New 1,2,4-oxadiazole nortopsentin derivatives with cytotoxic activity

Article abstract of DOI:10.3390/md17010035

New analogs of nortopsentin, a natural 2,4-bis(3⁰-indolyl)imidazole alkaloid, in which the central imidazole ring of the natural lead was replaced by a 1,2,4-oxadiazole moiety, and in which a 7-azaindole portion substituted the original indole moiety, were efficiently synthesized. Among all derivatives, prescreened against the HCT-116 colon rectal carcinoma cell line, the two most active compounds were selected and further investigated in different human tumor cells showing IC₅₀ values in the micromolar and submicromolar range. Flow cytometric analysis of propidium iodide-stained MCF-7 cells demonstrated that both the active derivatives caused cell cycle arrest in the G0-G1 phase. The cell death mechanism induced by the compounds was considered to be apoptotic by measuring the exposure of phosphatidylserine to the outer membrane and observed morphological evaluation using acridine orange/ethidium bromide double staining. Moreover, further tested on intestinal normal-like differentiated Caco-2 cell line, they exhibited preferential toxicity towards cancer cells.

Full text of DOI:10.3390/md17010035

marine drugs
Article
New 1,2,4-Oxadiazole Nortopsentin Derivatives with
Cytotoxic Activity
Stella Cascioferro 1 , Alessandro Attanzio 1, Veronica Di Sarno 2, Simona Musella 2,
Luisa Tesoriere 1, Girolamo Cirrincione 1, Patrizia Diana 1 and Barbara Parrino 1,
*
1
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF),
University of Palermo, via Archirafi 32, 90123 Palermo, Italy; stellamaria.cascioferro@unipa.it (S.C.);
alessandro.attanzio@unipa.it (A.A.); luisa.tesoriere@unipa.it (L.T.); girolamo.cirrincione@unipa.it (G.C.);
patrizia.diana@unipa.it (P.D.)
Department of Pharmacy, University of Salerno, Via G. Paolo II 132, 84084 Fisciano, Italy;
vdisarno@unisa.it (V.D.S.); smusella@unisa.it (S.M.)
2
*
Correspondence: barbara.parrino@unipa.it; Tel.: +39-091-238-97413
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 5 December 2018; Accepted: 4 January 2019; Published: 8 January 2019
Abstract: New analogs of nortopsentin, a natural 2,4-bis(30-indolyl)imidazole alkaloid, in which
the central imidazole ring of the natural lead was replaced by a 1,2,4-oxadiazole moiety, and in
which a 7-azaindole portion substituted the original indole moiety, were efficiently synthesized.
Among all derivatives, prescreened against the HCT-116 colon rectal carcinoma cell line, the two most
active compounds were selected and further investigated in different human tumor cells showing
IC50 values in the micromolar and submicromolar range. Flow cytometric analysis of propidium
iodide-stained MCF-7 cells demonstrated that both the active derivatives caused cell cycle arrest
in the G0–G1 phase. The cell death mechanism induced by the compounds was considered to be
apoptotic by measuring the exposure of phosphatidylserine to the outer membrane and observed
morphological evaluation using acridine orange/ethidium bromide double staining. Moreover,
further tested on intestinal normal-like differentiated Caco-2 cell line, they exhibited preferential
toxicity towards cancer cells.
Keywords: marine alkaloids; nortopsentin analogs; 1,2,4-oxadiazole derivatives; anti-cancer agents;
antiproliferative activity
1. Introduction
Natural products (NP) constitute a significant source of bioactive molecules and potential drug
leads due to their high chemical diversity, biochemical specificity, binding efficiency with biological
targets, and broad panel of bioactivities. About 60% of drugs currently on the market are of natural
origin and natural products screening still plays a fundamental role in the drug discovery process [1].
Marine natural products (MNP), in particular marine sponge-derived compounds, have attracted
considerable attention due to their unique biodiversity and structural differences as compared to
terrestrial natural products. Among MNP or marine-derived molecules, eight compounds are currently
on the market with application in different therapeutic areas and several compounds are in different
phases of the clinical pipeline, showing promising anticancer activity. Only a few of them are original
MNPs, while the majority are derivatives obtained through molecular lead optimization [2].
Considering the successful results obtained using MNP as leads for drug discovery and the
growing number of marine-derived molecules entering into clinical trials, researchers are still inspired
by their scaffold for the design of new active molecules.
Mar. Drugs 2019, 17, 35; doi:10.3390/md17010035
www.mdpi.com/journal/marinedrugs

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Among MNPs used as lead compounds for the synthesis of new anticancer agents,
nortopsentin, an alkaloid isolated from deep-sea sponge Spongsorites ruetzleri having a characteristic
2,4-bis(30-indolyl)imidazole skeleton, attracted remarkable attention due to its significant
antiproliferative activity against the P388 murine leukemia cell line [3]. Many derivatives in which the
central imidazole ring was replaced by several five-membered heterocycles were reported, most of
them showing antiproliferative activity, often reaching GI50 values in the low micromolar range or
even at the sub-micromolar level [3–12]. The thiazole nortopsentin analogs in particular, in which the
structural modification of the lead natural molecule also involved one or both indole portions, led to
potent compounds with effect against a wide range of cell lines, including diffuse malignant peritoneal
mesothelioma (DMPM), a fatal disease, poorly responsive to conventional therapies, and acted as
CDK1 inhibitors [13–16]. Moreover, the most active thiazole derivatives also bearing a 7-azaindole
substitution, in the mouse model, by intraperitoneal administration were effective with a significant
reduction of the DMPM and two complete responses at well-tolerated doses [13].
On the other hand, the 1,2,4-oxadiazole ring system is a five-membered heterocycle ring found
in many molecules with significant biological activity, especially antitumor [17–19], despite its
uncommon presence in MNP. To the best of our knowledge, phidianidines, isolated from the marine
opisthobranch mollusk Phidiana militaris, are a unique example of MNP possessing a 1,2,4-oxadiazole
ring system in their structure, and together with their derivatives they exhibit significant cytotoxic,
DAT inhibitory, or neuroprotective activities [20–22]. Moreover, the 1,2,4 oxadiazole ring is a bioisoster
of amides and esters and for this reason could improve bioavailability and physiochemical properties
of compounds bearing it.
Continuing our search for new anticancer compounds [23
of a new 1-methyl-3-[3-(1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1H-pyrrolo[2,3-b]pyridine
nortopsentin analog , designed on the basis of the potent activity shown by thiazole nortopsentin
–40], herein we report the synthesis
1
analogs with a 7-azaindole portion and considering the important characteristics of the 1,2,4-oxadiazole
ring found in compounds with promising biological activity. The new compounds, in which the central
imidazole ring of the natural lead was replaced by a 1,2,4-oxadiazole moiety, and in which a 7-azaindole
portion substituted the original indole, were prescreened against the HCT-116 colon rectal carcinoma
cell line and the two most active compounds were selected and further investigated in different human
tumor cell lines.
2. Results and Discussion
2.1. Chemistry
1-Methyl-3-[3-(1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1H-pyrrolo[2,3-b]pyridines
1 were
synthesized by reaction between two different key intermediates, N0-hydroxy-1-methyl-1H-indole-3-
carboximidamides
Table 1).
2 and methyl-1-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylates 3 (Scheme 1,
N0-hydroxy-1-methyl-1H-indole-3-carboximidamides 2a
–
e
, commercially unavailable, were
, prepared as previously reported [16].
The reaction of compounds 4a–e with chlorosulfonyl isocyanate (CSI) in acetonitrile, followed by the
addition of N,N-dimethylformamide (DMF), led to the corresponding carbonitriles 5a (90–98%),
easily converted (65–82%) to the corresponding carboximidamides 2a through reaction with
hydroxylamine hydrochloride in ethanol (EtOH), in the presence of diisopripylenethylamide (DIPEA).
Methyl-1-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylates 3a were in turn prepared from
their 1-methyl-7-azaindoles 6a–c 10 16] converted into the corresponding 2,2,2-trichloro-1-(1-methyl-
1H-pyrrolo[2,3-b]pyridin-3-yl)ethanones 7a (80–99%) by reaction with trichloroacetyl chloride in
dichloromethane (DCM) in the presence of aluminum chloride. Once obtained, compounds were
synthesized from the corresponding 1-methyl-indoles of type
4
–
e
–
e
–
c
[
,
–c
7
subjected to basic hydrolysis with potassium hydroxide solution (KOH 20%) to afford the desired
derivatives 3 (90–99%).

