued)
In vitro antitumor activity (GI50/μM) data for compounds 8b, 8d, 8g, 8h, 8k from the five dose 60 human cancer cell lines
Panel/Cell Line
8b
8d
8g
8h
8k
GI50 (μM)
MG-MID*
GI50 (μM)
MG-MID*
GI50 (μM)
MG-MID*
GI50 (μM)
MG-MID*
GI50 (μM)
MG-MID*
*
*
**
T-47D
DNS
2.47
DNS
3.29
1.87
3.65
0.851
1.28
MDA-MB-468
2.84
DNS**
MG-MID*: average sensitivity of all cell lines of a particular subpanel towards the testing agents in μM.
DNS : Data not shown.
*
*
4
.4.2. 7-chloro-3-phenyl-3,4-dihydroacridin-1(2H)-one (8b)
39.7, 27.5, 21.9.
Yellow solid, mp = 272–274 °C. IR (neat) 3061, 3033, 2959, 2895,
−
1 1
1
3
1
690, 832, 761 cm ; H NMR (DMSO) δ 2.81 (dd,2H), 2.9–3.4 (t, 3H),
.6 (d, 2H), 7.20 (d, 2H), 7.33 (d, 2H) 7.45 (d, 2H), 7.95 (d, 1H), 8.1 (d,
H), 8.2 (d, 1H), 8.91 (d, 1H). GCMS m/z; 51 (100%), 84 (45%), 271
4
.4.8. 7-methoxy-3-phenyl-3,4-dihydroacridin-1(2H)-one (8h)
Yellow solid, mp = 340–342 °C. IR (neat) 3061, 3028, 2895, 1665,
−1 1
1
2
3
194, 698 cm ; H NMR (DMSO) δ 2.20–2.70 (dd, 2H), 2.91–3.51 (dd,
H), 3.61 (m, 1H), 7.55 (d, 1H), 7.95 (d, 1H), 8.95 (s, 3H). GCMS m/z;
96 (100.0%), 267 (30%), 224 (5%), 51 (60%), 84 (20%), 128 (12%).
C (400 MHz, DMSO) δ 196.80, 157.0, 156.8, 143.5, 142.20, 135.70,
31.6, 129.40, 128.4, 126.1, 125.9, 55.8, 45.00, 42.5, 32.0.
1
3
(
44%), 299 (80%), 301(30%) (M++). C (400 MHz, DMSO) δ 197.2,
1
1
4
60.1, 157.99, 146.55, 143.43, 136.57, 130.78, 130.55, 129.10,
28.45, 127.58, 127.42, 127.13, 126.15, 123.03, 113.15, 103.67,
4.80, 39.83, 38.04.
1
3
1
4
.4.3. 7-bromo-3-(trifluoromethyl)-3,4-dihydroacridin-1(2H)-one (8c)
4.4.9. 5-methoxy-3-(trifluoromethyl)-3,4-dihydroacridin-1(2H)-one (8i)
Light Yellow solid, mp = 227–229 °C. IR (neat) 3051, 2435, 1589,
Yellow solid, mp = 262–263 °C. IR (neat) 3020, 2936, 2440, 1957,
1
−1 1
1
439, 977, 714 cm-1; H NMR (DMSO) δ 2.20–2.65 (dd, 2H), 2.88–3.31
1708, 1119 cm
; H NMR (DMSO) δ 2.20–2.49 (dd, 2H), 3.53–3.76
(
dd, 2H), 3.63 (m, 1H), 7.78 (d, 1H), 8.32 (d, 1H), 8.95 (s, 1H). GCMS
(dd, 2H), 3.91 (m, 1H), 7.88 (d, 1H), 7.91 (d, 1H), 8.95 (s, 3H). GCMS
m/z; 69 (20%), 140 (30%), 315 (50%), 344 (75%), (M++) 346
m/z; 51 (30%), 69 (20%), 140 (12%), 167 (5%), 266 (50%), 295
1
3
13
(
100%). C (400 MHz, DMSO) δ 194.34, 159.62, 148.03, 136.07,
(100%), 296 (70%) (M+). C (400 MHz, DMSO) δ 196.8, 158.3,
1
4
35.99, 132.29, 131.91, 130.72, 128.29, 127.38, 126.38, 120.30,
0.11, 37.16.
155.6, 137.2, 136.2, 131.4, 129.2, 126.4, 121.3, 109.2, 55.8, 39.7,
27.5, 21.9.
