Synthesis of 5H-indeno[1,2-b]pyridine derivatives: Antiproliferative and antimetastatic activities against two human prostate cancer cell lines

Article abstract of DOI:10.1002/ardp.202100092

This study describes the direct synthesis of 2-amino-4-(phenylsubstituted)-5H-indeno[1,2-b]pyridine-3-carbonitrile derivatives 5–21, through sequential multicomponent reaction of aromatic aldehydes, malononitrile, and 1-indanone in the presence of ammonium acetate and acetic acid (catalytic). The biological study showed that compound 10 significantly impeded proliferation of the cell lines PC-3, LNCaP, and MatLyLu. The antimetastatic effects of compound 10 could be related with inhibition of MMP9 in the PC-3 and LNCaP human cell lines. On the basis of a study of the structure–activity relationship of these compounds, we propose that the presence of two methoxy groups at positions 6 and 7 of the indeno nucleus and a 4-hydroxy-3-methoxy phenyl substitution pattern at position 4 of the pyridine ring is decisive for these types of molecules to exert?very good antiproliferative and antimetastatic activities.

Full text of DOI:10.1002/ardp.202100092

Received: 5 March 2021
Revised: 4 April 2021
Accepted: 6 April 2021
|
|
DOI: 10.1002/ardp.202100092
F U L L P A P E R
Synthesis of 5H‐indeno[1,2‐b]pyridine derivatives:
Antiproliferative and antimetastatic activities against
two human prostate cancer cell lines
Katiuska E. Charris1
Esteban Fernandez‐Moreira4
| Juan R. Rodrigues2
| Hegira Ramírez1,3
|
| Jorge E. Ángel5 | Jaime E. Charris1
1Organic Synthesis Laboratory, Faculty of
Pharmacy, Central University of Venezuela,
Caracas, Venezuela
Abstract
This study describes the direct synthesis of 2‐amino‐4‐(phenylsubstituted)‐5H‐
indeno[1,2‐b]pyridine‐3‐carbonitrile derivatives 5–21, through sequential multi-
component reaction of aromatic aldehydes, malononitrile, and 1‐indanone in the
presence of ammonium acetate and acetic acid (catalytic). The biological study
showed that compound 10 significantly impeded proliferation of the cell lines PC‐3,
LNCaP, and MatLyLu. The antimetastatic effects of compound 10 could be related
with inhibition of MMP9 in the PC‐3 and LNCaP human cell lines. On the basis of a
study of the structure–activity relationship of these compounds, we propose that
the presence of two methoxy groups at positions 6 and 7 of the indeno nucleus and
a 4‐hydroxy‐3‐methoxy phenyl substitution pattern at position 4 of the pyridine ring
is decisive for these types of molecules to exert very good antiproliferative and
antimetastatic activities.
2Departamento de Tecnología de Procesos
Biológicos y Bioquímicos, División de Ciencias
Biológicas, Universidad Simón Bolívar,
Caracas, Venezuela
3Facultad de Medicina, Universidad de Las
Américas, Quito, Ecuador
4Escuela de Medicina, Universidad Espíritu
Santo, Samborondón, Ecuador
5Organic Synthesis Laboratory, Design and
Pharmacological Evaluation of New Products,
Experimental Faculty of Science, Central
University of Venezuela, Zulia University,
Maracaibo, Venezuela
Correspondence
Jaime E. Charris, Organic Synthesis
Laboratory, Faculty of Pharmacy, Central
University of Venezuela, 47206, Los
Chaguaramos 1041‐A, Caracas, Venezuela.
Email: jaime.charris@ucv.ve
K E Y W O R D S
anticancer, antimetastatic, antiproliferative, indeno, prostate, pyridine
|
1
INTRODUCTION
Prostate cancer (PCa) is the second most common cancer among
men in the United States and the fifth most common cancer world-
Cancer is the second leading cause of death globally after ischemic
heart disease and stroke. In 2018, there were an estimated 18.1
million new cases and 9.6 million deaths from cancer, as per data
registered by the World Health Organization (WHO).[1] The most
frequently diagnosed cancer was of the lung, followed by female
breast cancer and colorectal cancer.
wide; however, incidence and mortality of this disease differ greatly
depending on the geographical area.[2] Although there are several
types of cancer treatment options in practice, their success depends
on the type and stage of cancer. However, they have limitations. To
date, chemotherapy has played a central role in the clinical treatment
of cancer, and numerous anticancer agents have been approved for
this purpose.[4‐6]
The American Cancer Society estimated that the number of new
cancer cases and deaths in the United States of America, by 2021,
will be in the order of 1,898,160 and 608,570, respectively.[2] On the
basis of expected population growth and aging along, the incidence
and mortality rates from cancer are expected to increase by 60% by
2040.[3]
In the clinical treatment of PCa, the strategy of inhibition of the
function of androgen receptors, established in the 1940s, is used.[7]
Although the disease is at first sensitive to androgen deprivation
therapy, the majority of patients eventually progress to a stage
termed castration‐resistant prostate cancer.[8] Therefore, a new
© 2021 Deutsche Pharmazeutische Gesellschaft
|
Arch Pharm. 2021;e2100092.
wileyonlinelibrary.com/journal/ardp
1 of 12
https://doi.org/10.1002/ardp.202100092

2 of 12
CHARRIS ET AL.
|
rational approach to finding new drugs and new targets of action for
PCa treatment is needed. Among those nucleos that have shown
promising results, we can point to the bicalutamide, isoxazolones,
pyrrole carboxamides, pyridines and N‐arylpiperazine derivatives,
indeno[1,2‐d]pyrimidine‐5‐one, imide‐based analogs, hydantoin,
benzochromene, benzoquinoline, quinoline, coumarin derivatives,
and organometallic‐containing derivatives.[9‐14]
cells as well as on clonogenic activity, possibly by inhibition of
MMP‐9 activity.
|
2
RESULTS AND DISCUSSION
Chemistry
|
2.1
One of those investigated targets with a renewed interest in
recent years is matrix metalloproteinases (MMPs), listed as a large
family of zinc‐dependent endoproteases known to exert multiple
regulatory roles in proliferation, adhesion, migration, and invasion of
cancer cells as well as in angiogenesis in the tumor.[15‐18] Two of
these MMPs, MMP2 and MMP9, known as gelatinases A and B, re-
spectively, are found in many human malignancies, including skin,
prostate, pancreas, breast, brain, colon, and lung, and are often
correlated with tumor aggressiveness and poor prognosis.[19‐21]
According to recent reports, it has been found that quinolinyl
acrylates,[22] 2‐amino‐5,6‐dihydro‐4‐phenylbenzo[h]quinoline‐3‐car-
bonitrile,[11] 2‐amino‐4‐benzylpyridine‐3‐carbonitrile,[9] 2‐amino‐4‐
quinolinyl‐naphthopyran‐3‐carbonitrile,[12] natural alkaloids like
onychnine and polyfothine or 2‐amino‐4‐phenyl‐5H‐indeno[1,2‐d]
pyrimidine‐5‐one derivatives,[10,23] and 6,7‐dimethyl‐4‐(3,4,5‐trimet-
hoxyphenyl)‐3,4‐dihydroquinolin‐2(1H)‐one (DTDQ)[24] are particu-
larly active as anticancer and MMP inhibitors. This literature reveals
that 1,2‐dihydronaphthalene and 1H‐indene‐coupled heterocyclic
compounds possess remarkable anticancer activity. On this basis,
and as a continuation of previous work,[11,22] we are reporting for
the first time the synthesis and characterization of 2‐amino‐4‐
Due to the importance of these kinds of compounds, the synthesis of
2‐aminopyridine derivatives has been largely investigated[25‐28]; how-
ever, the development of a general and efficient synthetic strategy to
obtain 2‐aminopyridine fused with a five‐membered ring of the indene
is still desired. In a pilot reaction, benzaldehyde‐substituted 1 (1 mmol),
malononitrile 2 (1 mmol), 1‐indanone‐substituted 3 (1 mmol), and am-
monium acetate (1.5 mmol) in toluene (10 ml), with catalytic amount of
acetic acid, were combined in a 20‐ml round‐bottom flask fitted with a
reflux condenser equipped with a Dean–Stark trap (Scheme 1a). The
resultant reaction may be assumed to proceed via the Knoevenagel
reaction with the formation of arylidenemalononitrile 4, which then
undergoes Michael addition with 1‐indanone respective 3, followed by
cyclization, isomerization, and aromatization to obtain the final pro-
ducts 5–21 (Scheme 2). The reaction mixture was then poured into
crushed ice. The solid product was filtered by suction and washed with
a mixture of water–ethanol. The analytical and spectral data of com-
pounds 5–21 were consistent with their respective structures.
The infrared (IR) spectra of the compounds show two char-
acteristic bands between 3392 and 3312, and 3328 and 2976 cm−1
,
confirming the presence of –OH and –NH2.
phenylsubstituted‐5H‐indeno[1,2‐b]pyridine‐3‐carbonitrile
deriva-
Furthermore, between 2224 and 2192 cm−1, stretching vibration
indicates the presence of –CN group, and between 1603 and
1596 cm−1, stretching vibration indicates the presence of –N═C– of
the heterocyclic group. In the 1H nuclear magnetic resonance (NMR)
spectrum, a singlet (s) is observed that appears between 3.35 and
3.86 ppm, assigned to the two protons at position 5 of this family
of compound 5H‐indeno[1,2‐b]pyridine. The remaining aromatic
tives, via a one‐pot synthesis from elementary starting materials as
1‐indanone methoxy‐substituted, aromatic aldehydes‐substituted,
malononitrile, ammonium acetate, and acetic acid (catalytic) in to-
luene, and their evaluation as potential anticancer agents against
human PCa cells PC‐3 and LNCaP in vitro. Inhibitory effects were
also observed on the adhesion, migration, and invasion of the PCa
(a)
SCHEME 1 (a) Three‐component process
for the synthesis of 5H‐indeno[1,2‐b]pyridine
derivatives 5–21. (b) Synthesis of 5H‐indeno
[1,2‐b]pyridines 5–21 in a stepwise manner.
Reagents and conditions: (i) Toluene,
AcONH4, AcOH, Δ, 5 h; (ii) EtOH, room
temperature, 24 h; (iii) toluene, AcONH4,
AcOH, Δ, 12 h
(b)