Mar. Drugs 2019, 17, 35
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Finally, the carboximidamides 2a
–
e
and the carboxylates 3a
–
c
were reacted in the presence of
sodium hydride in tetrahydrofuran (THF) to afford the final compounds 1a–o (55–85%).
OH
N
Cl
O
O
Cl
Cl
OCH3
CN
NH2
R1
R1
R2
R2
ii
iv
N
N
N
N
N
N
CH3
CH3
CH3
CH3
2a-e
5a-e
3a-c
7a-c
v
i
iii
N
O
R2
R1
R1
R2
N
N
N
N
N
N
N
CH3
CH3
CH3
CH3
4a-e
6a-c
1a-o
a R1 = H; b R1 = Br; c = F; d = Cl; e = OCH3
a R2 = H; b R2 = F; c R2 = Br
Scheme 1. Synthesis of 1-methyl-3- [3-(1-methyl-1H-indol-3-yl)- 1,2,4-oxadiazol-5-yl]-1H-pyrrolo [2,3-b]
pyridines 1a–o. Reagents and conditions: ( ) (
) CSI, MeCN, 0 ◦C, 2 h; ( ) DMF, 0 ◦C, 1 h, 90–98%;
ii) NH2NH2 HCl, DIPEA, EtOH, reflux, 4 h, 65–82%; (iii) trichloroacetyl chloride, AlCl3, DCM, 6 h,
80–99%; (iv) KOH 20%, rt, 90–99%; (v) NaH, EtOH, reflux, 4–6 h, 55–85%.
i
a
b
(
Table 1. New 1,2,4-oxadiazole derivatives 1a–o.
Compound
R1
R2
Yield (%)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
H
H
H
Br
Br
Br
F
F
F
Cl
Cl
Cl
OCH3
OCH3
OCH3
H
Br
F
H
Br
F
H
Br
F
H
Br
F
75
72
62
75
82
75
72
60
55
65
75
85
70
65
80
H
Br
F
2.2. Biology
In vitro cytotoxicity of the synthesized compounds 1a
–o was prescreened against the HCT-116
colon rectal carcinoma cell line. Monolayer cultures treated for 72 h with 10−8–10−4 µM concentrations
of the compounds were examined by MTT assay for the cell viability.
Among the synthesized nortopsentin analogs, the compounds bearing the 5-bromo-1-methyl-1H-
pyrrolo[2,3-b]pyridine moiety (see compounds 1k and 1n) showed the highest cytotoxic activity
(Figure 1B). Substitution of the bromine atom in this portion with a fluorine (see compounds 1o and 1l
)
or absence of the halogen atom (see compounds 1j and 1m) resulted in a drop of the antiproliferative
effect of the derivatives. The remaining compounds appeared poorly effective.

Mar. Drugs 2019, 17, 35
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Figure 1.
Effect of 1-methyl-3-[3-(1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1H-pyrrolo[2,3-b]
. on the growth of human tumor cells HCT-116. ( ) Compounds bearing the 1-methyl-1H-
) compounds bearing the 5-bromo-1-methyl-1H-pyrrolo[2,3-b]pyridine
) compounds bearing the 5-fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridine moiety. Cells were
treated with the compounds and cell viability was measured after 72 h by MTT assay in comparison
pyridines 1a
–o
A
pyrrolo[2,3-b]pyridine moiety; (
B
moiety; (
C
to cells treated with vehicle alone (control). Values are the mean
in triplicate.
±
SD of three separate experiments
The most active derivatives 1k and 1n were further investigated against MCF-7 (human breast
cancer), HeLa (cervix adenocarcinoma), and CaCo2 (colorectal carcinoma) cell lines (Figure 2), and
IC50 values in the micromolar and submicromolar range were calculated (Table 2). Compared to 1n
,
1k derivative, with the exception of HeLa cell line, appeared 2–4-fold more effective.
Figure 2. Effect of compounds 1k (left) and 1n (right) on the viability of different human tumor cell
lines. Cells were treated with the compounds and cell survival was measured after 72 h by MTT assay
in comparison to cells treated with vehicle alone (control), as reported in Section 3.2. Values are the
mean ± SD of three separate experiments carried out in triplicate.