4
.4.4. 7-bromo-3-phenyl-3,4-dihydroacridin-1(2H)-one (8d)
4.4.10. 5-methoxy-3-phenyl-3,4-dihydroacridin-1(2H)-one (8j)
Yellow solid, mp = 302–305 °C. IR (neat) 3062, 3029, 2947, 2897,
Yellow solid, mp = 341–343 °C. IR (neat) 3061, 3028, 2895, 1665,
−
1
1
−1 1
1
3
8
3
688, 823, 760 cm
;
H NMR (DMSO) δ 2.81 (dd,2H), 2.91–3.53 (t,
1194, 698 cm ; H NMR (DMSO) δ 2.20–2.90 (dd, 2H), 2.91–3.81 (dd,
2H), 3.94 (m, 1H), 7.60 (d, 1H), 7.97 (d, 1H), 8.90 (s, 3H). GCMS m/z;
H), 3.62 (d, 2H), 7.20 (d, 2H), 7.33 (d, 2H) 7.45 (d, 2H), 7.95 (d, 1H),
1
3
.1 (d, 1H), 8.2 (d, 1H), 8.91 (d, 1H). GCMS m/z; 51 (70%), 140 (35%),
303 (75%), 304 (40%), 305 (2.7%), 199 (22%) C (400 MHz, DMSO) δ
197.49, 160.12, 154.99, 143.93, 141.31, 136.28, 129.08, 127.95,
127.39, 127.14, 126.22, 121.70, 111.43, 56.18, 45.73, 40.56, 39.93,
39.52, 38.84.
1
3
17 (40%), 343 (80%), 345 (100%), 346 (12%). C (400 MHz, DMSO)
δ 197.19, 161.28, 161.03, 158.83, 146.47, 143.77, 136.58, 131.17,
1
1
29.13, 128.95, 127.67, 127.38, 127.24, 126.16, 123.08, 113.19,
03.98, 45.70, 39.87, 38.25.
4
.4.11. 5,7-dibromo-3-phenyl-3,4-dihydroacridin-1(2H)-one (8k)
4.4.5. 7-fluoro-3-(trifluoromethyl)-3,4-dihydroacridin-1(2H)-one (8e)
Slightly Yellow Solid; mp = 440–441 °C 1H NMR (400 MHz,
Yellow solid, mp = 168–170 °C. IR (neat) 3051, 2435, 1589, 1439,
CDCl3): δ 8.21–8.53, 2H on dibromobenzene), δ 8.95 94 pyridyl 1H), δ
7.20–7.39 (5H, phenyl), δ 3.28 91H, m), δ 2.52–2.90 (m, 2H), δ
−
1 1
9
77, 714 cm ; H NMR (DMSO) δ 2.20–2.49 (dd, 2H), 2.88–3.11 (dd,
H), 3.23 (m, 1H), 8.10 (d, 1H), 8.33 (d, 1H), 8.93 (s, 1H). GCMS m/z;
1 (52%), 69 (35%), 158 (30%), 185 (20%) 255 (70%), 283 (100%), (M
1
3
2
2.90–3.26 (2H, m); C NMR (400 MHZ, CDCl3) δ 32.1, 43.0, 44.8, 126,
5
128.2, 135.9, 137.2, 143.7, 124.0, 118.0, 132.2, 142.1, 160.3, 197.2.
1
3
+
+) 285 (25%), C (400 MHz, DMSO) δ 196.8, 160.1, 150.50,
1
36.30, 132.2, 129.1, 128.6, 123.7, 110.8, 39.7, 27.5, 21.9.
4.5. Biological activity
4
.4.6. 7-fluoro-3-phenyl-3,4-dihydroacridin-1(2H)-one (8f)
4.5.1. Interpretation of one-dose data
Yellow solid, mp = 249–250 °C. IR (neat) 3061, 3033, 2959, 2895,
The One-dose was reported as a mean graph of the percent growth
of treated cells and was similar in appearance to mean graphs from the
5-dose assay. The number reported for the One-dose assay was growth
relative to the no-drug control, and relative to the time zero number of
cells. This allowed detection of both growth inhibition (values between
0 and 100) and lethality (values less than 0). This was the same as for
the 5-dose assay, described below. For example, a value of 100 means
total growth inhibition. A value of 40 would mean 60% no growth
inhibition. A value of 0 means no net growth over the course of the
experiment. A value of +40 would mean 40% lethality. A value of
−
1 1
1
3
8
690, 832, 761 cm
; H NMR (DMSO) δ 2.81 (dd, 2H), 2.91–3.53 (t,
H), 3.62 (d, 2H), 7.20 (d, 2H), 7.33 (d, 2H) 7.45 (d, 2H), 7.95 (d, 1H),
.1 (d, 1H), 8.2 (d, 1H), 8.91 (d, 1H). GCMS m/z; 51 (60%), 104
1
3
(
100%), 145 (65%), 190 (40%), 291 (63%), 292 (15%) (M+)
C
(
400 MHz, DMSO) δ 197.45, 161.1, 157.36, 147.52, 144.43, 136.57,
1
1
31.58, 130.65, 129.30, 129.01, 128.86, 127.19, 127.05, 126.44,
23.73, 113.18, 103.87, 44.84, 39.93, 38.34.
