Design, synthesis, and antiproliferative activity of new 1H-pyrrolo[3,2-c]pyridine derivatives against melanoma cell lines

Article abstract of DOI:10.1016/j.ejmech.2011.04.031

Synthesis of a new series of diarylureas and diarylamides having 1H-pyrrolo[3,2-c]pyridine scaffold is described. Their in vitro antiproliferative activity against A375P human melanoma cell line was tested and the effect of substituents on pyrrolo[3,2-c]pyridine nucleus was investigated. The newly synthesized compounds, except three N-tolyl derivatives (8f, 9f, and 9h), generally showed superior activity against A375P to Sorafenib. Among all of these derivatives, compounds 8b, 8g, and 9a-e showed the highest potency against A375P with IC₅₀ in nanomolar range. In addition, compounds 8d, 8e, 8h, 9g, 9i, and 9j were more potent than Sorafenib but with IC ₅₀ in micromolar range. Compounds 8b, 8g, 9b-d, and 9i demonstrated higher selectivity towards A375P compared with NIH3T3 fibroblasts. The most potent diarylurea 8g and diarylamide 9d were further tested and showed high potency over nine melanoma cell lines at the NCI.

Full text of DOI:10.1016/j.ejmech.2011.04.031

European Journal of Medicinal Chemistry 46 (2011) 3218e3226
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, and antiproliferative activity of new 1H-pyrrolo[3,2-c]pyridine
derivatives against melanoma cell lines
,
Mohammed I. El-Gamal a b, Myung-Ho Jung a, Woong San Lee a, Taebo Sim a, Kyung Ho Yoo a,
,b,
*
Chang-Hyun Oh a
a Biomaterials Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea
b Department of Biomolecular Science, University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a new series of diarylureas and diarylamides having 1H-pyrrolo[3,2-c]pyridine scaffold is
described. Their in vitro antiproliferative activity against A375P human melanoma cell line was tested
and the effect of substituents on pyrrolo[3,2-c]pyridine nucleus was investigated. The newly synthesized
compounds, except three N-tolyl derivatives (8f, 9f, and 9h), generally showed superior activity against
A375P to Sorafenib. Among all of these derivatives, compounds 8b, 8g, and 9aee showed the highest
potency against A375P with IC50 in nanomolar range. In addition, compounds 8d, 8e, 8h, 9g, 9i, and 9j
were more potent than Sorafenib but with IC50 in micromolar range. Compounds 8b, 8g, 9bed, and 9i
demonstrated higher selectivity towards A375P compared with NIH3T3 fibroblasts. The most potent
diarylurea 8g and diarylamide 9d were further tested and showed high potency over nine melanoma cell
lines at the NCI.
Received 1 February 2011
Received in revised form
29 March 2011
Accepted 10 April 2011
Available online 16 April 2011
Keywords:
Pyrrolo[3,2-c]pyridine
A375P
Antiproliferative activity
Melanoma
Diarylurea
Ó 2011 Elsevier Masson SAS. All rights reserved.
Diarylamide
1. Introduction
both the FDA and EMEA for adjuvant treatment of melanoma
patients, and aldesleukin (Proleukin) [9,10] has been also approved
Melanoma is the most serious type of skin cancer as a malignant
tumor of melanocytes. Incidence of melanoma has tripled in the
last 4 decades, and more than 80% of skin cancer deaths are due to
melanoma. Generally, two major risk factors for melanoma devel-
opment are the individual’s family history and environmental
factors. The most important exogenous etiological factor is expo-
sure to solar ultraviolet irradiation [1].
Early stage melanoma can be cured surgically. However, mela-
noma metastasizing to major organs (stage IV) is virtually incurable
[2]. Patients with advanced melanoma have a median survival time
of less than one year, and the estimated 5-year survival rate is less
than 15% [3,4]. With the incidence of melanoma rapidly rising in
the United States and other developed countries, there is an urgent
need to develop more effective drugs [5e7].
for the treatment of metastatic melanoma in the USA.
A number of reports have recently highlighted diarylureas and
diarylamides as potential antiproliferative agents against mela-
noma cell line [11e18]. Sorafenib (Nexavar) is a diarylurea deriva-
tive that has been extensively used in clinical trials [19].
Encouraged by the interesting antiproliferative activity of diary-
lurea and diarylamide derivatives, a new series of diarylureas and
diarylamides possessing 1H-pyrrolo[3,2-c]pyridine scaffold was
synthesized (Fig. 1). Herein, we report the synthesis and anti-
proliferative activities of these compounds against human mela-
noma cell lines and NIH3T3 fibroblasts.
2. Results and discussion
The current treatments involve surgical removal of the tumor,
immunotherapy, radiotherapy, chemotherapy, various combina-
tions, or the use of new treatments in clinical trials. As for immu-
notherapy, interferon alfa-2b (Intron-A) [8] has been approved by
2.1. Chemistry
2.1.1. Synthesis of the target compounds 8aeg and 9aei (Scheme 1)
7-Hydroxy-1H-pyrrolo[2,3-b]pyridinium 3-chlorobenzoate (2)
was prepared by reacting 7-azaindole (1) with 3-chloroperbenzoic
acid [20,21]. Compound 2 was heated with phosphorus oxychloride
to produce 4-chloro-7-azaindole (3) [21]. Compounds 4a,b were
prepared according to the literature procedure [22,23]. Fusion of 3
* Corresponding author. Biomaterials Center, Korea Institute of Science and
Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea. Tel.: þ82
2 958 5160; fax: þ82 2 958 5189.