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Table 2. Anti-proliferative activity of compounds 1k and 1n against different human cell lines (IC50).
IC50 (µM) a
HCT-116 HeLa
Compound
MCF-7
CaCo2
1k
1n
0.65 ± 0.05
1.93 ± 0.06
10.56
13.96
±
0.98
1.41
1.06 ± 0.09
2.41 ± 0.23
3.55 ± 0.1
±
3.33 ± 0.25
a
IC50 value was calculated by plotting the percentage viability versus concentration on a logarithmic graph. Results
are the mean values SD (standard deviation) of three separated experiment carried out in triplicate.
Further studies on the mechanism of the cytotoxic activity of the active compounds were carried
out on MCF-7 cells, the tumor cell line more sensitive to both compounds. In order to investigate the
effect on the cell cycle, flow cytometric analysis on PI stained MCF-7 cells was carried out after 24 h
treatment with compounds 1k and 1n at their relevant IC50 values. As shown in Figure 3A, both the
synthesized derivatives caused an accumulation of treated cells in subG0/G1 phase and induced their
marked arrest (more than 60%) in G0–G1 phase of the cycle. Although changes in distribution within
mitotic phases cannot provide precise information on the mechanism of drug’s activity, these findings
suggest that compounds 1k and 1n can affect the cell machinery promoting DNA duplication.
In order to assess the cell death mechanism, apoptosis induction caused by compounds 1k and 1n
in MCF7 cells, after 24 h treatment, was investigated by means of Annexin V/PI dual staining method
followed by cytofluorimetric analysis. The biparametric analysis showed that both the compounds
induced early apoptosis without causing death by necrosis (Figure 3B).
Figure 3. Effect of compounds 1k and 1n on cell cycle distribution (
MCF-7 cells ( ). Cell monolayers were incubated for 24 h in the absence (control) or in the presence
of individual compounds and submitted to flow cytometric analysis after propidium iodide ( ) or
AnnexinV/PI double staining ( ) as reported in 3.2 Paragraph. ( ) Percentage of viable cells in the
different phases are given in each picture. Values are the mean ± SD of three separate experiments in
triplicate. Representative images of three experiments with comparable results; ( ) BU3, viable cells
(AnnexinV /PI ); BU4, cells in early apoptosis (AnnexinV+/PI ); BU2, cells in tardive apoptosis
/PI+). Representative images of three experiments
A) and PS externalization of
B
A
B
A
B
−
−
−
(AnnexinV+/PI+); BU1, necrotic cells (AnnexinV
with comparable results.
−

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It is of interest to emphasize that other previously synthesized nortopsentin analogs bearing a
7-azaindole substitution and in which a thiazole ring substituted the central ring of the lead compound,
showed cytotoxic activity towards MCF-7 cells associated with mitotic failure and accumulation of
cells in phase G2/M [12,16]. The substitution of the central ring with a 1,2,4 oxadiazole one provides
molecules of different biological activity that deserves to be investigated.
To further verify the pro-apoptotic effects of the synthesized derivatives, AO/EB staining was
also used. Morphological evaluation of the cells using AO and EB double staining allows to detect
early apoptotic cells stained green with yellow dots, with blebbing cytoplasm, while late apoptotic
cells stain orange with fragmented nuclei. Non-apoptotic cells stain green. As shown in Figure 4,
MCF-7 cells treated for 24 h with 1k or 1n showed morphological changes typical for early apoptosis
with condensation of nuclear material and formation of membrane blebbing.
Figure 4. Apoptosis by compounds 1k and 1n in MCF-7 cells assessed by acridine orange/ethidium
bromide (AO/EB) staining. MCF-7 cells were treated with the compounds for 24 h and compared with
the untreated cells. Fluorescence micrographs represent one of three independent experiments. Red
arrow: chromatin condensation; white arrow: membrane blebbing.
Additional experiments were conducted on intestinal normal-like differentiated Caco-2 cells.
As shown in Figure 5, at the concentrations effective to inhibit the growth of tumor cells,
the compounds 1k and 1n did not affect to a large extent the viability of the normal-like cells showing
selectivity towards cancer cells (Figure 4).
Figure 5. Effect of compounds 1k and 1n on the viability of intestinal normal-like differentiated Caco-2
cells. Fifteen-days post-confluence monolayers of Caco-2 were incubated for 24 h in the absence
(control) or in the presence of the compounds at the indicated concentrations and cell viability was
assessed by MTT test as reported in Section 3.2. Values are the mean
carried out in triplicate.
±
SD of two separate experiments

Mar. Drugs 2019, 17, 35
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3. Materials and Methods
3.1. Chemistry
3.1.1. General
All melting points were taken on a Büchi-Tottoly capillary apparatus (Büchi, Cornaredo, Italy)
and are uncorrected. IR spectra were determined in bromoform with a Shimadzu FT/IR 8400S
1
spectrophotometer (Shimadzu Corporation, Milan, Italy). H and 13C NMR spectra were measured
at 200 and 50.0 MHz, respectively, in DMSO-d6 solution, using a Bruker Avance II series 200 MHz
spectrometer (Bruker, Milan, Italy). Column chromatography was performed with Merck silica gel
230–400 mesh ASTM or with a Büchi Sepacor chromatography module (prepacked cartridge system).
Elemental analyses (C, H, N) were within
a VARIO EL III elemental analyzer (Elementar, Langenselbold, Germany). Purity of all the tested
compounds was greater than 95%, determined by HPLC (Agilent 1100 Series). Mass spectra of final
±
0.4% of theoretical values and were performed with
compounds were performed using a Mariner
™ mass spectrometer, Applied Biosystems (Foster City,
CA, USA). A Harvard model 11 syringe pump (Holliston, MA, USA) was used to infuse the sample
solutions. The ESI source was operated in positive ion mode with an electrospray voltage of 4.5 kV.
Compounds 1a–o were characterized only by 1H NMR spectra due to their poor solubility.
General Procedure for the Synthesis of 1-methyl-1H-indole-3-carbonitriles (5a–e)
The appropriate indole 4a
reacted with chlorosulfonyl isocyanate (CSI) (0.16 mL, 1.8 mmol, added dropwise at 0 C. The resulting
reaction mixture was stirred at 0 C for 2 h after which anhydrous dimethylformamide (DMF) (1.0 mL
106.3 mmol) was added dropwise. The mixture was stirred at 0 ◦C for 1 h and then it was poured
into ice-water. The obtained precipitate was filtered off, dried (Na2SO4), and purified by column
chromatography using dichloromethane (DCM) as the eluent.
–
e
(1.8 mmol) solubilized in anhydrous acetonitrile (2.0 mL), was
◦
◦
,
1-Methyl-1H-indole-3-carbonitrile (5a). White solid; yield 90% m.p. 61–62 ◦C; spectroscopic data are in
accordance to those reported in literature [41].
5-Bromo-1-methyl-1H-indole-3-carbonitrile (5b). White solid; yield 92%; m.p. 103–104 ◦C; spectroscopic
data are in accordance to those reported in literature [41].
5-Fluoro-1-methyl-1H-indole-3-carbonitrile (5c). White solid; yield 90%; m.p. 76.5–78 ◦C; spectroscopic
data are in accordance to those reported in literature [41].
5-Chloro-1-methyl-1H-indolo-3-carbonitrile (5d). White solid; yield: 91%; m.p. 103.8–104.5 ◦C;
spectroscopic data are in accordance to those reported in literature [42].
◦
5-Metoxy-1-metil-1H-indolo-3-carbonitrile (5e). White solid; yield 98%; m.p. 106.5–107.4 C; spectroscopic
data are in accordance to those reported in literature [41].
General Procedure for the Synthesis of N0-hydroxy-1-methyl-1H-indole-3-carboximidamides (2a–e)
To a solution of the appropriate 1-methyl-1H-indolo-3-carbonitrile
5
(0.9 mmol) in ethanol (15 mL
)
diisopripylenethylamide (DIPEA, 1.32 mL) and hydroxylamine hydrochloride (2.3 mmol, 158.0 mg
)
were added and the resulting reaction mixture was heated under reflux for 4 h. The solvent was
evaporated at reduced pressure and the obtained crude was suspended in water and extracted with
ethyl acetate (3
×
30 mL). The organic phases were dried (Na2SO4) and evaporated under reduced
pressure. The residue was purified by column chromatography using ethyl acetate as the eluent.