4.4.7. 7-methoxy-3-(trifluoromethyl)-3,4-dihydroacridin-1(2H)-one (8g)
+
100 means all cells are dead. Information from the One-dose mean
Dark Yellow solid, mp = 262–263 °C. IR (neat) 3020, 2936, 2440,
27
−
1
1
graph is available for COMPARE analysis.
1
3
3
957, 1708, 1119 cm
; H NMR (DMSO) δ 2.20–2.49 (dd, 2H),
.53–3.76 (dd, 2H), 3.91 (m, 1H), 7.85 (d, 1H), 7.90 (d, 1H), 8.95 (s,
H). GCMS m/z; 51 (13%), 78 (7%), 159 (10%), 187 (25%), 262
4.5.2. NCI 60 cell five-dose screen and assay
1
3
(
2.2%), 293 (100%), 294 (7%) (M + ). C (400 MHz, DMSO) δ 196.8,
Compounds which exhibit significant growth inhibition in the One-
Dose Screen are evaluated against the 60-cell panel at five
1
57.0, 156.8, 142.2, 137.2, 135.7, 131.6, 129.4, 124.4, 106.7, 55.80,
6

A.S. Oyedele, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
concentration levels.
4.6.1. Protein preparation
The human tumor cell lines of the cancer screening panel were
grown in RPMI 1640 medium containing 5% fetal bovine serum and
The crystal structure of human topo IIα ATPase/ADP was retrieved
2
9
from the rcsb protein data bank (PDB ID: 1zxm ) and was used as a
target for the screening of all nine compounds. 1ZXM is a well-estab-
lished target for catalytic inhibitors of topoisomerase. The above target
was chosen because acridone compounds previously synthesized in our
laboratory, that lacked a 9-amino functionality were inactive in a DNA-
topoisomerase II cleavage assay and as such could not act as poisons of
topoisomerase. All water molecules were removed. PyRx automates
many intermediate steps including addition of hydrogens, energy
minimization, removal of bound ligands and heteroatoms, including
setting of autogrid dimensions. The target is prepared as a pdb file but
was converted into a pdbqt file prior to docking simulation. At this
point we are only interested in the binding affinity of each derivative
with the protein.
2
mM L-glutamine. For a typical screening experiment, cells were in-
oculated into 96 well microtiter plates in 100 μl at plating densities
ranging from 5000 to 40,000 cells/well depending on the doubling time
of individual cell lines. After cell inoculation, the microtiter plates were
incubated at 37 °C, 5% CO , 95% air and 100% relative humidity for
2
2
4 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line were fixed in situ with TCA,
to represent a measurement of the cell population for each cell line at
the time of drug addition (Tz). Experimental drugs were solubilized in
dimethyl sulfoxide at 400-fold the desired final maximum test con-
centration and stored frozen prior to use. At the time of drug addition,
an aliquot of frozen concentrate was thawed and diluted to twice the
desired final maximum test concentration with complete medium
containing 50 μg/ml gentamicin. Additional four, 10-fold or ½ log se-
rial dilutions were made to provide a total of five drug concentrations
plus control. Aliquots of 100 μl of these different drug dilutions were
added to the appropriate microtiter wells already containing 100 μl of
medium, resulting in the required final drug concentrations.
4.6.2. Ligand preparation
For the construction of ligand files from chemical structures, all nine
compounds were drawn using ChemSketch, an open source program
3
0
available as a free download from the developers (ACD Labs). Once
the structure is drawn it is saved as a mol file and then imported into
3
1
Following drug addition, the plates were incubated for an additional
OpenBabel. OpenBabel is a versatile program that converts structural
files between a volume of different formats. The ligand file is saved as a
pdb file before importing into PyRx, which converts the pdb file into a
pdbqt file format for docking simulation.