E-mail address: choh@kist.re.kr (C.-H. Oh).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.031

M.I. El-Gamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3218e3226
3219
O
CF3
R3 NH
N
2.1.2. Synthesis of the target amino compounds 8h,i and 9j,k
(Scheme 2)
R1
H3C
O
Cl
N
H
O
N
N
Refluxing the nitro compound 5a with stannous chloride in
ethanol led to simultaneous reduction of the nitro group and
hydrolysis of the benzamido moiety to produce the diamino
compound 10. Interaction of the aniline amino group of 10 with
1-(trifluoromethyl)-3-isocyanatobenzene or 1-chloro-2-(trifluoro-
methyl)-4-isocyanato-benzene gave the target diarylurea derivatives
8h,i, respectively, and the amino group at position 4 on the pyrrolo
[3,2-c]pyridine nucleus remained unaffected. Preparation of
compounds 9j,k was carried out in a similar way as described for
preparation of compounds 9aei andtheamino group at position 4 on
the pyrrolo[3,2-c]pyridine nucleus remained unaffected also.
O
N
H
N
H
R2
N
H
1H-pyrrolo[3,2-c]pyridine derivatives
Sorafenib
R1 = H, CH3
R2 = Ar, ArNH
R3 = H, PhCO
Fig. 1. Structures of Sorafenib and 1H-pyrrolo[3,2-c]pyridine derivatives.
with p-nitroaniline or 2-methyl-4-nitrobenzenamine neat led to
nucleophilic displacement of the 4-chloro group by the aromatic
amino group, followed by rearrangement of the resulting
secondary amine to give the amine hydrochloride salts 4a,b, albeit
in low yields. The benzamido derivatives 5a,b were obtained by
reaction of the amino group of 4a,b with benzoyl chloride in the
presence of diisopropylamine as a base. Reduction of the nitro
group of 5a,b using PdeC/H2 gave the corresponding amino
compounds 6a,b. Treatment of 6a,b with the appropriate aryl
isocyanates produced the corresponding diarylurea derivatives
8aef. The bisamide derivatives 9aei were obtained by condensa-
tion of 6a,b with the corresponding aromatic carboxylic acids in the
presence of HOBt/EDCI/triethylamine.
2.2. Biological activity
2.2.1. Antiproliferative activity against A375P human melanoma
cell line
The antiproliferative activity of the newly synthesized
compounds against A375P human melanoma cell line was tested.
The ability of pyrrolo[3,2-c]pyridine diarylureas and diarylamides
to inhibit the growth of A375P cell line is summarized in Tables 1
and 2. Sorafenib was selected as the reference standard.
The newly synthesized compounds, except three N-tolyl deriv-
atives (8f, 9f, and 9h), generally showed superior activity against
A375P to Sorafenib. Among all of these derivatives, compounds
8b, 8g, and 9aee showed the most potent antiproliferative
activity against A375P with IC50 in nanomolar range. In addition,
The N-ethylpiperazinyl diarylurea derivative 8g was prepared by
a modified method through heating the aniline derivative 7 with
p-nitrophenyl chloroformate in the presence of triethylamine as
a base to form the corresponding carbamate intermediate, and
subsequent heating with compound 6a.
R
Cl
O
N
O-
b
H2N
HCl
a
c
NO2
N
H
N
H
N
H
N
N
N
OH
N
Cl
4a,b
1
3
2
4a: R = H
4b: R = CH3
R
R
H
N
H
N
e
N
d
N
NO2
NH2
O
N
O
N
5a,b
6a,b
5a: R = H
5b: R = CH3
6a: R = H
6b: R = CH3
g
f
R
R
O
O
H
N
H
N
N
N
Ar
Ar
N
H
N
H
N
H
O
N
O
N
9a-i
8a-f
R = H, CH3
F3C
NH2
N
N
h
O
N
H
N
CF3
N
N
H
H
N
O
N
N
8g
7
Scheme 1. Reagents and conditions: (a) 3-chloroperoxybenzoic acid, DME:heptane (1:2), rt; (b) POCl3, 55 ꢀC then rt then 85e90 ꢀC; (c) appropriate nitroaniline, 180 ꢀC; (d) benzoyl
chloride, diisopropylamine, CH3CN, rt; (e) Pd/C, H2, THF, rt; (f) aryl isocyanate, THF, rt; (g) carboxylic acid derivative, HOBt, EDCI, TEA, DMF, 80 ꢀC; (h) (i) 4-nitrophenyl chloro-
formate, TEA, 1,4-dioxane, 60 ꢀC, (ii) 6a in 1,4-dioxane, 90 ꢀC.

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M.I. El-Gamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3218e3226
O
Ar
H
N
N
N
N
H2N
H2N
N
H
b
a
N
NO2
NH2
H
N
O
N
N
8h,i
5a
10
c
O
N
H2N
Ar
N
H
N
9j,k
Scheme 2. Reagents and conditions: (a) SnCl2$H2O, EtOH, reflux; (b) aryl isocyanate, THF, rt; (c) benzoic acid derivative, HOBt, EDCI, TEA, DMF, 80 ꢀC.
compounds 8d, 8e, 8h, 9g, 9i, and 9j were more potent than
Sorafenib against A375P but with IC50 in micromolar range.
Compounds 9a, 9b, and 9d with 20-unsubstituted phenyl ring at
position 1 on the pyrrolo[3,2-c]pyridine nucleus were more potent
than compounds 9f, 9g, and 9h having a tolyl ring against A375P.
This suggests that introduction of 20-methyl group on this phenyl
ring is unfavorable.
The effect of substituents on the phenyl ring of the tail was also
investigated. The introduction of para-chloro, meta-trifluoromethyl
groups, or para-(4-ethylpiperazinyl)methyl moiety on the 30-tri-
fluoromethylphenyl ring of tail (compounds 8d, 8e, and 8g,
respectively) significantly enhanced the antiproliferative acti-
vity compared with compound 8c with 40,50-unsubstituted
trifluoromethylphenyl ring. This may be attributed to the enhanced
binding affinity produced by these substituents tothe target protein.
The enhanced antiproliferative activity produced by para-chloro
substituent on the phenyl ring of the tail can be confirmed by
comparing the IC50 values of compounds 9b and 9g with those of
compounds 9a and 9f. In addition, compound 8b with 30,40-
dichlorophenyl moiety demonstrated more potent antiproliferative
activity than compound 8a with 20,30-dichlorophenyl moiety
against A375P cell line. This also assures that para-chloro substit-
uent on the phenyl ring of the tail is favorable.