CHARRIS ET AL.
3 of 12
|
SCHEME 2 A plausible mechanism for the synthesis of compounds 5–21
TABLE 1 Various substitutions, yield, and melting point data of synthesized derivatives 5–21
Compound no.
Ar
R1
R2
R3
R4
H
Yield %b
78
Yield %a
91
m.p. (°C)
228–230
304–306
286–288
272–274
300–302
276–278
288–290
258–260
278–280
262–264
>310
5
3‐OCH3‐4OH–C6H3
3‐OCH3‐4‐OH–C6H3
3‐OCH3‐4‐OH–C6H3
3‐OCH3‐4‐OH–C6H3
3‐OCH3‐4‐OH–C6H3
3‐OCH3‐4‐OH–C6H3
(3,4,5‐OCH3)–C6H2
(3,4,5‐OCH3)–C6H2
(3,4,5‐OCH3)–C6H2
(3,4,5‐OCH3)–C6H2
(3,5‐OCH3)‐4‐OH–C6H2
(3,5‐OCH3)‐4‐OH–C6H2
(3,5‐OCH3)‐4‐OH–C6H2
(3,4‐OCH3)–C6H3
H
H
H
6
H
H
H
OCH3
H
74
93
7
H
OCH3
H
H
70
92
8
OCH3
H
H
H
67
88
9
OCH3
OCH3
H
OCH3
H
H
74
90
10
11
12
13
14
15
16
17
18
19
20
21
OCH3
H
H
67
83
H
H
74
90
H
H
H
OCH3
H
74
87
H
OCH3
OCH3
H
OCH3
H
73
84
OCH3
H
H
78
85
H
H
65
86
H
OCH3
OCH3
H
OCH3
H
H
72
89
>310
OCH3
H
H
68
79
246–248
218–220
234–236
240–242
272–274
H
OCH3
H
83
92
(3,4‐OCH3)–C6H3
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
77
83
(2,4‐OCH3)–C6H3
H
H
72
89
4‐OCH3‐3‐OH–C6H3
OCH3
H
75
84
aThree‐component process yield % for compounds 5–21.
bStepwise yield % for compounds 5–21.

4 of 12
CHARRIS ET AL.
|
protons of the indeno and aldehydic moiety are reported according
to the substitution pattern, respectively, whereas two protons of
–NH2 group appear as a broad singlet (brs) between 5.20 and
6.90 ppm in each compound. The 13C NMR spectrum of the same
compounds exhibits signals between 31 and 39 ppm, assigned to C5,
between 85 and 88 ppm, assigned to C–CN, and at 118 ppm, as-
signed to –CN, which were also confirmed by DEPT 135°, HETCOR,
and FLOCK experiments (see the Supporting Information). The
analytical data for all compounds are summarized in Section 4.
To compare the effect of multicomponent strategy on the re-
action yield, it was considered worthwhile to attempt the synthesis
of 5–21 in a stepwise manner (Scheme 1b).
cells derived from the left supraclavicular lymph node metastasis
(LNCaP), and prostate tumor cells derived from rats (MatLyLu). From a
total of 17 compounds, 7 were cytotoxic (IC50 <200 µM), inhibiting PC‐
3, LNCaP, and MatLyLu cell viability, and the most active compound 10
was also active against nontumor prostate cells, benign hyperplastic
prostatic epithelial cells (BPH‐1); however, it showed a more specific
response to tumor cells than dequalinium,
a drug used as a
reference.[33,34] The results are summarized in Table 2.
The effect of compound 10 on cell viability showed a dose‐
dependent response in all the cell lines tested with inhibition in
viability from 15 µM onward. The activity of this compound con-
centration also followed a time‐dependent manner at its cytotoxic
IC50 (Figure 1).
The 2‐arylidenemalononitile derivatives 4a–f were achieved ac-
cording to published procedures[29–32] (see the Supporting Information),
and the addition of 1‐indanone respective 3 in the presence of am-
monium acetate and catalytic amount of acetic acid in toluene at reflux
temperature yielded the 5H‐indeno[1,2‐b]pyridines 5–21. The results
reveal that there was a considerable improvement in the yields ob-
tained when the multicomponent strategy was used (see Table 1). The
products were identified by spectra data, and the structures were
further confirmed by comparison with data obtained previously.
|
2.2.2
Effect, in vitro, on adhesion, propagation,
migration, and invasion of PCa cells after exposure to
compound 10
The effect of compound 10 on cell attachment and spreading at their
cytotoxic IC50 concentration is shown in Figure 2. These results
clearly demonstrated the extent of cell attachment and spreading at
an incubation period of 0–3 h, showing that compound 10 is an in-
hibitor of cell adhesion. The measurements were performed on the
real‐time analyzer xCELLigence.
|
2.2
Biological evaluation
|
2.2.1
Effect on cell viability and cell growth of
The wound‐healing assay was used to examine the effect of
compound 10 on the migration properties of the tumor cells.[35]
Compound‐free cultures of PC‐3 cell line used as controls largely
displayed wound recovery within 24 h and cells migrated to the
wound (Figure 3). As shown in Figure 4, compound 10 after 18‐h
incubation period also significantly decreases the invasion of LNCaP
cell line using the Boyden chamber coated with Matrigel.
derivatives 5–21
The synthesized 5H‐indeno[1,2‐b]pyridine derivatives 5–21 were
tested for their ability to inhibit the viability of human and murine PCa
cell lines: advanced androgen‐independent bone metastasis prostate
cancer (PC‐3), androgen‐sensitive human prostate adenocarcinoma
TABLE 2 The antiproliferative activity
BPH‐1
Compound no.
PC‐3
LNCaP
MatLyLu
of compounds 5–21 (µM) against PC‐3,
9
119.16 1.54
45.25 0.23**
111.39 0.04
58.56 1.39*
88.75 0.47
56.78 1.23**
66.30 0.17**
46.62 4.21**
136.94 1.18
18.35 0.08**
196.39 0.28
111.18 0.05
67.11 0.93
60.10 0.11**
37.01 1.97**
9.36 1.25**
131.26 0.77
201.59 1.63
LNCaP, MatLyLu, and BPH‐1 cell lines
10
13
14
16
17
21
DQA
26.24 1.13**
>250
92.87 0.15
>250
>50 1.37
>250
85.77 0.27
185.04 1.51
58.98 0.17
122.63 1.29
19.58 4.11
40.08 0.03**
31.66 1.75**
>50
Note: For 5–8, 11, 12, 15, 18–20 IC50 >200 (µM). Results are expressed as the mean SEM of the
half‐maximal inhibitory concentration or IC50 (µM). Each experiment was performed three times in
five different wells.
Abbreviations: BPH‐1, benign hyperplastic prostatic epithelial cells; DQA, dequalinium; LNCaP,
androgen‐sensitive human prostate adenocarcinoma cells derived from the left supraclavicular
lymph node metastasis; MatLyLu, prostate tumor cells derived from rats; PC‐3, advanced
androgen‐independent bone metastasis prostate cancer.
*p < .05.
**p < .001 compared with BPH‐1 cells treated with the compounds.