Mar. Drugs 2019, 17, 35
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0
◦
N -hydroxy-1-methyl-1H-indole-3-carboximidamide (2a). solid; yield 65%; m.p. 110.2–111.6 C; spectroscopic
data are in accordance to those reported in literature [41].
0
5-Bromo-N -hydroxy-1-methyl-1H-indole-3-carboximidamide (2b). White solid; yield 75%; m.p. 103.0–104.0 ◦C
IR (cm−1): 3465 (OH), 3360 (NH2), 1669 (C=N); 1H NMR (200 MHz, DMSO-d6) δ: 3.79 (s, 3H, CH3), 5.64
(s, 2H, NH2), 7.30 (d, 1H, J = 8.7 Hz, H-6), 7.44 (d, J = 8.7 Hz, 1H, H-7), 7.79 (s, 1H, H-4), 8.27 (s, 1H, H-2),
9.34 (1H, s, OH); 13C NMR (50 MHz, DMSO-d6)
δ: 32.9 (q), 107.8 (s), 111.9 (d), 112.4 (s), 124.0 (d), 124.4 (d),
126.4 (s), 130.0 (d), 135.6 (s), 148.6 (s); Anal. Calculated for C10H10BrN3O (MW: 268.11): C, 44.80; H, 3.76;
N, 15.67%. Found: C, 45.11; H, 4.00; N, 15.48%.
5-Fluoro-N0-hydroxy-1-methyl-1H-indole-3-carboximidamide (2c). Gray solid; yield 80%; m.p. 114.0–115.0 ◦C
IR (cm−1): 3465 (OH), 3370 (NH2), 1640 (C=N); 1H NMR (200 MHz, DMSO-d6)
: 3.79 (s, 3H, CH3), 5.62
(s, 2H, NH2), 6.98–7.09 (td, J = 9.2, 9.1, 2.5 Hz, 1H, H-6), 7.45 (dd, J = 9.1, 4.4 Hz, 1H, H-7), 7.74–7.80 (m,
2H, H-4 and H-2), 8.27 (s, 1H, OH); 13C NMR (50 MHz, DMSO-d6)
:33.0 (q), 106.7 (d, JC4-F = 24.8 Hz),
;
δ
δ
108.5 (s, JC7a-F = 4.6 Hz), 109.7 (d, JC6-F = 26.1 Hz), 111.0 (d, JC7-F = 9.9 Hz), 125.0 (s, JC3a-F = 11.0 Hz), 130.3
(d), 133.6 (s), 148.8 (s), 157.3 (s, JC5-F = 231.9 Hz); Anal. Calculated for C10H10FN3O (MW: 207.20): C, 57.97;
H, 4.86; N, 20.28%. Found: C, 57.88; H, 4.62; N, 20.31%.
5-Chloro-N0-hydroxy-1-methyl-1H-indole-3-carboximidamide (2d). Gray solid; yield 82%; m.p. 170.7–172.0
◦C; IR (cm−1): 3461 (OH), 3364 (NH2), 1652 (C=N); 1H NMR (200 MHz, DMSO-d6)
δ
3.79 (s, 3H, CH3),
5.66 (s, 2H, NH2), 7.19 (dd, J = 8.7, 2.2 Hz, 1H, H-6), 7.47 (d, J = 8.7 Hz, 1H, H-7), 7.81 (s, 1H, H-2),
8.13 (d, J = 2.2 Hz, 1H, H-4), 9.35 (s, 1H, OH). 13C NMR (50 MHz, DMSO-d6)
32.89 (q), 107.84 (s),
δ
111.47 (d), 121.31 (d), 121.52 (d) 124.38 (s), 125.75 (s), 130.15 (d), 135.36 (s), 148.63 (s). Anal. Calculated for
C10H10ClN3O (MW: 223.66): C, 53.70; H, 4.51; N, 18.79%. Found: C, 53.82; H 4.67; N, 19.00%.
5-Methoxy-N0-hydroxy-1-methyl-1H-indole-3-carboximidamide (2e). White solid; yield 75%; m.p.
1
165.9–166.5 ◦C; IR (cm−1): 3447 (OH), 3356 (NH2), 1652 (C=N); H NMR (200 MHz, DMSO-d6)
δ
3.75 (s, 3H, CH3x2), 5.58 (s, 2H, NH2), 6.82 (dd, J = 8.9, 2.5 Hz, 1H, H-6), 7.33 (d, J = 8.9 Hz, 1H, H-7),
7.59 (d, J = 2.5 Hz, 1H, H-4), 7.69 (s, 1H, H-2), 9.28 (s, 1H, OH).13C NMR (50 MHz, DMSO-d6)
32.79
δ
(q), 55.22 (q), 103.67 (d), 107.64 (s), 110.49 (d), 111.77 (d), 125.22 (s), 129.07 (d), 132.09 (s), 149.29 (s),
153.86 (s). Anal. Calculated for C10H10N2O (MW: 219.24): C, 60.26; H, 5.98; N, 19.17%. Found: C, 60.22;
H, 5.67; N, 19.25%.
General Procedure for the Synthesis of 2,2,2-trichloro-1-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-
yl)ethanones (7a–c)
To a solution of the appropriate 1-methyl-1H-pyrrolo[2,3-b]pyridine
6 (10.15 mmol) in DCM
(
(
24 mL), aluminum chloride (35 mmol, 4.7 g) and a solution of trichloroacetyl chloride in DCM
10.15 mmol, 1.1 mL in 5 mL di DCM), were added. The resulting mixture was stirred at room
temperature for 6 h, poured into ice-water and extracted with DCM (3
×
20 mL). The organic phases
were washed with HCl 10% (2 30 mL), dried (Na2SO4), and evaporated under reduced pressure,
×
to afford the desired derivatives as pure compounds.
2,2,2-Trichloro-1-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (7a). White solid; yield 80%; m.p.
99–100 ◦C; IR cm−1: 1690 (CO);1H NMR (200 MHz, DMSO-d6)
δ
4.00 (s, 3H, CH3). 7.42 (dd, J = 8.0
4.8 Hz, 1H, H-5), 8.49 (dt, J = 4.8, 2.0 Hz, 1H, H-6), 8.54 (d, J = 2.0 Hz, 1H, H-4), 8.90 (s, 1H, H-2). 13C
NMR (50 MHz, DMSO-d6) 31.95 (q), 95.84 (s), 102.37 (s), 119.40 (d), 119.82 (s), 129.97 (d), 140.09 (d),
,
δ
144.90 (d), 147.64 (s), 176.30 (s). Anal. Calculated for C10H7Cl3N2O (MW: 277.53): C, 43.28; H, 2.54; N,
10.09%. Found: C, 43.32; H 2.67; N, 9.85%.