Upon comparing the activities of compounds 9cee with that of
compound9a, substitution of the 30-trifluoromethylphenylringofthe
tail with a morpholine moietyat position 40 (compound 9d)improved
Table 1
Antiproliferative activity of 1H-pyrrolo[3,2-c]pyridine ureas (8aei).
Structure
Comp. no.
R1
R2
R3
IC50 (m
M)a
A375P
NIH3T3
8a
8b
8c
H
H
H
PhCO
PhCO
PhCO
>10
e
0.2
7.0
11.48
e
8d
H
PhCO
2.4
e
R3 NH
N
R1
N
8e
8f
H
PhCO
PhCO
1.8
e
e
O
R2
N
H
N
H
CH3
>10
8g
8h
H
PhCO
95 ꢁ 10ꢂ3
0.98
H
H
H
H
5.4
e
8i
8.0
5.6
e
Sorafenib
24.75
a
Values are expressed as mean IC50 of the triplicate experiment.

M.I. El-Gamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3218e3226
3221
Table 2
Antiproliferative activity of 1H-pyrrolo[3,2-c]pyridine amides (9aek).
Structure
Comp. no.
R1
R2
R3
IC50 (m
M)a
A375P
NIH3T3
9a
9b
H
H
PhCO
PhCO
0.1
0.05
5.65
16.4 ꢁ 10ꢂ3
9c
H
H
PhCO
PhCO
0.8
5.57
9d
7.0 ꢁ 10ꢂ3
1.74
0.27
9e
9f
H
PhCO
PhCO
0.3
R3 NH
N
R1
N
O
CH3
>10
e
R2
N
H
9g
CH3
PhCO
2.7
e
9h
9i
CH3
H
PhCO
PhCO
H
>10
1.0
e
15.91
e
9j
H
3.8
9k
H
H
6.2
5.6
e
Sorafenib
24.75
a
Values are expressed as mean IC50 of the triplicate experiment.
the potency. On the other hand, introduction of methylimidazole or
morpholine moieties at meta position on the same tail 30-tri-
fluoromethylphenylring(compounds9cand9e, respectively)slightly
decreased the potency. This can be attributed to the influence of the
substituents orientation at the receptor site on the activity.
2.2.2. Antiproliferative activity against NIH3T3 fibroblasts
Compounds 8b, 8g, 9aee, and 9i, which showed the highest
potency against A375P melanoma cell line, were tested against
NIH3T3 fibroblasts in order to determine their selectivity for
melanoma cells compared with normal cells. Sorafenib was
considered as a reference drug in this experiment also.
Compounds 8b, 8g, 9bed, and 9i showed higher selectivity for
A375P cells than for NIH3T3 fibroblasts. Their selectivity indices
were higher than that of Sorafenib. These compounds are prom-
ising leads for design of highly potent and highly selective anti-
proliferative agents for treatment of melanoma.
On the other hand, compounds 9a and 9e demonstrated low
selectivity towards melanoma cells despite of theirhigh potency. So it
can be concluded that para-chloro, meta-methylimidazole, and para-
morpholino substituents on the terminal 30-trifluoromethylphenyl
ring increased the selectivity of compounds 9b, 9c and 9d, respec-
tively, compared with9a. Inaddition, para-morpholino compound9d
showed higher selectivity than meta-morpholino derivative 9e.
Regarding the substituents on the 4-position of pyrrolo[3,2-c]
pyridine, compounds 8d, 9b, and 9d having benzoylamino moiety
were more potent than the corresponding compounds 8i, 9j, and 9k
with unsubstituted amino group against A375P. This suggests that
the aromatic amide substituent at this position is more favorable.
By comparing the activities of derivatives with amide and urea
moieties at pyrrolo[3,2-c]pyridine side chain as a linker, it was found
that the derivatives with amide moieties (9a, 9b, and 9j) were more
potent than those with urea moieties (8c, 8d, and 8i). Moreover,
most of the newly synthesized amide derivatives (9aee, 9g, 9i, and
9j) demonstrated more potent antiproliferative activity against
A375P than that of Sorafenib with urea moiety. These results were
seemed to indicate the effect of the linker on the activity.

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M.I. El-Gamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3218e3226
Table 3
spectra (MS) were taken in ESI mode on a Waters 3100 Mass
IC50 values in mM of compounds 8g and 9d over 9 melanoma cell lines.
Detector (Waters, Milford, MA, USA). Nuclear magnetic resonance
(NMR) spectroscopy was performed using a Bruker ARX-300,
300 MHz spectrometer (Bruker Bioscience, Billerica, MA, USA)
with TMS as an internal standard. Purities of the target compounds
(>95%) were determined by LCeMS analysis using the following
system: Waters 2998 photodiode array detector, Waters 3100 mass
detector, Waters SFO system fluidics organizer, Waters 2545 binary
gradient module, Waters reagent manager, Waters 2767 sample
Melanoma cell line
Compound no.
8g
9d
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
0.481
1.54
1.22
0.894
3.62
1.29
0.764
1.59
1.27
45.6 ꢁ 10ꢂ3
11.5
0.763
0.119
0.637
0.292
0.138
1.98
manager, SunfireÔ C18 column (4.6 ꢁ 50 mm, 5
mm particle size);
Solvent gradient ¼ 95% A at 0 min, 1% A at 5 min; solvent A: 0.035%
trifluoroacetic acid (TFA) in water; solvent B: 0.035% TFA in CH3OH;
flow rate ¼ 3.0 mL/min; the AUC was calculated using Waters
MassLynx 4.1 software. All reagents and solvents were purchased
from Aldrich chemical Co. and Tokyo Chemical Industry (TCI) Co.,
and used without further purification.