CHARRIS ET AL.
5 of 12
|
FIGURE 1 Time–response effects of compound 10 at its cytotoxic IC50 (Table 2) concentration on PC‐3, LNCaP, and MatLyLu cell growth.
Results are expressed as the mean SEM. *p < .05; **p < .01; and ***p < .001
FIGURE 2 Effect of compound 10 on cell
adhesion and spreading. PC‐3 cells were
incubated with the compound at its cytotoxic
IC50 (Table 2). The cells were applied in
triplicate to the fibronectin‐coated sensors,
and their attachment and spreading were
quantified by real‐time cell electronic sensing
as cell index values (CI) after treatments.
Medium–dimethyl sulfoxide (0.2%) alone
served as the control (vehicle). The results
represent the mean SEM of three
independent experiments.
***p < .01 compared with control vehicle
|
2.2.3
Inhibition of the activity of MMP‐9 against
(Figure 5a,b). We could not detect any MMP‐2 activity in both cell
PCa cell lines for compound 10
lines tested, and gelatin zymography was used to observe activities.
MMP‐2 and MMP‐9 are the two major gelatinases expressed in PCa
cells.[24,36] We hypothesized that inhibited migration and invasion of
PC‐3 and LNCaP cell lines may be originated from the inhibition of
those MMPs. The result shows the stronger effect of compound
10 inhibiting the activity of MMP‐9 in PC‐3 and LNCaP cell lines
|
2.2.4
Effect on colony formation for compound 10
The ability of PC‐3 and LNCaP cell lines to grow in a semi‐soft agar
medium was examined, using the anchorage‐independent growth in

6 of 12
CHARRIS ET AL.
|
FIGURE 3 Effect of compound 10 on PC‐3
wound closure in a single scrape wound
model; cells were incubated with the
compound at its cytotoxic IC50 (Table 2).
Representative images of PC‐3 cells were
captured at the time of wounding and 24 h
after that to illustrate recovery from a scrape
wound. The images presented are from a
representative experiment carried out
independently three times
FIGURE 4 Effect of compound 10 on the
invasion of LNCaP cells. LNCaP cells were
incubated with the compound at its cytotoxic
IC50 (Table 2). Cells treated with compound
10 or the vehicle were seeded on a Matrigel‐
coated 0.8‐µm porous membrane for 18 h, and
the inhibition of invasion, relative to the
control vehicle‐treated cells, was determined.
The results represent the mean SEM of three
independent experiments. ***p < .01
compared with control vehicle
vitro that correlates with tumorigenesis in nude mice.[37] Cells
treated with compound 10 showed a significant decrease in the
number and size of the colonies (Figure 6a,b). A significant growth in
colonies formed in soft agar was shown by the control vehicle on Day
14 of the incubation period, as previously reported by others.[38]
2.2.5
Toxicity of compound 10 on brine shrimps
|
The brine shrimp toxicity bioassay was used to determine the toxi-
city of compound 10.[39] The results show that 10 was less toxic to
brine shrimps as compared with PC‐3, LNCaP, and MatLyLu tumor

CHARRIS ET AL.
7 of 12
|
TABLE 3 Toxicological effect of compound 10 on brine shrimp
TAR
TAR
TAR
Compound no. LC50 (µM)
10
PC‐3
LNCaP
MatLyLu
302.12 1.93 6.7
16.5
11.5
Note: The toxicological effect was tested by the brine shrimp bioassay.
LC50 represents the half lethal concentration in the brine shrimp assay,
respectively. The toxicity‐to‐antitumor ratio (TAR) is defined by the value
resulted of the LC50 divided by the IC50 values (Table 2) of the
corresponding compound for PC‐3, LNCaP, or MatLyuLu cell lines.
|
3
CONCLUSION
FIGURE 5 Activity of MMP‐9 metalloproteinase by gelatin
zymography in PC‐3 (a) and in LNCaP cells (b) when exposed to
compound 10 for 24 h. Cell lines were incubated with the compound
at its cytotoxic IC50 (Table 2). Conditioned medium prepared from
subconfluent cultures was collected, resolved in nonreducing gels
containing gelatin (1 mg/ml), and processed for zones of gel
degradation activity. Only the results in PC‐3 cells were quantified in
relation to control vehicle and are presented as the mean SEM of
the percentage of activity in three different experiments (b).
***p < .001 compared with control vehicle
In summary, our study describes the synthesis of 2‐amino‐4‐
(phenylsubstituted)‐5H‐indeno[1,2‐b]pyridine‐3‐carbonitrile deriva-
tives through sequential multicomponent reaction of aromatic alde-
hydes, malononitrile, and 1‐indanones, as well as their anticancer
evaluation. The effect of one‐pot method in the generation of the
target product has been studied by thin‐layer chromatography, IR,
1H NMR, 13C NMR, DEPT 130°, HETCOR, FLOCK. However, the
biological study showed that compound 10 significantly impeded the
proliferation of PC‐3, LNCaP, and MatLyLu cell lines. The antimeta-
static effects of 10 could be related with the inhibition of MMP9 in
PC‐3 and LNCaP human cell lines; these results coincide with the
results previously reported for analog molecules.[11,24,40]
cell lines (Table 3). The toxicity‐to‐antitumor ratio (TAR) was used to
confirm the lower toxicological effect of compound 10. The TAR is
defined as the ratio of the LC50 concentration in the brine shrimp
assay to the IC50 antitumor cytotoxicity concentration, which was
always higher than 1; thus, the tumor cells were more sensitive to
compound 10.
A
study of the structure–activity relationship of these
compounds allows us to propose that the presence of two
methoxy groups at positions 6, 7 of the indeno nucleus and a
(a)
(b)
FIGURE 6 Effect of compound 10 at its
cytotoxic IC50 (Table 2) on the formation of
colonies in soft agar. PC‐3 (a) and LNCaP cells
(b) were plated over a semi‐solid layer of soft
agar treated with the compound and
incubated for 14 days. The results represent
standard images of three different
experiments