Mar. Drugs 2019, 17, 35
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2,2,2-Trichloro-1-(5-fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (7b). Yellow solid; yield 99%;
m.p. 110–111 ◦C; IR cm−1: 1710 (CO); 1H NMR (200 MHz, DMSO-d6)
3.98 (s, 3H, CH3). 8.29 (dd,
J = 9.0, 2.8 Hz, 1H, H-4), 8.51 (dd, J = 2.8, 1.7 Hz, 1H, H-6), 8.99 (s, 1H, H-2). 13C NMR (50 MHz
DMSO-d6) 32.31 (q), 99.49 (s), 120.43 (s), 102.29 (s), 115.74 (d, JC4-F = 29.3 Hz), 133.54 (d, JC6-F
δ
,
δ
=
29.3 Hz), 141.80 (d), 140.22 (s), 144.40 (s), 160.20 (d, JC5-F = 70.4 Hz). Anal. Calculated for C11H6Cl3FN2O
(MW: 295.52): C, 40.64; H, 2.05; N, 9.48%. Found: C, 40.55; H 1.98; N, 9.68%.
2,2,2-Trichloro-1-(5-bromo-1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (7c). Orange solid; yield 99%; m.p.
123–124 ◦C; IR cm−1: 1725 (CO);1H NMR (200 MHz, DMSO-d6)
δ
3.97 (s, 3H, CH3), 8.57 (d, J = 2.2 Hz,
1H, H-4), 8.63 (d, J = 2.2 Hz, 1H, H-6), 8.96 (s, 1H, H-2).13C NMR (50 MHz, DMSO-d6)
δ
32.21 (s),
101.97 (s), 115.00 (s), 121.41 (s), 131.69 (d), 141.40 (d), 145.23 (d), 146.24 (s), 176.32 (s). Anal. Calculated for
C10H6BrCl3N2O (MW: 356.43): C, 33.70; H, 1.70; N, 7.86%. Found: C, 34.01; H 1.55; N, 7.68%.
General Procedure for the Synthesis of methyl-1-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate (3a–c)
To a mixture of the appropriate ethanone
7 (1.1 mmol) in methanol, KOH 20% solution (0.33 mL)
was added. The reaction mixture was stirred at room temperature for 3 h. Then HCl 6N (0.2 mL) was
added and the mixture was extracted in ethyl acetate (3 × 30 mL).
◦
Methyl-1-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate (3a). White solid; yield 99%; m.p. 135–136 C; IR
cm−1: 1730 (CO); 1H NMR (200 MHz, DMSO-d6)
δ
3.83 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 7.30 (dd, J =
7.2, 4.8 Hz, 1H, H-5), 8.35 (m, 3H, H-2, H-4, H-6). 13C NMR (50 MHz, DMSO-d6)
δ
31.41 (q), 50.91 (q),
103.75 (s), 117.87 (d), 118.23 (s), 128.95 (d), 136.38 (d), 143.73 (d), 147.56 (s), 163.99 (s). Anal. Calculated
for C10H10N2O2 (MW: 190.20): C, 63.15; H, 5.30; N, 14.73%. Found: C, 63.28; H 5.15; N, 14.58%.
Methyl-5-fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridine-3- carboxylate (3b). White solid; yield 90%; m.p.
◦
128–129 C; IR cm−1: 1738 (CO); 1H NMR (200 MHz, DMSO-d6)
δ 3.83 (s, 3H, CH3), 3.88 (s, 3H, OCH3),
8.05 (dd, J = 9.2, 2.8 Hz, 1H, H-4), 8.39 (dd, J = 2.8, 1.8 Hz, 1H, H-6), 8.43 (s, 1H, H-2). 13C NMR
(50 MHz, DMSO-d6) δ 31.73 (q), 51.02 (q), 103.76 (d, JC7a-F = 4.1 Hz), 114.48 (d, JC4-F = 21.8 Hz), 118.41
(d, JC3-F = 7.9 Hz), 132.19 (d, JC6-F = 29.4 Hz), 138.27 (d), 144.32 (s), 156.26 (d, JC5a-F = 242.4 Hz), 163.66
(s). Anal. Calculated for C10H9FN2O2 (MW: 208.19): C, 57.69; H, 4.36; N, 13.46%. Found: C, 57.53; H
4.15; N, 13.38%.
Methyl-5-bromo-1-methyl-1H-pyrrolo[2,3-b]pyridine-3- carboxylate (3c). White solid; yield 99%; m.p.
144–145 ◦C; IR cm−1: 1715 (CO); 1H NMR (200 MHz, DMSO-d6)
δ
3.84 (s, 3H, CH3), 3.86 (s, 3H, CH3),
8.39 (d, J = 2.0 Hz, 2H, H-2, H-4), 8.45 (d, J = 2.0 Hz, 1H, H-6). 13C NMR (50 MHz, DMSO-d6)
δ
31.64
(q), 51.10 (q), 103.45 (s), 113.36 (s), 119.73 (s), 130.63 (d), 137.85 (d), 143.88 (d), 146.03 (s) 163.57 (s). Anal.
Calculated for C10H9BrN2O2 (MW: 269.09): C, 44.63; H, 3.37; N, 10.41%. Found: C, 44.87; H 3.51; N,
10.12%.
General Procedure for the Synthesis of 1-methyl-3-[3-(1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-
1H-pyrrolo[2,3-b]pyridines (1a–o)
To a solution of the proper derivative
(3.0 mmol, 79.3 mg) and molecular sieves were added. The reaction mixture was heated at 60 ◦C for
30 min. Then, the appropriate derivative (1.3 mmol) was added and the mixture was heated under
2 (2.6 mmol) in tetrahydrofuran (10 mL), sodium hydride
3
reflux for 4–6 h. The organic solvent was evaporated at reduced pressure and the resulting crude was
purified by column chromatography using dichloromethane/ethyl acetate 1:1 as the eluent.
1-Methyl-3-[3-(1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1H-pyrrolo[2,3-b]pyridine (1a). White solid;
yield 75%; m.p. 203.2–203.9 ◦C;1H NMR (200 MHz, DMSO-d6)
δ
3.94 (s, 3H, CH3), 3.98 (s, 3H, CH3),
00
00
00
00
7.24–7.36 (m, 2H, H-6 , H-7 ), 7.40–7.46 (m, 1H, H-4 ), 7.58–7.62 (m, 1H, H-5 ), 8.12–8.16 (m, 1H, H-5),
8.24 (s, 1H, H-200), 8.48–8.51 (m, 1H, H-6), 8.60 (dd, J = 2.1, 8.0 Hz, 1H, H-4), 8.65 (s, 1H, H-2). Anal.
Calculated for C19H15N5O (MW: 329.36): C, 69.29; H, 4.59; N, 21.26%. Found: C, 69.55; H 4.51; N,
21.21%. HRMS: [MH]+, found 330.1351 C19H15N5O requires 330.1349.