0.485
2.2.3. Antiproliferative activities of compounds 8g and 9d against
nine human melanoma cell lines at the NCI
The most potent diarylurea and diarylamide derivatives, 8g and
9d, respectively, against A375P cell line were selected by the
National Cancer Institute (NCI) [24], Bethesda, Maryland, USA, for
evaluation of their antineoplastic activity. After initial single dose
screening of these two compounds, they were further tested in
a five-dose testing mode in order to determine their potency. For
each of these compounds, the IC50 value was recorded. The anti-
proliferative activities of 8g and 9d over 9 melanoma cell lines are
summarized in Table 3.
4.2. 7-Hydroxy-1H-pyrrolo[2,3-b]pyridinium 3-chlorobenzoate (2)
It was prepared following the literature procedure [20,21]. Mp:
141e143 ꢀC (Lit. mp: 144.1e146 ꢀC [21]).
4.3. 4-Chloro-7-azaindole (3)
It was prepared following the literature procedure [21]. Mp:
176e177 ꢀC (Lit. mp: 175.3e177 ꢀC [21]).
As shown in Table 3, both the tested compounds showed
good potency over the 9 tested cell lines. The IC50 values of the
tested compounds were less than 4 mM over all tested cell lines,
4.4. General procedure for preparation of compounds 4a,b
except for the IC50 of compound 9d against MALME-3M cell line.
The IC50 of the diarylurea compound 8g was in nanomolar range
against 3 cell lines. In addition, compound 9d with amide linker and
4-morpholino-3-(trifluoromethyl)phenylterminalmoietygenerally
showed higher potency against the 9-melanoma cell line panel,
compared with 8g. The IC50 value of compound 9d was in nanomolar
range over 7 cell lines. Its highest potency was recorded against LOX
IMVI cell line (IC50 ¼ 45.6 nM). This high potency together with its
result against A375P cell line makes compound 9d a promising lead
for further design of antiproliferative agents for melanoma.
A mixture ofcompound 3 (230 mg, 2.0 mmol) and the appropriate
nitroaniline derivative (10.0 mmol) was fused at 180 ꢀC for 2e5 h
with stirring. The reaction mixture was cooled to room temperature
and dissolved in ethanol (150 mL). The resulting suspension was
filtered to remove the insoluble material and the filtrate was evapo-
rated under reduced pressure. The residue was purified by column
chromatography (silica gel, ethyl acetateemethanol 10:1 v/v then
switching to ethyl acetateemethanol 5:1 v/v) to obtain the desired
purified products.
3. Conclusions
4.4.1. 1-(4-Nitrophenyl)-1H-pyrrolo[3,2-c]pyridin-4-amine
hydrochloride (4a)
Yield 18%; 1H NMR (DMSO-d6):
d 13.40 (brs, 1H), 8.42 (d, 2H,
A new series of diarylureas and diarylamides containing 1H-pyr-
rolo[3,2-c]pyridine scaffold was synthesized based on our previous
literature studies. Among all of these derivatives, compounds 8g, 9b,
and 9d demonstrated the highest potency against A375P human
J ¼ 8.9 Hz), 7.90 (d, 2H, J ¼ 9.0 Hz), 7.80 (d, 1H, J ¼ 5.9 Hz), 7.68 (d,
1H, J ¼ 6.8 Hz), 7.34 (d, 1H, J ¼ 6.3 Hz), 7.01 (d, 1H, J ¼ 7.2 Hz); MS
m/z: 255.95 (Mþ þ 2, 90.57%), 254.65 (Mþ, 100%).
melanoma cell line with IC50 less than 0.1
compounds, 8b, 9a, 9c, and 9e, were more potent than Sorafenib
against A375P with IC50 ranging from 0.1 to 0.8 M. In addition,
compounds 8d, 8e, 9g, and 9i were more potent than Sorafenib
against A375P with IC50 in the range of 1.0e2.7 M. Compounds 8b,
mM. Another four
4.4.2. 1-(2-Methyl-4-nitrophenyl)-1H-pyrrolo[3,2-c]pyridin-4-
m
amine hydrochloride (4b)
Yield 9.5%; 1H NMR (DMSO-d6):
d 8.28e8.27 (m, 1H), 8.08 (d, 1H,
m
J ¼ 5.2 Hz), 7.53e7.50 (m, 2H), 7.43e7.36 (m, 2H), 7.30e7.25 (m, 3H),
6.76 (d, 1H, J ¼ 5.2 Hz), 6.03e6.01 (m, 1H), 2.37 (s, 3H); MS m/z:
270.00 (Mþ þ 2, 90%), 268.69 (Mþ, 100%).
8g, 9bed, and 9i showed superior selectivity towards melanoma cells
than NIH3T3 fibroblasts, compared with Sorafenib.
The most potent diarylurea 8g and diarylamide 9d were further
tested over nine melanoma cell lines at the NCI. Both compounds
demonstrated high potency over the 9-cell line panel. The IC50
values of compounds 8g and 9d were in nanomolar range over 3 and
7 cell lines, respectively. Further modifications of these compounds
in order to improve their potency are currently in progress.
4.5. General procedure for preparation of compounds 5a,b
To a stirred solution of compound 4a,b (1.6 mmol) in acetonitrile
(25 mL) at room temperature, diisopropylamine (0.7 mL, 4.0 mmol)
was slowly added under nitrogen atmosphere. Benzoyl chloride
(0.2 mL, 1.9 mmol) was slowly added and the reaction mixture was
stirred at room temperature for 8 h. The reaction mixture was
concentrated under reduced pressure, and water (20 mL) and
CH2Cl2 (20 mL) were added to the residue. The organic layer was
separated and the aqueous layer was extracted with CH2Cl2
(2 ꢁ 20 mL). The combined organic layer extracts were washed
with brine, 1 N HCl, and then aqueous NaHCO3, dried over
4. Experimental
4.1. General
All melting points were obtained on a Walden Precision Appa-
ratus Electrothermal 9300 apparatus and are uncorrected. Mass

M.I. El-Gamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3218e3226
3223
anhydrous MgSO4, and filtered. The organic solvent was evaporated
4.7.1. 1-(4-(4-Benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-3-
under reduced pressure and the residue was purified by column
chromatography to give the desired products.