8 of 12
CHARRIS ET AL.
|
4‐hydroxy‐3‐methoxy phenyl substitution pattern at position 4 of the
pyridine ring is decisive for these types of molecules to exert a very
good antiproliferative activity. The activity is affected when the
substitution pattern in the phenyl group at position 4 of pyridine
changes to 3‐hydroxy‐4‐methoxy, compound 21, and is marginalized
when another methoxy group is introduced at position 5. Other
substitution patterns with these same groups in both the indeno ring
and the phenyl ring significantly affect the antiproliferative activity
of these types of compounds. This study shows the importance of
5H‐indeno[1,2‐b]pyridine derivatives as potential anticancer agents,
to improve PC chemotherapy by inhibiting the metastasis process.
Other studies will be required to explore an exact mechanism of
action that allows us to clarify the roles of functional groups in
anticancer activity in this type of nucleus.
2‐Amino‐4‐(4ʹ‐hydroxy‐3ʹ‐methoxyphenyl)‐5H‐indeno[1,2‐b]-
pyridine‐3‐carbonitrile (5)
IR KBr (cm−1): 3372, 3326, 2205, 1554, 1510; 1H NMR (CDCl3) δ
ppm: 3.85 (s, 2H, H5), 3.95 (s, 3H, OCH3), 5.83 (s, 2H, OH), 7.04–7.08
(m, 3H, Ar–H), 7.36–7.52 (m, 4H, Ar–H), 8.40 (brs, 2H, NH2); 13C
NMR (DMSO‐d6) δ ppm: 39.5, 56.50, 113.3, 116.2, 120.8, 121.9,
125.9, 127.6, 127.9, 128.8, 133.9, 141.3, 143.8, 144.9, 147.4, 148.3;
anal. calcd. for C20H15N3O2: % C, 72.94; H, 4.59; N, 12.76. Found: %
C, 73.01; H, 4.63; N, 12.95.
2‐Amino‐9‐methoxy‐4‐(4ʹ‐hydroxy‐3ʹ‐methoxyphenyl)‐5H‐indeno-
[1,2‐b]pyridine‐3‐carbonitrile (6)
IR KBr (cm−1): 3356, 3312, 2224, 1593, 1555; 1H NMR (DMSO‐d6) δ
ppm: 3.74 (s, 2H, H5), 3.83 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.60 (s,
2H, NH2), 6.92 (d, 1H, H5ʹ, J = 8.2 Hz), 7.01–7.05 (m, 2H, Ar–H),
7.14–7.18 (m, 2H, Ar–H), 7.38 (t, 1H, H7, J = 7.9 Hz), 9.42 (s, 1H, OH);
13C NMR (DMSO‐d6) δ ppm: 34.0, 56.2, 56.4, 85.8, 110.6, 113.4,
116.1, 118.1, 118.6, 122.2, 123.6, 127.1, 127.7, 131.6, 148.0, 148.1,
148.9, 149.6, 157.0, 162.0, 162.8; anal. calcd. for C21H17N3O3: % C,
70.18; H, 4.77; N, 11.69. Found: % C, 70.19; H, 4.77; N, 11.81.
|
4
EXPERIMENTAL
Chemistry
|
4.1
4.1.1
|
General
2‐Amino‐7‐methoxy‐4‐(4ʹ‐hydroxy‐3ʹ‐methoxyphenyl)‐5H‐indeno-
[1,2‐b]pyridine‐3‐carbonitrile (7)
Thin‐layer chromatography was carried out on Merck silica F254
0.255‐mm plates, and spots were visualized by UV fluorescence at
254 nm. Elemental analyses were performed using a PerkinElmer
2400 CHN elemental analyzer. The results were within 0.4% of the
predicted values. NMR spectra were obtained using a JEOL Eclipse™
at 270 MHz for 1H NMR and at 67.9 MHz for 13C NMR using CDCl3
or DMSO‐d6, and are reported in ppm downfield from the residual
IR KBr (cm−1): 3376, 3299, 2208, 1600, 1561; 1H NMR (DMSO‐d6) δ
ppm: 3.73 (s, 2H, H5), 3.83 (s, 6H, OCH3), 6.75 (brs, 2H, NH2), 6.91 (d,
1H, H5ʹ, J = 8.2 Hz), 7.01–7.07 (m, 2H, Ar–H), 7.17–7.20 (m, 2H,
Ar–H), 7.76 (d, 1H, H9, J = 8.4 Hz), 9.46 (s, 1H, OH); 13C NMR
(DMSO‐d6) δ ppm: 33.9, 56.0, 56.4, 85.6, 110.9, 113.4, 114.7, 116.1,
118.7, 122.2, 122.9, 123.3, 127.1, 133.0, 148.0, 148.1, 148.9, 148.9,
150.1, 162.4, 162.6; anal. calcd. for C21H17N3O3: % C, 70.18; H, 4.77;
N 11.69. Found: % C, 70.25; H, 4.83; N, 11.89.
CHCl3 or DMSO (δ 7.25 or 2.50 for 1H NMR and 77.0 or 39.8 for 13
C
NMR, respectively) (see the Supporting Information for the original
spectra). A Nicolet™ IS5 FT‐IR (ID3 Zn‐Se) spectrophotometer was
used to determine the IR spectra. A Thomas micro hot‐stage device
was used to determine the melting points (mp). All organic products
or solvents (from Sigma‐Aldrich Group) were used directly or
distilled and dried in the usual manner, respectively.
2‐Amino‐6‐methoxy‐4‐(4ʹ‐hydroxy‐3ʹ‐methoxyphenyl)‐5H‐indeno-
[1,2‐b]pyridine‐3‐carbonitrile (8)
IR KBr (cm−1): 3378, 3312, 2210, 1558, 1507; 1H NMR (DMSO‐d6) δ
ppm: 3.73 (s, 2H, H5), 3.87 (s, 6H, OCH3), 6.96–7.06 (m, 4H, Ar–H,
NH2), 7.20 (dd, 1H, H6ʹ, J1 = 1.7 Hz, J2 = 8.0 Hz), 7.34 (d, 1H, H2ʹ,
J = 1.7 Hz), 7.4 (t, 1H, H8, J1 = 7.4 Hz), 7.67 (d, 1H, H9, J = 7.4 Hz),
9.33 (s, 1H, OH); 13C NMR (DMSO‐d6) δ ppm: 31.8, 55.8, 56.6,
85.8, 110.9, 110.9, 111.1, 113.4, 113.5, 116.2, 116.9, 118.6, 122.1,
129.5, 131.7, 133.8, 142.8, 148.3, 156.5, 161.9, 162.7; anal. calcd.
for C21H17N3O3: % C, 70.18; H, 4.77; N, 11.69. Found: % C, 70.22;
H, 4.79; N, 11.91.
The InChI codes of the investigated compounds, together with
some biological data, are provided as Supporting Information.
|
4.1.2
General procedure for the synthesis of
2‐amino‐4‐(phenylsubstituted)‐5H‐indeno[1,2‐b]-
pyridine‐3‐carbonitrile 5–21
Benzaldehyde‐substituted
1
(1 mmol), malononitrile
2
(1 mmol),
2‐Amino‐7,8‐dimethoxy‐4‐(4ʹ‐hydroxy‐3ʹ‐methoxyphenyl)‐5H‐
indeno[1,2‐b]pyridine‐3‐carbonitrile (9)
1‐indanone 3 (1 mmol), and ammonium acetate (1.5 mmol), in 10 ml
of toluene, with catalytic amount of acetic acid, were combined
in a 20‐ml round‐bottom flask fitted with a reflux condenser and
Dean–Stark trap. The mixture was heated under reflux for 5 h and
IR KBr (cm−1): 3374, 3340, 2215, 1605, 1504; 1H NMR (DMSO‐d6) δ
ppm: 3.66 (s, 2H, H5), 3.82 (s, 6H, OCH3), 3.83 (s, 3H, OCH3), 6.72
(brs, 2H, NH2), 6.91 (d, 1H, H5ʹ, J = 8.2 Hz), 7.03 (dd, 1H, H6ʹ,
J1 = 1.9 Hz, J2 = 8.2 Hz), 7.19 (d, 1H, H2ʹ, J = 1.9 Hz), 7.22 (s, 1H, H6),
7.31 (s, 1H, H9), 9.46 (s, 1H, OH); 13C NMR (DMSO‐d6) δ ppm: 34.5,
56.1, 56.3, 56.4, 85.2, 103.8, 109.0, 113.4, 116.0, 118.8, 122.2, 123.8,
127.1, 132.4, 140.2, 147.9, 148.1, 149.4, 151.7, 162.4, 163.1; anal.
the solvent was then evaporated using
a rotary evaporator.
The residue was then poured into crushed ice. The desired product
was isolated, filtered, and washed with cold water–ethanol (1:1).
The solid was recrystallized from ethanol.