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5-Bromo-1-methyl-3-[3-(1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1H-pyrrolo[2,3-b]pyridine (1b). White
◦
solid; yield 72% m.p. 230.7–231.9 C; 1H NMR (200 MHz, DMSO-d6)
δ 3.95 (s, 6H, 2xCH3), 7.31 (s, 2H,
H-600, H-700), 7.59–7.63 (m, 1H, H-500), 8.11–8.30 (m, 2H, H-200, H-400), 8.58–8.71 (m, 3H, H-2, H-4, H-6).
Anal. Calculated for C19H14BrN5O (MW: 408.25): C, 55.90; H, 3.46; N, 17.15%. Found: C, 56.13; H 3.25;
N, 16.84%. HRMS: [MH]+, found 408.0449 C19H14BrN5O requires 408.0454.
5-Fluoro-1-methyl-3-[3-(1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1H-pyrrolo[2,3-b]pyridine (1c). White
solid; yield 62 %; m.p. 227.4–228.4 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ 3.94 (s, 3H, CH3), 3.97 (s, 3H,
CH3), 7.24–7.36 (m, 2H, H-600, H-700), 7.60 (d, J = 7.4 Hz, 1H, H-400), 8.12 (d, J = 7.4 Hz, 1H, H-500), 8.30
(s, 1H, H-200), 8.38 (m, 1H, H-4) 8.51 (s, 1H, H-6), 8.74 (s, 1H, H-2). Anal. Calculated for C19H14FN5O
(MW: 347.35): C, 65.70; H, 4.06; N, 20.16%. Found: C, 65.87; H 4.32; N, 20.44%. HRMS: [MH]+, found
348.1259 C19H14FN5O requires 348.1255.
1-Methyl-3-[3-(5-bromo-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1H-pyrrolo[2,3-b]pyridine (1d). Light
brown solid; yield 75%; m.p. 248.2–248.9 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ 3.95 (s, 3H, CH3), 3.99
(s, 3H, CH3), 7.40–7.47 (m, 2H, H-400, H-600), 7.61 (d, J = 8.8 Hz, 1H, H-700) 8.25 (s, 1H, H-4), 8.30 (s, 1H,
H-200), 8.50 (d, J = 6.2 Hz, 1H, H-5), 8.60 (d, J = 6.2 Hz, 1H, H-6), 8.68 (s, 1H, H-2). Anal. Calculated for
C19H14BrN5O (MW: 408.25): C, 55.90; H, 3.46; N, 17.15%. Found: C, 56.08; H 3.31; N, 17.25%. HRMS:
[MH]+, found 408.0456 C19H14BrN5O requires 408.0454.
5-Bromo-3-[3-(5-bromo-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (1e).
White solid; yield 82%; m.p. 264.2–265.9 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ 3.94 (s, 3H, CH3), 3.96
(s, 3H, CH3), 7.46 (d, J = 8.1 Hz, 1H, H-600) 7.61 (d, J = 8.1 Hz, 1H, H-700), 8.25 (s, 1H, H-400), 8.36 (s,
1H, H-200), 8.58 (s, 1H, H-2), 8.72 (m, 2H, H-4, H-6). Anal. Calculated for C19H13Br2N5O (MW: 487.15):
C, 46.84; H, 2.69; N, 14.38%. Found: C, 46.97; H 3.00; N, 14.12%. HRMS: [MH]+, found 485.9561
C19H13Br2N5O requires 485.9559.
5-Fluoro-3-[3-(5-bromo-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (1f)
.
◦
White solid; yield 75%; m.p. 255.3–256.2 C; 1H NMR (200 MHz, DMSO-d6)
δ
3.94 (s, 3H, CH3), 3.97 (s,
3H, CH3), 7.46 (d, J = 8.7 Hz, 1H, H-600), 7.61 (d, J = 8.7 Hz, 1H, H-700), 8.23 (s, 1H, H-200), 8.35–8.37 (m,
2H, H-400, H-4), 8.51 (s, 1H, H-6), 8.76 (s, 1H, H-2). Anal. Calculated for C19H13BrFN5O (MW: 426.24):
C, 53.54; H, 3.07; N, 16.43%. Found: C, 53.68; H 2.98; N, 16.71%. HRMS: [MH]+, found 426.0358
C19H13BrFN5O requires 426.0360.
3-[3-(5-Fluoro-1-methyl-1H-indol-3-yl)-1,2,◦4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (1g). Light
brown solid; yield 72%; m.p. 236.0–236.7 C; 1H NMR (200 MHz, DMSO-d6)
δ 3.95 (s, 3H, CH3), 3.99 (s,
3H, CH3), 7.19 (t, J = 8.8 Hz, 1H, H-600), 7.41–7.48 (m, 1H, H-700), 7.63 (m, 1H, H-400), 7.80 (d, J = 8.0 Hz
,
1H, H-4), 8.33 (s, 1H, H-200), 8.51 (d, J = 3.4 Hz, 1H, H-6), 8.60 (d, J = 8.0 Hz, 1H, H-5), 8.68 (s, 1H, H-2).
Anal. Calculated for C19H14FN5O (MW: 347.35): C, 65.70; H, 4.06; N, 20.16%. Found: C, 65.55; H 4.31;
N, 20.25%. HRMS: [MH]+, found 348.1253 C19H14FN5O requires 348.1255
5-Bromo-3-[3-(5-fluoro-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (1h).
Light brown solid; yield 60%; m.p. 255.2–256.8 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ 3.95 (s, 3H, CH3),
3.96 (s, 3H, CH3), 7.19 (t, J = 8.3 Hz, 1H, H-700), 7.64 (m, 1H, H-400), 7.77 (d, J = 8.3, 1.7 Hz, 1H, H-600),
8.37 (s, 1H, H-200), 8.58 (s, 1H, H-2), 8.72 (m, 2H, H-4, H-6). Anal. Calculated for C19H13BrFN5O (MW:
426.24): C, 53.54; H, 3.07; N, 16.43%. Found: C, 53.28; H 3.15; N, 16.26%. %. HRMS: [MH]+, found
426.0363 C19H13BrFN5O requires 426.0360
5-Fluoro-3-[3-(5-fluoro-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (1i).
White solid; yield 55%; m.p. 265.2–266.8 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ 3.95 (s, 3H, CH3), 3.98
(s, 3H, CH3), 7.20 (td, J = 2.6, 9.0 Hz, 1H, H-600), 7.61–7.68 (m, 1H, H-400), 7.79 (d, J = 2.6 Hz, 1H,
H-600), 8.35–8.41 (m, 2H, H-200, H-4), 8.51–8.53 (m, 1H, H-6), 8.76 (s, 1H, H-2). Anal. Calculated for
C19H13F2N5O (MW: 365.34): C, 62.46; H, 3.59; N, 19.17%. Found: C, 62.68; H 3.38; N, 19.42%. HRMS:
[MH]+, found 366.1157 C19H13F2N5O requires 366.1160