(3,4-dichloro-phenyl)urea (8b)
Yield 14%; mp: 172e175 ꢀC;1H NMR (DMSO-d6):
d
10.87 (brs,
1H), 9.26e9.16 (m, 2H), 8.12e8.09 (m, 3H), 7.92 (d, 1H, J ¼ 2.4 Hz),
4.5.1. N-(1-(4-nitrophenyl)-1H-pyrrolo[3,2-c]pyridin-4-yl)
benzamide (5a)
7.74e7.61 (m, 3H), 7.57e7.48 (m, 5H), 7.41e7.31 (m, 2H), 6.64 (d, 1H,
J ¼ 3.2 Hz); 13C NMR (DMSO-d6):
d 170.8, 152.8, 140.4, 139.2, 131.5,
It was purified by column chromatography (silica gel,
131.1, 129.8, 129.2, 128.9, 128.6, 127.4, 127.0, 125.2, 124.8, 123.7,
122.9, 121.8, 120.0, 119.9, 118.9, 104.7; MS m/z: 518.38 (Mþ þ 2, 34%),
517.42 (Mþ þ 1, 89%), 516.45 (Mþ, 100%).
hexaneeethyl acetate 3:1 v/v); yield 85%; 1H NMR (DMSO-d6):
d
8.49 (d, 2H, J ¼ 5.0 Hz), 8.10 (d, 1H, J ¼ 6.0 Hz), 7.88e7.82 (m, 6H),
7.63 (d, 1H, J ¼ 6.0 Hz), 7.55e7.50 (m, 2H), 7.43e7.38 (m, 2H); MS m/
z: 359.11 (Mþ þ 1, 100%), 358.24 (Mþ, 27.71%).
4.7.2. 1-(4-(4-Benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-
3-(3-trifluoromethyl-phenyl)urea (8c)
4.5.2. N-(1-(2-methyl-4-nitrophenyl)-1H-pyrrolo[3,2-c]pyridin-4-
yl)benzamide (5b)
Yield 35%; mp: 158e160 ꢀC; 1H NMR (DMSO-d6):
d 11.71 (brs,
1H), 10.83 (brs, 1H), 8.59e8.57 (m, 2H), 8.11e8.09 (m, 4H), 7.74 (d,
2H, J ¼ 7.8 Hz), 7.66 (d, 2H, J ¼ 3.2 Hz), 7.62e7.59 (m, 2H), 7.58e7.52
(m, 4H), 7.39 (d, 1H, J ¼ 5.4 Hz), 6.66 (brs, 1H); MS m/z: 517.33
(Mþ þ 2, 46.6%), 516.36 (Mþ þ 1, 100%), 515.38 (Mþ, 21%).
It was purified by column chromatography (silica gel,
hexaneeethyl acetate 1:1 v/v); yield 15%; 1H NMR (DMSO-d6):
d
11.86 (brs, 1H), 8.28 (d, 1H, J ¼ 2.6 Hz), 8.09 (d, 1H, J ¼ 5.2 Hz),
8.04e8.01 (m, 2H), 7.54e7.51 (m, 2H), 7.43e7.37 (m, 2H), 7.31e7.26
(m, 3H), 6.77 (d, 1H, J ¼ 5.1 Hz), 6.02 (d, 1H, J ¼ 2.8 Hz), 2.37 (s, 3H);
MS m/z: 373.42 (Mþ þ 1, 100%), 372.40 (Mþ, 42%).
4.7.3. 1-(4-(4-Benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-
3-(4-chloro-3-trifluoromethylphenyl)urea (8d)
Yield 25%; mp: 204e206 ꢀC; 1H NMR (DMSO-d6):
d 10.83 (brs,
4.6. General procedure for preparation of compounds 6a,b
1H), 9.37 (brs, 1H), 9.24 (brs, 1H), 8.15 (d, 1H, J ¼ 2.0 Hz), 8.11e8.08
(m, 3H), 7.73e7.65 (m, 5H), 7.57e7.54 (m, 5H), 7.39 (d,1H, J ¼ 5.8 Hz),
A mixture of compound 5a,b (1.3 mmol) and Pd/C (10%) in
anhydrous THF (20 mL) was stirred in hydrogen atmosphere at
room temperature for 2 h. The reaction mixture was filtered
through celite and the filtrate was concentrated under reduced
pressure. The residue was purified by column chromatography to
give the purified desired products.
6.65 (d, 1H, J ¼ 3.2 Hz); 13C NMR (DMSO-d6):
d 164.8, 152.9, 141.0,
139.8,139.0,132.8,132.5,132.3,129.7,129.1,128.8,128.7,127.3,127.0,
125.7, 125.2, 124.7, 123.6, 122.8, 121.9, 120.1, 119.5, 117.3, 104.6; MS
m/z: 552.22 (Mþ þ 2, 38%), 551.18 (Mþ þ 1, 95%), 550.14 (Mþ, 100%).
4.7.4. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-(4-benzamido-1H-
pyrrolo[3,2-c] pyridine-1-yl)phenyl)urea (8e)
4.6.1. N-(1-(4-aminophenyl)-1H-pyrrolo[3,2-c]pyridin-4-yl)
benzamide (6a)
Yield 18%; mp: >280 ꢀC; 1H NMR (DMSO-d6):
d 10.84 (brs, 1H),
9.50 (brs, 1H), 9.29 (brs, 1H), 8.17e8.03 (m, 4H), 7.73e7.67 (m, 4H),
7.62e7.52 (m, 5H), 7.39 (d, 1H, J ¼ 5.3 Hz), 6.86 (s, 1H), 6.65 (brs,
It was purified by column chromatography (silica gel,
hexaneeethyl acetate 1:2 v/v); yield 48%; 1H NMR (DMSO-d6):
1H); 13C NMR (DMSO-d6):
d 168.1, 153.0, 141.1, 139.1, 132.2, 131.9,
d
10.78 (brs, 1H), 8.12e7.93 (m, 3H), 7.63e7.46 (m, 5H), 7.20 (d, 2H,
129.0, 128.6, 127.8, 126.8, 125.3, 124.9, 124.3, 123.1, 122.3, 121.6,
J ¼ 8.6 Hz), 6.75e6.62 (m, 3H), 5.39 (brs, 2H); 13C NMR (DMSO-d6):
120.1,117.4,104.9; MS m/z: 584.52 (Mþ þ 1,100%), 583.56 (Mþ, 31%).