CHARRIS ET AL.
9 of 12
|
calcd. for C22H19N3O4: % C, 67.86; H, 4.92; N, 10.79. Found: % C,
67.86; H, 4.94; N, 10.93.
NMR (DMSO‐d6) δ ppm: 31.8, 56.7, 56.8, 60.3, 60.6, 85.1, 106.5,
113.2, 116.5, 118.6, 132.5, 135.3, 136.9, 143.7, 145.8, 153.0, 153.8,
159.5, 160.1, 162.3; anal. calcd. for C24H23N3O5: % C, 66.50; H, 5.35;
N, 9.69. Found: % C, 66.56; H, 5.41; N, 9.91.
2‐Amino‐6,7‐dimethoxy‐4‐(4ʹ‐hydroxy‐3ʹ‐methoxyphenyl)‐5H‐
indeno[1,2‐b]pyridine‐3‐carbonitrile (10)
IR KBr (cm−1): 3356, 3344, 2211, 1603, 1555; 1H NMR (DMSO‐d6) δ
ppm: 3.35 (s, 2H, H5), 3.79 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.83 (s,
3H, OCH3), 6.75 (brs, 2H, NH2), 6.93 (d, 1H, H5ʹ, J = 8.0 Hz), 7.03 (dd,
1H, H6ʹ, J1 = 1.0 Hz, J2 = 8.0 Hz), 7.18 (m, 2H, H2ʹ, 6), 7.57 (d, 1H, H9,
J = 8.4 Hz), 9.46 (s, 1H, OH); 13C NMR (DMSO‐d6) δ ppm: 31.0, 56.4,
56.6, 60.3, 85.9, 113.4, 113.4, 116.1, 117.7, 122.2, 123.3, 126.9,
134.1, 138.7, 145.6, 148.0, 148.2, 150.5, 154.1, 162.3, 162.4; anal.
calcd. for C22H19N3O4: % C, 67.86; H, 4.92; N, 10.79. Found: % C,
67.90; H, 4.97; N, 10.89.
2‐Amino‐4‐(4ʹ‐hydroxy‐3ʹ,5ʹ‐dimethoxyphenyl)‐5H‐indeno[1,2‐b]-
pyridine‐3‐carbonitrile (15)
IR KBr (cm−1): 3388, 3317, 2219, 1603, 1552; 1H NMR (DMSO‐d6) δ
ppm: 3.86 (s, 3H, OCH3), 3.87 (s, 2H, H5), 3.96 (s, 3H, OCH3), 7.03 (s,
2H, H2ʹ, 6ʹ), 7.41–7.52 (m, 4H, Ar–H, NH2), 7.60 (d, 1H, H6, J = 7.2 Hz),
8.10 (d, 1H, H9, J = 7.2 Hz), 8.66 (s, 1H, OH); 13C NMR (DMSO‐d6) δ
ppm: 34.5, 56.6, 56.9, 85.6, 106.8, 118.4, 120.8, 125.9, 127.6, 128.8,
133.9, 141.4, 144.9, 148.8, 152.8, 160.1, 162.0; anal. calcd. for
C21H17N3O3: % C, 70.18; H, 4.77; N, 11.69. Found: % C, 70.24; H,
4.80; N, 11.85.
2‐Amino‐4‐(3ʹ,4ʹ,5ʹ‐trimethoxyphenyl)‐5H‐indeno[1,2‐b]pyridine‐3‐
carbonitrile (11)
2‐Amino‐7,8‐dimethoxy‐4‐(4ʹ‐hydroxy‐3ʹ,5ʹ‐dimethoxyphenyl)‐5H‐
indeno[1,2‐b]pyridine‐3‐carbonitrile (16)
IR KBr (cm−1): 3343, 3260, 2207, 1638, 1561; 1H NMR (CDCl3) δ
ppm: 3.86 (s, 2H, H5), 3.94 (s, 3H, OCH3), 3.99 (s, 6H, OCH3),
7.03–713 (m, 3H, H2ʹ, 6ʹ, Ar), 7.37–7.53 (m, 3H, Ar–H), 8.41 (brs, 2H,
NH2); 13C NMR (DMSO‐d6) δ ppm: 39.5, 57.0, 61.2, 106.7, 106.9,
121.0, 126.2, 127.9, 129.1, 134.3, 137.9, 141.4, 143.9, 145.2, 147.4,
153.7, 153.9, 159.9; anal. calcd. for C22H19N3O3: % C, 70.76; H, 5.13;
N, 11.25. Found: % C, 70.78; H, 5.17; N, 11.57.
IR KBr (cm−1): 3392, 3317, 2215, 1616, 1518; 1H NMR (DMSO‐d6) δ
ppm: 3.71 (s, 2H, H5), 3.81 (s, 6H, OCH3), 3.82 (s, 3H, OCH3), 3.84 (s,
3H, OCH3), 6.73 (s, H, NH2), 6.89 (s, 2H, H2ʹ, 6ʹ), 7.23 (s, 1H, H6), 7.32
(s, 1H, H9), 8.81 (s, 1H, OH); 13C NMR (DMSO‐d6) δ ppm: 34.5, 56.1,
56.3, 56.8, 85.3, 103.8, 107.0, 109.0, 118.8, 123,9, 126.1, 132.4,
137.1, 140.3, 148.4, 149.4, 149.9, 151.7, 162.3, 163.2; anal. calcd. for
C23H21N3O5: % C, 65.86; H, 5.05; N, 10.02. Found: % C, 65.89; H,
2‐Amino‐9‐methoxy‐4‐(3ʹ,4ʹ,5ʹ‐trimethoxyphenyl)‐5H‐indeno[1,2‐b]-
pyridine‐3‐carbonitrile (12)
5.09; N, 9.97.
IR KBr (cm−1): 3392, 3312, 2210, 1602, 1494; 1H NMR (DMSO‐d6) δ
ppm: 3.74 (s, 2H, H5), 3.77 (s, 3H, OCH3), 3.83 (s, 6H, OCH3), 3.92 (s,
3H, OCH3), 6.69 (brs, 2H, NH2), 6.91 (s, 2H, H2ʹ, 6ʹ), 7.03 (d, 1H, H6,
2‐Amino‐6,7‐dimethoxy‐4‐(4ʹ‐hydroxy‐3ʹ,5ʹ‐dimethoxyphenyl)‐5H‐
indeno[1,2‐b]pyridine‐3‐carbonitrile (17)
IR KBr (cm−1): 3324, 3234, 2215, 1609, 1558; 1H NMR (DMSO‐d6) δ
ppm: 3.76 (s, 2H, H5), 3.80 (s, 6H, OCH3), 3.81 (s, 6H, OCH3), 3.87 (s,
3H, OCH3), 6.76 (brs, 2H, NH2), 6.89 (s, 2H, H2ʹ, 6ʹ), 7.19 (d, 1H, H8,
J = 8.2 Hz), 7.58 (d, 1H, H9, J = 8.2 Hz), 8.81 (brs, 1H, OH); 13C NMR
(DMSO‐d6) δ ppm: 31.3, 56.7, 56.9, 60.3, 86.1, 107.0, 113.4, 117.7,
118.6, 123.4, 126.0, 134.1, 137.2, 138.6, 145.6, 148.5, 150.7, 154.1,
162.3, 162.4; anal. calcd. for C23H21N3O5: % C, 65.86; H, 5.05; N,
10.02. Found: % C, 65.89; H, 5.07; N, 10.27.
J = 7.6 Hz), 7.16 (d, 1H, H8, J = 7.6 Hz), 7.40 (t, 1H, H7, J = 7.6 Hz); 13
C
NMR (DMSO‐d6) δ ppm: 33.9, 56.1, 56.7, 60.6, 85.6, 106.7, 110.5,
118.1, 118.4, 123.6, 126.4, 127.6, 131.7, 138.4, 148.9, 149.4, 154.5,
157.0, 161.9, 163.0; anal. calcd. for C23H21N3O4: % C, 68.47; H, 5.25;
N. 10.42. Found: % C, 68.51; H, 5.27; N, 10.61.
2‐Amino‐7,8‐dimethoxy‐4‐(3ʹ,4ʹ,5ʹ‐trimethoxyphenyl)‐5H‐indeno-
[1,2‐b]pyridine‐3‐carbonitrile (13)
IR KBr (cm−1): 3317, 3290, 2203, 1632, 1552; 1H NMR (DMSO‐d6) δ
ppm: 3.69 (s, 2H, H5), 3.75 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.83 (s,
6H, OCH3), 3.84 (s, 3H, OCH3), 6.80 (brs, 2H, NH2), 6.93 (s, 2H,
H2ʹ, 6ʹ), 7.22 (s, 1H, H6), 7.33 (s, 1H, H9); 13C NMR (DMSO‐d6) δ ppm:
34.5, 56.0, 56.3, 56.7, 60.7, 85.1, 103.8, 106.8, 108.9, 118.6, 123.9,
131.7, 132.7, 138.4, 140.3, 149.4, 150.2, 151.8, 153.4, 162.3, 163.3;
anal. calcd. for C24H23N3O5: % C, 66.50; H, 5.35; N, 9.69. Found: % C,
66.53; H, 5.38; N, 9.87.
2‐Amino‐9‐methoxy‐4‐(3ʹ,4ʹ‐dimethoxyphenyl)‐5H‐indeno[1,2‐b]-
pyridine‐3‐carbonitrile (18)
IR KBr (cm−1): 3340, 3328, 2208, 1593, 1504; 1H NMR (DMSO‐d6) δ
ppm: 3.73 (s, 2H, H5), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.92 (s,
3H, OCH3), 6.65 (s, 2H, NH2), 7.04 (d, 1H, H5ʹ, J = 8.2 Hz), 7.09–7.19
(m, 4H, Ar–H), 7.42 (t, 1H, H7, J = 7.8 Hz); 13C NMR (DMSO‐d6) δ
ppm: 33.9, 56.1, 56.3, 56.9, 85.7, 103.8, 110.6, 112.2, 112.9, 118.1,
118.4, 121.9, 123.7, 127.5, 128.6, 131.7, 148.9, 149.1, 149.9, 157.0,
161.9, 163.9; anal. calcd. for C22H19N3O3: % C, 70.76; H, 5.13; N,
11.25. Found: % C, 70.80; H, 5.16; N, 11.42.
2‐Amino‐6,7‐dimethoxy‐4‐(3ʹ,4ʹ,5ʹ‐trimethoxyphenyl)‐5H‐indeno-
[1,2‐b]pyridine‐3‐carbonitrile (14)
IR KBr (cm−1): 3324, 3298, 2208, 1615, 1488; 1H NMR (DMSO‐d6) δ
ppm: 3.74 (s, 2H, H5), 3.76 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.83 (s,
6H, OCH3), 3.88 (s, 3H, OCH3), 6.91 (brs, 2H, NH2), 6.99 (s, 2H,
H2ʹ, 6ʹ), 7.17 (d, 1H, H8, J = 8.2 Hz), 7.75 (d, 1H, H9, J = 8.2 Hz); 13C
2‐Amino‐7,8‐dimethoxy‐4(3ʹ,4ʹ‐dimethoxyphenyl)‐5H‐indeno[1,2‐b]-
pyridine‐3‐carbonitrile (19)
IR KBr (cm−1): 3312, 3283, 2212, 1635, 1552; 1H NMR (DMSO‐d6) δ
ppm: 3.67 (s, 2H, H5), 3.81 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.84 (s,