4
8 h at 37 °C, 5% CO , 95% air, and 100% relative humidity. For ad-
2
herent cells, the assay was terminated by the addition of cold TCA. Cells
were fixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA
(
final concentration, 10% TCA) and incubated for 60 min at 4 °C. The
supernatant was discarded, and the plates were washed five times with
tap water and air dried. Sulforhodamine B (SRB) solution (100 μl) at
Declaration of Competing Interest
0
.4% (w/v) in 1% acetic acid was added to each well, and plates were
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
incubated for 10 min at room temperature. After stained, unbound dye
was removed by washing five times with 1% acetic acid and the plates
were air dried. Bound stained was subsequently solubilized with 10 mM
trizma base, and the absorbance was read on an automated plate reader
at a wavelength of 515 nm. For suspension cells, the methodology was
the same except that the assay was terminated by fixing settled cells at
the bottom of the wells by gently adding 50 μl of 80% TCA (final
concentration, 16% TCA). Used the seven absorbance measurements
Acknowledgement
We are grateful to the United States Department of Education Title
III grant, Tennessee State University for their financial support. We are
also thankful to the National Institute of Health (NIH), National Cancer
Institute Branch for the opportunity to use their Developmental
Therapeutic Program (DTP) for the timely screening of our compounds
using 60 Human cancer cell lines.
[
time zero, (Tz), control growth, (C), and test growth in the presence of
drug at the five concentration levels (Ti)], the percentage growth was
calculated at each of the drug concentrations levels. Percentage growth
inhibition was calculated as:
Appendix A. Supplementary material
[
(Ti Tz)/(C Tz)] × 100 for concentrations for which Ti > /=Tz
Three dose response parameters were calculated for each experi-
[
(Ti Tz)/Tz] × 100 for concentrations for which Ti < Tz.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115426.
mental agent. Growth inhibition of 50% (GI50) was calculated from
References
[
(Ti-Tz)/(C-Tz)] × 100 = 50, which was the drug concentration re-
sulting in a 50% reduction in the net protein increase (as measured by
SRB staining) in control cells during the drug incubation. The drug
concentration resulting in total growth inhibition (TGI) was calculated
from Ti = Tz. The LC50 (concentration of drug resulting in a 50% re-
duction in the measured protein at the end of the drug treatment as
compared to that at the beginning) indicating a net loss of cells fol-
lowing treatment was calculated from [(Ti-Tz)/Tz] × 100 = −50.
Values were calculated for each of these three parameters if the level of
activity was reached; however, if the effect is not reached or was ex-
1
.
World Health Organization. Cancer: Key facts. http://www.who.int/news-room/fact-
sheets/detail/cancer.
2. Gao F, Zhang X, Wang TF, Xiao JQ. Quinolone hybrids and their anti-cancer activ-
ities: an overview. Eur J Med Chem. 2019;165:59–79.
3
.
World Health Organization. Latest global cancer data: Cancer burden rises to 18.1
million new cases and 9.6 million cancer deaths in 2018.
4. Cholewiñski Grzegorz, Dzierzbicka Krystyna, Koodziejczyk Aleksander M. Natural
and synthetic acridines/acridones as antitumor agents: their biological activities and
methods of synthesis. Pharmacol Rep. 2011;63:305–336.
5. Michel S, Gaslonde T, Tillequin F. Eur J Med Chem. 2004;39:649–655.
6. Tillequin F. Phytochemistry Rev. 2003;1:355–368.
7
.
Mazerska Z, Sowinski P, Konopa J. Biochem Pharmacol. 2003;66:1727–1736.
Atwell GJ, Cain BF, Seelye RN. Potential antitumor agents. 9-Anilinoacridines. J Med
Chem. 1972;15:611–615.
ceeded, the value for that parameter was expressed as greater or less
than the maximum or minimum concentration tested.27
8.
9
.
Denny WA. Acridine derivatives as chemotherapeutic agents. Curr Med Chem.
2
002;9:1655–1665.
4.6. Virtual screening
1
0. Finlay GJ, Atwell GJ, Baguley BC. Inhibition of the action of the topoisomerase II
poison amsacrine by simple aniline derivatives: evidence for drug-protein interac-
tions. Oncol Res Featuring Preclin Clin Cancer Therap. 1999;11:255–264.