d
164.4, 152.9, 145.0, 141.0, 133.4, 132.8, 132.5, 132.3, 129.0, 127.3,
124.7, 120.7, 117.5, 117.3, 104.7; MS m/z: 330.24 (Mþ þ 2, 23.65%),
4.7.5. 1-(4-(4-Benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)-3-
329.21 (Mþ þ 1, 100%).
methylphenyl)-3-(2,3-dichlorophenyl)urea (8f)
Yield 53%; mp: 181e184 ꢀC; 1H NMR (DMSO-d6):
d 11.75 (brs,
4.6.2. N-(1-(4-amino-2-methylphenyl)-1H-pyrrolo[3,2-c]pyridin-
4-yl)benzamide (6b)
1H), 9.68 (brs, 1H), 8.64 (brs, 1H), 8.12e8.04 (m, 2H), 7.50e7.45 (m,
2H), 7.40e7.23 (m, 7H), 7.04e6.96 (m, 1H), 6.74e6.65 (m, 2H), 6.00
(d, 1H, J ¼ 2.4 Hz), 2.17 (s, 3H); MS m/z: 532.15 (Mþ þ 2, 59.63%),
531.17 (Mþ þ 1, 30.42%), 530.19 (Mþ, 100%).
It was purified by column chromatography (silica gel,
hexaneeethyl acetate 1:1 v/v); yield 32%; 1H NMR (DMSO-d6):
d
11.68 (brs, 1H), 7.94 (d, 2H, J ¼ 6.9 Hz), 7.58e7.47 (m, 7H), 6.89 (d,
1H, J ¼ 8.2 Hz), 6.45e6.39 (m, 2H), 4.94 (brs, 2H), 2.06 (s, 3H); MS
4.8. 1-(4-(4-Benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-3-(4-
((4-ethyl-piperazin-1-yl)methyl)-3-trifluoromethylphenyl)urea (8g)
m/z: 344.50 (Mþ þ 2, 27%), 343.45 (Mþ þ 1, 100%).
4.7. N-(1-(4-[3-(2,3-dichlorophenyl)urea]phenyl)-1H-pyrrolo[3,2-
A mixture of 4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoro-
c]pyridin-4-yl) benzamide (8a)
methyl)aniline (7, 17.5 mg, 0.06 mmol), triethylamine (0.014 mL,
0.1 mmol), and p-nitrophenyl chloroformate (12.4 mg, 0.06 mmol)
in 1,4-dioxane (5 mL) was heated at 60 ꢀC for 2 h. A solution of 6a
(20 mg, 0.06 mmol) in 1,4-dioxane (5 mL) was slowly added
thereto. The reaction mixture was heated at 90 ꢀC overnight. The
reaction mixture was concentrated under reduced pressure and
then partitioned between water (5 mL) and ethyl acetate (5 mL).
The organic layer was separated and the aqueous layer was then
extracted with ethyl acetate (3 ꢁ 3 mL). The combined organic
extracts were washed with brine and dried over anhydrous Na2SO4.
After evaporation of the organic solvent, the residue was purified
by column chromatography (silica gel, hexaneeethyl acetate 3:1
v/v then switching to hexaneeethyl acetate 1:1 v/v) to yield
compound 8g (6.0 mg, 15%). Mp: 265e267 ꢀC; 1H NMR (DMSO-d6):
To a solution of compound 6a (22.0 mg, 0.06 mmol) in anhy-
drous THF (10 mL), 2,3-dichlorophenyl isocyanate (13.7 mg,
0.06 mmol) was added. The reaction mixture was stirred under
nitrogen atmosphere at room temperature for 8 h. The solvent was
evaporated under reduced pressure and the residue was purified by
column chromatography (silica gel, hexaneeethyl acetate 3:1 v/v
then switching to hexaneeethyl acetate 1:1 v/v) to obtain the
purified product 8a (9.0 mg, 26%). Mp: >280 ꢀC; 1H NMR (DMSO-
d6): d 11.71 (brs, 1H), 10.84 (brs, 1H), 9.74 (brs, 1H), 8.50 (s, 1H), 8.20
(dd,1H, J ¼ 2.0 Hz, J ¼ 2.0 Hz), 8.12e8.09 (m, 2H), 7.69e7.63 (m, 3H),
7.58e7.52 (m, 5H), 7.45e7.30 (m, 3H), 6.66 (d, 1H, J ¼ 3.2 Hz); MS
m/z: 518.38 (Mþ þ 2, 60%), 517.42 (Mþ þ 1, 80%), 516.45 (Mþ, 100%).
Synthesis of compounds 8beg was achieved by the same
procedure as described for compound 8a.
d
10.85 (brs, 1H), 9.19e9.14 (m, 2H), 8.11e8.08 (m, 3H), 8.02e7.97
(m, 2H), 7.72e7.66 (m, 3H), 7.63e7.53 (m, 3H), 7.42 (dd, 1H,

3224
M.I. El-Gamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3218e3226
J ¼ 6.5 Hz, J ¼ 5.6 Hz), 2.73 (s, 4H), 2.38e2.27 (m, 6H), 1.23 (s, 4H),
0.98 (q, 2H, J ¼ 7.2 Hz), 0.83 (t, 3H, J ¼ 7.3 Hz); MS m/z: 642.75
(Mþ þ 1, 38%), 641.72 (Mþ, 100%).