10 of 12
CHARRIS ET AL.
|
6H, OCH3), 6.76 (brs, 2H, NH2), 7.09 (d, 1H, H5ʹ, J = 8.2 Hz), 7.15 (dd,
1H, J1 = 1.1 Hz, J2 = 8.2 Hz H6ʹ), 7.22 (s, 2H, H2ʹ, 6), 7.32 (s, 1H, H9);
13C NMR (DMSO‐d6) δ ppm: 31.3, 56.1, 56.3, 56.6, 57.2, 85.2, 103.8,
108.9, 112.2, 112.8, 118.7, 121.9, 123.9, 128.6, 132.3, 140.2, 149.1,
149.4, 149.9, 162.3, 163.2; anal. calcd. for C23H21N3O4: % C, 68.47;
H, 5.25; N, 10.42. Found: % C, 68.48; H, 5.26; N, 10.57.
The time‐dependent effects of the best compound on cell growth
were measured according to a previous procedure described.[42] The
PC‐3 (1 × 105), LNCaP (2.4 × 105), or MatLyLu (1.5 × 104) cell lines
were seeded in six‐well plates in RPMI containing 10% fetal bovine
serum (FBS) and the compound at its cytotoxic IC50 (37°C, 95%
O2, 5% CO2). Cells were collected from culture dishes after
trypsin–EDTA treatment for 7 min at 37°C. A hemocytometer was
used for counting the number of viable cells at 24 h intervals for a
period of 96 h.
2‐Amino‐7,8‐dimethoxy‐4(2ʹ,4ʹ‐dimethoxyphenyl)‐5H‐indeno[1,2‐b]-
pyridine‐3‐carbonitrile (20)
IR KBr (cm−1): 3372, 3296, 2208, 1596, 1571; 1H NMR (DMSO‐d6) δ
ppm: 3.77 (s, 2H, H5), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.85 (s,
6H, OCH3), 6.64–6.68 (m, 3H, H5ʹ, NH2), 6.72 (d, 1H, H3ʹ, J = 2.2 Hz),
7.18 (s, 1H, H6), 7.26 (d, 1H, H6ʹ, J = 8.40 Hz), 7.30 (s, 1H, H9); 13C
NMR (DMSO‐d6) δ ppm: 31.3, 55.9, 56.1, 56.3, 87.1, 99.4, 103.8,
105.9, 109.1, 117.4, 118.3, 125.2, 131.3, 132.4, 139.8, 147.2, 149.5,
151.6, 157.8, 161.8, 161.9, 162.8; anal. calcd. for C23H21N3O4: % C,
68.47; H, 5.25; N, 10.42. Found: % C, 68.51; H, 5.29; N, 10.61.
|
4.2.2
Cell adhesion assay
An xCELLigence Real‐Time Cell Analyzer from Applied Science
Roche was used to perform measurements. The system monitors the
biological status of cells as cell number and adhesion by measuring
electrical impedance via microelectrodes fixed at the bottom of
special 96‐well tissue plates. The analyzer automatically measures
the electrical impedance as cell index (CI), which is transferred,
analyzed, and processed by the integrated software.[43] Under the
same physiological conditions, a larger impedance would result in a
larger CI number, a consequence that more cells have been attached
to the electrodes in the plate. Results are expressed as CI and re-
lative attachment and spreading. For the measurements, the special
96‐well ACEA E‐plates® were coated with fibronectin (20 µg/ml), 1 h
at 37°C. With a phosphate‐buffered saline (PBS), the plates were
washed and coated with bovine serum albumin solution in PBS
(0.5%) for 20 min at 37°C, after which each well was washed with
PBS. Next, 50 µl of the medium was added to record the background,
and then 100 μl of PC‐3 cell suspension (5 × 105 cells) was trans-
ferred to each well of the ACEA E‐plates® and 50 µl of the most
active compound 10 at its cytotoxic IC50 (Table 2) was added.
The spreading and adhesion of the cells were monitored every
minute using this real‐time cell electronic sensing system according
to the manufacturer's instructions for a period up to 4 h.[22,35]
2‐Amino‐6,7‐dimethoxy‐4‐(3‐hydroxy‐4‐methoxyphenyl)‐5H‐indeno-
[1,2‐b]pyridine‐3‐carbonitrile (21)
IR KBr (cm−1): 3376, 2208, 1616, 1555, 1497; 1H NMR (DMSO‐d6) δ
(ppm): 3.59 (s, 2H, H5), 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.85
(s, 3H, OCH3), 6.76 (brs, 2H, NH2), 7.02–7.09 (m, 3H, H2ʹ, 5ʹ, 6ʹ),
7.21 (d, 1H, H8, J = 8.2 Hz), 7.28 (d, 1H, H9, J = 8.2 Hz), 9.32 (s, 1H,
OH); 13C NMR (DMSO‐d6) δ (ppm): 33.6, 56.1, 56.3, 56.7, 86.1,
103.8, 108.9, 112.6, 116.3, 118.7, 120.4, 123.6, 128.7, 132.3,
140.1, 146.9, 148.9, 149.5, 151.6, 162.2, 163.0; anal. calcd. for
C
22H19N3O4: % C, 67.86; H, 4.92; N, 10.79. Found: % C, 67.88; H,
4.95; N, 11.01.
|
4.2
Biology
|
4.2.1
Effect of compounds 5–21 on cell viability
and cell growth
A 96‐well microtiter plate (tissue culture grade) containing 0.1 ml of
growth medium/well (RPMI) was seeded with PC‐3 (5 × 103), LNCaP
(1.2 × 104), or MatLyLu (5 × 102) prostate tumor cell lines. The cells
were exposed after 24 h of culture to the cytotoxic action of com-
pounds 5–21 dissolved in dimethylsulfoxide (DMSO) for 72 h at
concentrations ranging from 15 to 250 µM. The final concentration
of DMSO in the culture medium was always lower than 0.2%.
After the incubation time, cells were incubated with sodium
3‐[1‐(phenylaminocarbonyl)‐3,4‐tetrazolium]‐bis(4‐methoxy‐6‐nitro)
benzene sulfonic acid hydrate (XTT)[11,41] at 37°C for 4 h, and the
colorimetric detection of the orange product formazan was regis-
tered at 492 nm (Tecan SpectraFluor). The cytotoxic IC50 value ob-
tained with the XTT assay was defined as the concentration of tested
compounds resulting in a 50% reduction of viability as compared
with vehicle‐treated cells. The experiments were carried out in tri-
plicates. The most active compound was chosen for further proce-
dures using the IC50 values obtained by this evaluation.
|
4.2.3
Cell migration and invasion evaluation
Cell migration was determined by the scraping wound repair test.[35]
8 × 104 PC‐3 cells were grown to confluence on 24‐well plates (48 h,
37°C). The sterilized micropipette tip was used to produce a wound
throughout the entire cell monolayer and then the medium was
disposed of. After being washed with PBS, compound 10 at its cy-
totoxic IC50 (Table 2) was added in a newly prepared medium and, in
the presence of endothelial growth factor (1 pg/ml), was incubated
for 24 h. Coverslips were placed in a light microscope, and the images
of the wounds were captured through a computer system using a
digital camera immediately after wounding (0 h) and after 24 h of
incubation. The results were expressed as percentage of wound
closure and the number of migrated cells/mm2.
LNCaP human tumor cells (1 × 105 cells/ml) were used for cell
invasion test; these were pretreated with compound 10 at its