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3-[3-(5-Chloro-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (1j). White
solid; yield 65%; m.p. 242.8–243.7 ◦C; 1H NMR (200 MHz, DMSO-d6)
3.94 (s, 3H, CH3), 3.97 (s, 3H,
δ
CH3), 7.51–7.20 (m, 1H, H-600), 7.64 (d, J = 9.3 Hz, 1H, H-700),8.65 (s, 1H, H-400), 8.57 (d, J = 7.1 Hz, 1H,
H-5), 8.49 (d, J = 2.5 Hz, 1H, H-4), 8.30 (s, 1H, H-200), 8.09 (s, 1H, H-2), 7 3.95 (d, J = 7.1 Hz, 1H, H-6).
Anal. Calculated for C19H14ClN5O (MW: 363.80): C, 62.73; H, 3.88; N, 19.25%. Found: C, 62.58; H 3.71;
N, 19.37%. HRMS: [MH]+, found 364.0962 C19H14ClN5O requires 364.0960.
5-Bromo-3-[3-(5-chloro-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine
(
1k). White solid; yield 75%; m.p. 270.2–271.3 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ
3.95 (s, 3H, CH3),
3.97 (s, 3H, CH3), 7.35 (dd, J = 8.7, 2.0 Hz, 1H, H-600), 7.66 (d, J = 8.7 Hz, 1H, H-700), 8.09 (d, J = 2.0 Hz,
00
00
1H, H-4 ), 8.38 (s, 1H, H-2 ), 8.58 (d, J = 2.2 Hz, 1H, H-4), 8.70 (d, J = 2.2 Hz, 1H, H-6), 8.74 (s, 1H, H-2).
Anal. Calculated for C19H13BrClN5O (MW: 442.70): C, 51.55; H, 2.96; N, 15.82%. Found: C, 51.28; H
3.11; N, 16.02%. HRMS: [MH]+, found 442.0068 C19H13BrClN5O requires 442.0065.
5-Fluoro-3-[3-(5-chloro-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (1l).
White solid; yield 85%; m.p. 258–259 ◦C; 1H NMR (2000M0 Hz, DMSO-d6)
δ 3.94 (s, 3H, CH3), 3.97 (s,
00
00
3H, CH3), 7.34 (d, J = 8.3 Hz, 1H, H-6 ), 8.07 (m, 1H, H-7 ), 7.64 (m, 1H, H-4 ), 8.39 (m, 2H, H-6, H-4’),
8.52 (s, 1H, H200), 8.77 (s, 1H, H-2). Anal. Calculated for C19H13ClFN5O (MW: 381.79): C, 59.77; H,
3.43; N, 18.34%. Found: C, 59.68; H 3.25; N, 18.56%. HRMS: [MH]+, found 382.0867 C19H13ClFN5O
requires 382.0865.
3-[3-(5-Methoxy-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (1m).
White solid; yield 70%; m.p. 197–198 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ 3.86 (s, 3H, CH3), 3.90 (s,
3H, CH3), 3.98 (s, 3H, OCH3), 6.96 (d, J = 9.0 Hz, 1H, H-600), 7.61 (s, 1H, H-200), 7.55–7.32 (m, 2H, H-700,
H-400), 8.17 (s, 1H, H-2), 8.49 (d, J = 4.0 Hz, 1H, H-6), 8.60 (m, 2H, H-4, H-5). Anal. Calculated for
C20H17N5O2 (MW: 359.38): C, 66.84; H, 4.77; N, 19.49%. Found: C, 66.59; H 4.89; N, 19.23%. HRMS:
[MH]+, found 360.1456 C20H17N5O2 requires 360.1455.
5-Bromo-3-[3-(5-methoxy-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine
(
1n). White solid; yield 65%; m.p. 229.6–230.2 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ 3.88 (s, 3H, CH3).
3.90 (s, 3H, CH3), 3.97 (s, 3H, OCH3), 6.95 (d, J = 8.3 Hz, 1H, H-600), 7.50 (d, J = 8.3 Hz, 1H, H-700), 7.62
(d, J = 1.3 Hz, 1H, H-400), 8.21 (s, 1H, H-200), 8.58 (s, 1H, H-2), 8.71 (m, 2H, H-4, H-6). Anal. Calculated
for C20H16BrN5O2 (MW: 438.28): C, 54.81; H, 3.68; N, 15.98%. Found: C, 54.78; H 3.95; N, 15.78%.
HRMS: [MH]+, found 438.0562 C20H16BrN5O2 requires 438.0560.
5-Fluoro-3-[3-(5-methoxy-1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine
(1o). White solid; yield 80%; m.p. 218–219 ◦C 1H NMR (200 MHz, DMSO-d6)
δ 3.78 (s, 3H, CH3), 3.90
(s, 3H, CH3), 3.96 (s, 3H, OCH3), 6.95 (dd, J = 8.9, 2.4 Hz, 1H, H-600), 7.54 (m, 2H, H-700, H-400), 8.22 (m,
1H, H-4), 8.50 (s, 1H, H-200), 8.37 (dd, J = 8.9, 2.7 Hz, 1H, H-6), 8.72 (s, 1H, H-2). Anal. Calculated for
C20H16FN5O2 (MW: 377.37): C, 63.65; H, 4.27; N, 18.56%. Found: C, 63.78; H 4.10; N, 18.63%. HRMS:
[MH]+, found 378.1359 C20H16FN5O2 requires 378.1361.
3.2. Biology
Compounds 1a–o, prepared as described above, were dissolved in dimethyl sulfoxide (DMSO)
and then diluted in culture medium to have a DMSO concentration not exceeding 0.1%. MCF-7 (human
breast cancer), HeLa (cervix adenocarcinoma), HCT-116 (human colorectal carcinoma), and CaCo2
(colorectal carcinoma) cell lines were obtained from American Type Culture Collection, Rockville, MD,
USA and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal, 10%
fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 µg/mL), and gentamicin (5 µg/mL).
All the cells were maintained in log phase by seeding twice a week at a density of 3 × 108 cells/L in
a humidified 5% CO2 atmosphere, at 37 ◦C. In all experiments, cells were left to incubate overnight
to allow adhesion before treatment with the compounds or vehicle alone (control cells). In selected