4.9.5. N-[4-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)-3-
methylphenyl]-3-trifluoromethyl-benzamide (9f)
Yield 6.7%; mp: 48e51 ꢀC; 1H NMR (DMSO-d6):
d 11.77 (brs, 1H),
10.50 (brs, 1H), 8.25 (dd, 1H, J ¼ 5.0 Hz, J ¼ 8.3 Hz), 8.08 (d, 1H,
J ¼ 5.2 Hz), 7.98 (d, 1H, J ¼ 7.8 Hz) 7.81e7.72 (m, 2H), 7.58e7.49 (m,
3H), 7.40e7.34 (m, 2H), 7.26e7.10 (m, 2H), 7.00 (d, 1H, J ¼ 9.0 Hz),
6.69 (d, 2H, J ¼ 5.2 Hz), 6.01 (brs, 1H), 2.21 (s, 3H); MS m/z: 515.20
(Mþ þ 1, 100%), 514.27 (Mþ, 28.7%).
4.9. N-[4-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)-phenyl]-3-
trifluoromethyl-benzamide (9a)
A
mixture of compound 6a (20.0 mg, 0.06 mmol), 3-tri-
fluoromethyl-benzoic acid (23.11 mg, 0.12 mmol), HOBt (18.1 mg,
0.13 mmol), and EDCI (29.1 mg, 0.15 mmol) in dry DMF (1.0 mL) was
cooled to 0 ꢀC under nitrogen atmosphere. To the reaction mixture,
triethylamine (0.002 mL, 0.015 mmol) was added at 0 ꢀC. The
mixture was then stirred at 80 ꢀC for 12 h. The reaction mixture was
cooled and then partitioned between water (5 mL) and ethyl
acetate (5 mL) and the organic layer was separated. The aqueous
layer was then extracted with ethyl acetate (3 ꢁ 3 mL) and the
combined organic extracts were washed with brine and dried over
anhydrous Na2SO4. After evaporation of the organic solvent, the
residue was purified by column chromatography (silica gel,
hexaneeethyl acetate 3:1 v/v then switching to hexaneeethyl
acetate 1:1 v/v) to yield compound 9a (5.0 mg, 16.4%). Mp:
4.9.6. N-[4-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)-3-
methylphenyl]-4-chloro-3-trifluoromethyl-benzamide (9g)
Yield 28%; mp: 105e106 ꢀC; 1H NMR (DMSO-d6):
d 11.78 (brs,1H),
10.53 (brs, 1H), 8.42e8.22 (m, 2H), 8.08 (d, 1H, J ¼ 5.2 Hz), 7.93 (d, 1H
J ¼ 5.6 Hz), 7.73e7.62 (m, 2H), 7.54e7.49 (m, 3H), 7.42e7.26 (m, 2H),
7.13e7.08(m,2H),6.69(d,1H,J ¼ 5.2 Hz),6.01(brs,1H),2.21(s,3H);MS
m/z: 551.00 (Mþ þ 2, 88%), 550.00 (Mþ þ 1,100%), 549.00 (Mþ, 33%).
4.9.7. N-[4-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)-3-
methylphenyl]-4-morpholino-3-trifluoromethyl-benzamide (9h)
Yield 26%; mp: 237e240 ꢀC; 1H NMR (DMSO-d6):
d 11.77 (brs,
1H), 11.68 (brs, 1H), 8.21 (d, 1H, J ¼ 7.0 Hz), 8.09e8.05 (m, 1H),
7.70e7.64 (m, 1H), 7.58e7.44 (m, 3H), 7.39e7.23 (m, 5H), 6.75e6.63
(m, 2H), 6.27 (d, 1H, J ¼ 2.6 Hz), 6.00 (d, 1H, J ¼ 3.3 Hz), 3.72 (t, 4H,
J ¼ 3.8 Hz), 2.96 (t, 4H, J ¼ 3.2 Hz), 1.99 (s, 3H); MS m/z: 600.63
(Mþ þ 1, 100%), 599.68 (Mþ, 39.5%).
166e169 ꢀC (dec.); 1H NMR (DMSO-d6):
d 10.95e10.85 (m, 2H), 8.51
(s, 1H), 8.43e8.37 (m, 2H), 8.17e7.97 (m, 4H), 7.82e7.53 (m, 5H),
7.33e7.26 (m, 3H), 6.80 (d, 2H, J ¼ 8.5 Hz); MS m/z: 501.50 (Mþ þ 1,
80%), 500.46 (Mþ, 100%).
Synthesis of compounds 9bei was achieved by the same
procedure as described for compound 9a.
4.9.8. N-(4-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-
5-(2-chloro-5-trifluoromethylphenyl)furan-2-carboxamide (9i)
Yield 29%; mp: 103e105 ꢀC (dec.); 1H NMR (DMSO-d6):
d 10.84
4.9.1. N-[4-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)-phenyl]-
(brs, 1H), 10.61 (brs, 1H), 8.27e8.14 (m, 4H), 8.02 (d, 2H, J ¼ 8.9 Hz),
7.97e7.87 (m, 2H), 7.72e7.46 (m, 8H), 7.45 (d, 1H, J ¼ 5.9 Hz),
6.75e6.67 (m, 1H); MS m/z: 603.07 (Mþ þ 2, 90%), 602.05 (Mþ þ 1,
100%), 601.03 (Mþ, 28%).
4-chloro-3-trifluoromethyl-benzamide (9b)
Yield 21%; mp: 133e136 ꢀC; 1H NMR (DMSO-d6):
d 10.86 (brs,
1H), 10.51 (brs, 1H), 8.43e8.30 (m, 2H), 8.13e7.96 (m, 7H),
7.68e7.45 (m, 6H), 6.74e6.67 (m, 1H); MS m/z: 537.02 (Mþ þ 2,
44%), 536.00 (Mþ þ 1, 90%), 535.00 (Mþ, 100%).
4.10. 1-(4-Aminophenyl)-1H-pyrrolo[3,2-c]pyridin-4-amine (10)
4.9.2. N-[4-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)-phenyl]-
A mixture of 6a (0.47 g, 1.31 mmol) and SnCl2$H2O (1.48 g,
6.55 mmol) in ethanol (20 mL) was heated under reflux for 2 h. The
solvent was evaporated under reduced pressure and the residue
was partitioned between aqueous NaHCO3 and ethyl acetate. The
organic layer was separated and the aqueous layer was extracted
with ethyl acetate. The combined organic extracts were washed
with brine and dried over anhydrous Na2SO4. After evaporation of
the organic solvent, the residue was purified by column chroma-
tography (silica gel, ethyl acetate) to yield compound 8 (0.12 g,
40.8%). MS m/z: 225.27 (Mþ þ 1, 100%), 224.25 (Mþ, 90%).