CHARRIS ET AL.
11 of 12
|
|
cytotoxic IC50 for 24 h. After this time, the cells were seeded at the
top of the Boyden chamber (Matrigel‐coated membrane) in 50 µl of
serum‐free media and incubated for 18 h at 37°C. The bottom of
the chamber contained 500 µl of standard medium with 20% FBS.
The cells that had invaded into the lower surface of the chamber
were reacted with calcein (4 µg/ml) in Hank's Buffered Salt Solution
(HBSS) for 1 h at 37°C. The fluorescence generated by the cells was
read at 485/530 nm.[11,22]
4.2.6
Toxicological effect on brine shrimps
The toxicity bioassay was carried out using brine shrimp (Artemia
salina) according to modification of method previously
a
a
described.[39] In brief, artificial seawater was prepared by dissolving
sea salt in distilled water (0.65 M). Then, seawater was added and a
teaspoon of brine shrimp eggs was added and deposited in a small
tank covered in one half. The other side of the tank was not covered
to allow light, which attracted the hatched shrimps. The tank with
the brine shrimp eggs was kept at room temperature for 24 h to
promote the hatching of the eggs. Compound 10 was dissolved in
DMSO, diluted with artificial seawater, so that the final concentra-
tion of DMSO did not exceed 0.05%. Different concentrations (50 µl,
15 μM–1.0 mM) of the compound were prepared; the test was
performed in triplicate, in 96‐well plates.[47] The brine shrimp larvae
(nauplii, 10–20, 150 µl) were added to each well, covered with par-
afilm, leaving at room temperature for 24 h, after which the number
of dead and surviving brine shrimps was determined using a light
microscope. The experiment was replicated three times. Results are
expressed as 50% lethal concentration (LC50) values for the brine
shrimp assay using probit analysis. The mortalities were corrected
for the natural mortality observed in the negative controls using
Abbott's formula, p = pi − C/1 − C, where pi denotes the observed
control mortality rate and C denotes the natural mortality in treated
shrimps.[11,46,47] The TAR is defined by the value resulting from the
LC50 divided by the IC50 cytotoxic value of compound 10 for PC‐3,
LNCaP, or MatLyLu cells.
|
4.2.4
MMP zymography
The gelatinolytic activities of MMP‐2 and MMP‐9 were assayed by
electrophoresis on 10% polyacrylamide gels containing 1 mg/ml ge-
latin in the conditioned culture medium.[44] In brief, PC‐3 and LNCaP
cells (80% confluent in six‐well plates) were washed twice with PBS
and treated with the most active compound at its cytotoxic IC50
(Table 2) in 2.5 ml of serum‐free medium using the following condi-
tions: 24 h at 37°C in a humidified atmosphere of 95% O2 and 5%
CO2. Then, 20 µl of a mixture composed of the conditioned medium
and sample buffer (without β‐mercaptoethanol, 0.75:0.25) was
subjected to electrophoresis on 10% sodium dodecyl sulfate
(SDS)–polyacrylamide gel electrophoresis copolymerized with
1 mg/ml of gelatin as a substrate. After electrophoresis was com-
pleted at 100 V, to remove SDS, the gel was washed with a 2%
solution of Triton X‐100 and incubated for 20 h at 37°C in a buffer
made up of 50 mM Tris‐HCl, pH 7.4, 0.2 mM NaCl, 10 mM CaCl2.
Gels were then dyed with 0.5% Coomassie brilliant blue and subse-
quently faded in water–methanol–acetic acid (50:40:10). Undyed
regions of the gel corresponding to gelatinase activity were quanti-
fied using ImageJ software for Windows. As a positive control, pure
human proteins MMP‐2 and MMP‐9 were used.[11,22]
|
4.3
Statistical analysis
One‐way analysis of variance and t tests for specific group compar-
isons were used for data analysis. Significance was only considered
when p < .05 for all analyses. Data were expressed as mean (SD). The
software used was GraphPad Prism 4.02.[48]
|
4.2.5
Measurement of clonogenic potential
To test for anchorage, independent growth cells were grown in 0.6%
agar according to a modification of the method previously described.[45]
Briefly, a stock of 1.2% Agar Noble (Gibco) was autoclaved and then the
solution was equilibrated at 37°C for 60 min. The agar was diluted in
RPMI medium (1:1), to form a basal layer, and 1 ml of the mixture was
added to each well of a six‐well plate. The basal layer solidified at 4°C in
15 min, which was subsequently rebalanced at room temperature for
30 min. PC‐3 and LNCaP cell lines (1 × 105) suspended in com-
plete RPMI medium with 20% FBS containing 0.3% agar noble were laid
on the semi‐solid bottom layer. For 15 min, the plates were kept at
room temperature and then incubated for 24 h (37°C, 95% O2, 5%
CO2). The next day, 1 ml of medium with compound 10 at its cytotoxic
IC50 concentrations was added to each well (Table 2). The cells were
dyed with crystal violet (0.01%) for 18 h at 37°C, after 2 weeks of
incubation. A light microscope was used to take the pictures. The
experiments were performed in triplicate.[11,22,46]
ACKNOWLEDGMENTS
The authors thank the Instituto de Investigaciones Farmacéuticas
(IIF) and Consejo de Desarrollo Científico y Humanístico de la
Universidad Central de Venezuela (CDCH‐UCV).
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
Katiuska E. Charris
Juan R. Rodrigues
Hegira Ramírez
http://orcid.org/0000-0002-8040-8318
http://orcid.org/0000-0003-2399-6165
http://orcid.org/0000-0003-1916-6118
Esteban Fernandez‐Moreira
http://orcid.org/0000-0003-
1892-5370
Jorge E. Ángel
http://orcid.org/0000-0002-2423-3285
http://orcid.org/0000-0003-4404-2619
Jaime E. Charris