Mar. Drugs 2019, 17, 35
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experiments CaCo2 cells were treated 15 days after confluence, at which time the cells are differentiated
in normal intestinal-like cells [43].
No differences were found between cells treated with DMSO 0.1% and untreated cells in terms of
cell number and viability.
3.2.1. Viability Assay in Vitro
Cytotoxic activity of compounds 1k and 1n against human tumor cell lines (MCF-7, HeLa, Caco-2,
and HCT-116) and intestinal-like differentiated cells was determined by the MTT colorimetric assay
commonly used to study inhibition of cellular proliferation. Briefly, cells were seeded at 2
cells/well in 96-well plates containing 200 L DMEM. When appropriated, cells were treated with
vehicle alone (0.1% DMSO, control) or various concentrations (0.01–100 M) of the drugs in DMEM
×
104
µ
µ
and let them incubate for 72 h. Then cells were washed, and 50
µ
L of FBS-free ◦medium containing
5 mg/mL MTT was added. The medium was discarded after 2 h incubation at 37 C by centrifugation,
and formazan blue formed in the cells was dissolved in DMSO. The absorbance, measured at 570 nm
on a microplate reader (Bio-RAD, Hercules, CA, USA), of MTT formazan of control cells were taken as
100% of viability.
Cytotoxicity of the compounds was defined as the IC50 value which represents the molar
concentration of the compound that inhibits 50% cell viability. IC50 values were calculated by the
dose-response inhibition model using GraphPad Prism 5.02 from GraphPad Software (San Diego, CA,
USA). Each experiment was repeated at least three times in triplicate to obtain the mean values.
3.2.2. Cell Cycle Analysis
MCF-7 cells (5.0
overnight incubation, the cells were washed with fresh medium and incubated with the compounds or
vehicle alone (control cells) in DMEM for 24 h. After trypsinization, aliquots of 1.0
106 cells were
washed with PBS and incubated in the dark in PBS containing 20 g/mL propidium iodide (PI) and
200 g/mL RNase, for 30 min, at room temperature. Then samples of at least 1.0
104 cells were
flow cytometer using
×
104 cells/cm2) were seeded in triplicate in 24-wells culture plates. After an
×
µ
µ
×
subjected to fluorescence-activated cell sorting (FACS) analysis by Epics XL
™
Expo32 software (Beckman Coulter, Fullerton, CA, USA).
3.2.3. Cell Death Detection
Cell death was assessed by staining the cells with AnnexinV-FITC and propidium iodide (PI)
(Sigma-Aldrich, Steinheim, Germany) according to the manufacturer’s instructions (eBioscience, San
Diego, CA, USA) and subsequent analysis by flow cytometry. MCF-7 cells (5.0
×
104 cells/cm2)
were seeded in triplicate in 24-wells culture and after an overnight incubation, washed with fresh
medium and incubated for 24 h with the compounds or vehicle alone (control cells) in DMEM. After
trypsinization, 1.0
×
106 cells/mL in combining buffer were incubated with Annexin V-FITC and PI
solution at room temperature in the dark for 15 min. Then samples of at least 1.0
subjected to FACS analysis using appropriate 2-bidimensional gating method.
×
104 cells were
Acridine orange/ethidium bromide (AO/EB) fluorescence staining was also used to identify
cell apoptosis. After 24 h treatment, the cells were washed with PBS and stained with AO/EB
solution (100 µg/mL, 1:1). After 20 s the AO/EB solution was discarded and cells were immediately
visualized by fluorescence microscopy (Leica; Wetzlar, Germany). Multiple photos were taken at
randomly-selected areas of the well to obtain representative data.
4. Conclusions
New analogs of nortopsentin, in which the central imidazole ring of the natural lead was replaced
by a 1,2,4-oxadiazole moiety and in which a 7-azaindole portion substituted the original indole
moiety, were efficiently synthesized. Among the synthesized derivatives, 1k and 1n showed the best
cytotoxic activity against several human cancer cell lines. The mechanism of the anti-proliferative

Mar. Drugs 2019, 17, 35
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effect on MCF-7 was pro-apoptotic, being associated with externalization of plasma membrane
phosphatidylserine, chromatin condensation, and membrane blebbing. Finally, the compounds
induced an accumulation of cells in G0–G1 phase suggesting that they can affect the cell mechanisms
promoting the DNA duplication.
Author Contributions: S.C., V.D.S., S.M., and B.P. performed the chemical research and analyzed the data. A.A.
and L.T. performed the biological research and analyzed the data. G.C., P.D., L.T., and B.P. participated in the
design of the research and the writing of the manuscript. All authors read and approved the final manuscript.
Funding: This project was supported by a 2014–2020 PON Ricerca e Innovazione grant from the Italian Ministry
of Education, University and Research, entitled “PROGEMA—Processi Green per l’Estrazione di Principi Attivi e
la Depurazione di Matrici di Scarto e Non” (ARS01_00432) to P.C.
Acknowledgments: The authors acknowledge the 2014–2020 PON Ricerca e Innovazione grant from the Italian
Ministry of Education, University and Research, entitled “PROGEMA—Processi Green per l’Estrazione di Principi
Attivi e la Depurazione di Matrici di Scarto e Non” (ARS01_00432) to P.C.
Conflicts of Interest: The authors declare no conflict of interest.
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