1. Su TL, Kohler B, Chou TC, Chun MW, Watanabe KA. Synthesis of the acridone al-
kaloids glyfoline and congeners structure-activity relationship studies of cytotoxic
acridones. J Med Chem. 1992;35:2703–2710.
The nine acridone derivatives were screened virtually using PyRx,
1
an open source program that combines several softwares, such as
AutoDock Vina, autodock 4.2, and OpenBabel. It uses both Vina and
AutoDock 4.2 as docking softwares.28
12. Gniazdowski M, Szmigiero L. Nitracrine and its congeners-an overview. Gen
7

A.S. Oyedele, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Pharmacol. 1995;26:473–481.
Ojima I, McCarthy JR, Welch JT, editors. ACS Symp. Ser. 639, Washington, DC;
1
1
3. Cholewiñski G, Dzierzbicka K, Koodziejczyk AM. Natural and synthetic acridines/
1996; pp. 158–81.
acridones as antitumor agents: their biological activities and methods of synthesis.
Pharmacol Rep. 2011;63:305–336.
(b) Ojima I. ChemBioChem. 2004;5:628–635
22. Lipinski Christopher A, Lombardo Franco, Dominy Beryl W, Feeney Paul J.
Experimental and computational approaches to estimate solubility and permeability
in drug discovery and development q settings. Adv Drug Deliv Rev. 2001;46:3–26.
23. Palm K, Stenberg P, Luthman K, Artursson P. Polar molecular surface properties
predict the intestinal absorption of drugs in humans. Pharm Res. 1997;14:568–571.
24. Zhao Yuan H, Abraham Michael H, Le Joelle, et al. Rate-limited steps of human oral
absorption and QSAR studies. Pharm Res. 2002;19.
4. Skladanowski A, Plisov SY, Konopa J, Larsen AK. Inhibition of DNA topoisomerase II
by imidazoacridinones, new antineoplastic agents with strong activity against solid
tumors. Mol Pharmacol. 1996;49:772–780 www.clinicaltrial.gov [NCT00004151],
[
NCT00004937].
1
1
5. Friedländer P, Gohring CF. Ber. 1833;1883:16.
6. Fadeyi Olugbeminiyi O, Adamson Saudat T, Lewis Myles E, Okoro Cosmas O. Novel
fluorinated acridone derivatives. Part 1: synthesis and evaluation as potential an-
ticancer agents. Bioorg Med Chem Lett. 2008;18:4172–4176.
25. http://www.swissadme.ch/index.php.
26. Rubinstein LV, Shoemaker RH, Paull KD, et al. J Natl Cancer Inst.
1990;82:1113–1118.
1
1
1
7. Fadeyi OO, Okoro CO. Synthesis of 5-(trifluoromethyl)cyclohexane-1,3-dione and 3-
amino-5-(trifluoromethyl)cyclohex-2-en-1-one: new trifluoromethyl building block.
Tetrahedron Lett. 2008;49:4725–4727.
27. https://dtp.cancer.gov/discovery_development/nci-60/methodology.htm.
28. https://pyrx.sourceforge.net/downloads.
8. Belmont P, Bosson J, Godet T, Tiano M. Acridine and acridone derivatives, anticancer
properties and synthetic methods: where are we now? Anti-Cancer Agents Med Chem.
29. https://www.rcsb.org.
30. http://www.acdlabs.com/resources/freeware/chemsketch/.
31. http://openbabel.org/wiki/category:installation.
2
007;7:139–169.
9. Harvey VJ, Hardy JR, Smith S, Grove W, Baguley BC. Phase II study of the amsacrine
32. Kenneth Horner J, Henry David. Analogs of 3-amino-7-chloro-l,2,4-benzotriazine1
-oxide as antimalarial agents. J of Med Chem. 1968;11:946–949.
33. Haiqing Yu, Li Yanxia, Ge Yang, et al. Novel 4-anilinoquinazoline derivatives fea-
turing an 1-adamantylmoiety as potent EGFR inhibitors with enhanced activity
against NSCLC cell lines. Eur J Med Chem. 2016;110:195–203.
analogue CI-921 (NSC 343499) in non-small cell lung cancer. Eur J Cancer.
1
991;27:1617–1620.
2
2
0. Böhm HJ, Banner D, Bendels S, et al. ChemBioChem. 2004;5:637.
1. (a) Ojima I, Kuduk SD, Slater JC, et al. Biomedical frontiers of fluorine chemistry. In:
8