12 of 12
CHARRIS ET AL.
|
REFERENCES
[28] L. Rong, H. Han, H. Jiang, S. Tu, J. Heterocycl. Chem. 2009, 46, 465.
[29] X. Wang, X. Zeng, Y. Lie, D. Shi, S. Tu, X. Wei, Z. Zong, Synth.
Commun. 2005, 35, 1915.
[1] WHO report on cancer: Setting priorities, investing wisely and
providing care for all. Geneva: World Health Organization; 2020.
Licence: CC BY‐NC‐SA 3.0 IGO.
[2] R. Siegel, K. Miller, H. Fuchs, A. Jemal, CA–Cancer J. Clin. 2021, 71, 7.
[3] J. Ferlay, M. Colombet, I. Soerjomataram, C. Mathers, D. Parkin,
M. Piñeros, A. Znaor, F. Bray, Int. J. Cancer 2019, 144, 1941.
[4] Ä. Rana, J. Alex, M. Chauhan, G. Joshi, R. Kumar, Med. Chem. Res.
2015, 24, 903.
[30] Z. Ren, W. Cao, W. Tong, Synth. Commun. 2002, 32, 3475.
[31] B. Karmakar, B. Chowdhury, J. Banerji, Catal. Commun. 2010, 11,
601.
[32] B. Lee, K. Moon, J. Lim, Y. Park, D. Kim, S. Son, H. Jeong, D. Kim,
E. Lee, K. Chung, H. An, P. Chun, A. Seo, J. Yang, B. Lee, J. Ma,
W. Cho, H. Moon, H. Chung, Oncotarget 2017, 8, 91481.
[33] K. Makowska, M. Estañ, I. Gañán‐Gómez, M. Boyano‐Adánez,
A. García‐Pérez, P. Sancho, Mol. Biol. 2014, 48, 359.
[34] Y. Pan, S. Zhao, F. Chen, Clin. Exp. Pharmacol. Physiol. 2021, 48, 445.
[35] L. Rodríguez, X. Wu, J. Guan, Methods Mol. Biol. 2005, 294, 23.
[36] G. Morgia, M. Falsaperla, G. Malaponte, M. Madonia, M. Indelicato,
S. Travali, M. Mazzarino, Urol. Res. 2005, 33, 44.
[5] W. Zheng, Y. Zhao, Q. Luo, Y. Zhang, K. Wu, F. Wang, Curr. Top. Med.
Chem. 2017, 17, 3084.
[6] M. Islam, C. Wang, Z. N. Paudyal, Y. Zhu, H. Sun, Eur. J. Med. Chem.
2019, 162, 109.
[7] C. Huggins, C. Hodges, Cancer Res. 1941, 1, 293.
[8] I. Asangani, V. Dommeti, X. Wang, R. Malik, M. Cieslik, R. Yang,
J. Escara‐Wilke, K. Wilder‐Romans, S. Dhanireddy, C. Engelke,
M. Iyer, X. Jing, Y. Wu, X. Cao, Z. Qin, S. Wang, F. Feng,
A. Chinnaiyan, Nature 2014, 510, 278.
[37] V. Freedman, S. Shin, Cell 1974, 3, 355.
[38] U. Kelavkar, J. Nixon, C. Cohen, D. Dillehay, T. Eling, K. Badr,
Carcinogenesis 2001, 22, 1765.
[39] J. McLaughlin, C. Chang, D. Smith, Studies in Natural Product Chemistry
(Ed: A. Rahman), Vol. 9, Elsevier, Amsterdam 1991, pp. 383.
[40] S. Park, J. Lee, S. Lee, Cell Biochem. Biophys. 2018, 76, 255.
[41] F. Denizot, R. Lang, J. Immunol. Methods 1986, 89, 271.
[42] Y. Ruiz, J. Rodrigues, F. Arvelo, A. Usubillaga, M. Monsalve, N. Diez,
I. Galindo‐Castro, Phytochemistry 2008, 69, 432.
[9] B. Liu, L. Su, J. Geng, J. Liu, G. Zhao, ChemMedChem 2010, 5, 1651.
[10] A. Patravale, A. Gore, D. Patil, G. Kolekar, M. Deshmukh,
P. Anbhule, Ind. Eng. Chem. Res. 2014, 53, 16568.
[11] G. Mata, J. Rodrigues, N. Gamboa, K. Charris, G. Lobo,
M. Monasterios, M. Avendaño, M. Lein, K. Jung, C. Abramjuk,
B. Nitzsche, M. Hopfner, J. Charris, Lett. Drug Des. Discov. 2017, 14,
398.
[43] J. Atienza, J. Zhu, X. Wang, X. Xu, Y. Abassi, J. Biomol. Screening
2005, 10, 795.
[12] E. Nasr, A. Mostafa, M. El‐Sayed, M. Massoud, Arch. Pharm. Chem.
Life Sci. 2019, 352, e1800355.
[44] X. Qu, Y. Yuan, W. Xu, M. Chen, S. Cui, H. Meng, Y. Li, M. Makuuchi,
M. Nakata, W. Tang, Anticancer Res. 2006, 26, 3573.
[45] A. Powell, S. Akare, W. Qi, P. Herzer, S. Jean‐Louis, R. Feldman,
J. Martinez, BMC Cancer 2006, 6, 219.
[13] X. Song, J. Fan, L. Liu, X. Liu, F. Gao, Arch. Pharm. Chem. Life Sci.
2020, 353, e2000025.
[14] J. Nemeth, R. Yousif, M. Herzog, M. M. Che, J. Upadhyay,
B. Shekarriz, S. Bhagat, C. Mullins, R. Fridman, M. Cher, J. Natl.
Cancer Inst. 2002, 94, 17.
[46] J. Rodrigues, J. Charris, J. Camacho, A. Barazarte, N. Gamboa,
F. Antunes, Anticancer Res. 2012, 32, 2721.
[47] P. Solis, C. Wright, M. Anderson, M. Gupta, J. Phillipson, Planta Med.
1993, 59, 250.
[15] M. Burg‐Roderfeld, M. Roderfeld, S. Wagner, C. Henkel,
J. Grotzinger, E. Roeb, Int. J. Oncol. 2007, 30, 985.
[16] L. Yang, W. Zeng, D. Li, R. Zhou, Oncol. Rep. 2009, 22, 121.
[17] A. Laghezza, G. Luisi, A. Caradonna, A. Di Pizio, L. Piemontese,
F. Loiodice, M. Agamenonne, P. Tortorella, Bioorg. Med. Chem. 2020,
28, 115257.
[48] GraphPad Prism Software Inc. 4.02 for Windows. May 17th
(1992–2004).
[18] E. Lencia, A. Continib, A. Trabocchi, Bioorg. Med. Chem. Lett. 2020,
30, 127467.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
[19] T. Turpeenniemi‐Hujanen, Biochimie 2005, 87, 287.
[20] B. Bauvois, Biochim. Biophys. Acta 2012, 1825, 29.
[21] W. Stetler‐Stevenson, A. Yu, Semin. Cancer Biol. 2001, 11, 143.
[22] J. Rodrigues, J. Charris, R. Ferrer, N. Gamboa, J. Ángel, B. Nitzsche,
M. Hoepfner, M. Lein, K. Jung, C. Abramjuk, Invest. New Drugs 2012,
30, 1426.
How to cite this article: K. E. Charris, J. R. Rodrigues, H.
Ramírez, E. Fernandez‐Moreira, J. E. Ángel, J. E. Charris.
Synthesis of 5H‐indeno[1,2‐b]pyridine derivatives:
Antiproliferative and antimetastatic activities against
two human prostate cancer cell lines. Arch. Pharm.
2021;e2100092. https://doi.org/10.1002/ardp.202100092
[23] G. Kumar, B. Nair, J. Perry, D. Martin, Med. Chem. Commun. 2019,
10, 2024.
[24] J. Son, S. Lee, Bioorg. Med. Chem. Lett. 2020, 30, 127223.
[25] S. Kambe, K. Saito, A. Sakurai, H. Midorikawa, Synthesis 1980, 1980,
366.
[26] F. Shi, S. Tu, F. Fang, T. Li, ARKIVOC 2005, 2005, 137.
[27] M. Jachak, D. Kendre, A. Avhale, R. Toche, R. Sabnis, J. Heterocycl.
Chem. 2007, 44, 1525.