3-(4-methyl-1H-imidazol-1-yl)-5-trifluoromethyl-benzamide (9c)
Yield 11%; mp: 206e207 ꢀC; 1H NMR (DMSO-d6):
d 8.35 (brs,1H),
8.31 (brs, 1H), 8.30e8.28 (m, 2H), 8.23 (d, 1H, J ¼ 1.4 Hz), 8.16e8.02
(m, 5H), 7.93 (brs, 1H), 7.74e7.71 (m, 2H), 7.65e7.54 (m, 5H), 7.47 (d,
1H, J ¼ 5.5 Hz), 6.89 (d, 1H, J ¼ 3.2 Hz), 2.02 (s, 3H); MS m/z: 582.47
(Mþ þ 2, 44.7%), 581.45 (Mþ þ 1, 100%), 580.43 (Mþ, 91%).
4.9.3. N-[4-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)-phenyl]-
4-morpholino-3-trifluoromethyl-benzamide (9d)
Yield 11%; mp: 220e223 ꢀC (dec.); 1H NMR (DMSO-d6):
d 10.92
(brs,1H),10.62(brs,1H), 8.43e8.28(m, 2H), 8.11e8.05(m, 2H), 8.01(d,
2H, J ¼ 8.8 Hz), 7.71 (d, 2H, J ¼ 2.6 Hz), 7.68e7.52 (m, 4H), 7.50e7.44
(m, 2H), 7.29 (brs, 1H), 6.74e6.69 (m,1H), 3.76 (t, 4H, J ¼ 3.1 Hz), 2.96
4.11. Synthesis of compounds 8h,i
Synthesis of compounds 8h,i was achieved by the same proce-
dure as described for compound 8a.
(t, 4H, J ¼ 3.3 Hz); 13C NMR (DMSO-d6):
d 164.5, 155.0, 141.0, 140.2,
134.3, 133.8, 133.5, 132.4, 130.9, 129.0, 128.8, 128.6, 127.5, 125.8, 124.8,
124.3,123.1, 122.0, 119.8,114.7, 104.8, 66.9, 53.6; MS (ESI) m/z: 587.63
(Mþ þ 2, 38.7%), 586.60 (Mþ þ 1, 100%), 585.56 (Mþ, 87%).
4.11.1. 1-(4-(4-Amino-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-3-(3-
trifluoromethyl-phenyl)urea (8h)
It was purified by column chromatography (silica gel,
hexaneeethyl acetate 2:1 v/v then switching to ethyl acetate); yield
4.9.4. N-[4-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)-phenyl]-
3-morpholino-5-trifluoromethyl-benzamide (9e)
8%; mp > 280 ꢀC; 1H NMR (DMSO-d6):
d
8.56 (d, 1H, J ¼ 6.6 Hz),
Yield 8%; mp: 233e234 ꢀC; 1H NMR (DMSO-d6):
d
10.86 (brs,
7.93e7.72 (m, 3H), 7.54e7.44 (m, 3H), 7.43e7.30 (m, 5H), 7.15 (d, 1H,
1H),10.59 (brs,1H), 8.11e8.06 (m, 2H), 8.05e7.99 (m, 2H), 7.72e7.61
(m, 6H), 7.57e7.54 (m, 2H), 7.47e7.42 (m, 2H), 7.21 (d, 1H,
J ¼ 8.3 Hz), 6.73 (d,1H, J ¼ 8.8 Hz), 3.78 (t, 4H, J ¼ 4.3 Hz), 2.74 (t, 4H,
J ¼ 6.4 Hz); MS m/z: 412.40 (Mþ þ 1, 100%), 411.43 (Mþ, 30.5%).
4.11.2. 1-(4-(4-Amino-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-3-(4-
chloro-3-trifluoro-methylphenyl)urea (8i)
J ¼ 4.1 Hz); 13C NMR (DMSO-d6):
d 164.5, 155.0, 141.1, 140.2, 134.3,
133.8, 133.5, 132.3, 130.9, 129.0, 128.8, 128.6, 127.5, 125.8, 124.8,
124.3, 123.0, 122.0, 119.7, 114.7, 104.7, 66.9, 53.6; MS m/z: 586.60
(Mþ þ 1, 100%), 585.61 (Mþ, 47%).
It was purified by column chromatography (silica gel,
hexaneeethyl acetate 2:1 v/v then switching to hexaneeethyl
acetate 1:3 v/v); yield 27%; mp > 280 ꢀC; 1H NMR (CD3OD):
d 8.06

M.I. El-Gamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3218e3226
3225
(d, 1H, J ¼ 2.4 Hz), 7.95 (d, 1H, J ¼ 2.2 Hz), 7.72e7.64 (m, 4H),
7.59e7.51 (m, 3H), 7.44 (d, 2H, J ¼ 3.6 Hz), 7.40e7.32 (m, 2H), 6.98
(d,1H, J ¼ 3.1 Hz), 6.87 (brd,1H, J ¼ 6.8 Hz); MS m/z: 447.90 (Mþ þ 2,
60%), 446.86 (Mþ þ 1, 43.7%), 445.82 (Mþ, 100%).
incubated at 37 ꢀC, 5% CO2, 95% air and 100% relative humidity for
24 h prior to addition of experimental drugs. After 24 h, two plates of
each cell line are fixed in situ with TCA, to represent a measurement
of the cell population for each cell line at the time of drug addition
(Tz). Experimental drugs are solubilized in dimethyl sulfoxide at
400-fold the desired final maximum test concentration and stored
frozen prior to use. At the time of drug addition, an aliquot of frozen
concentrate is thawed and diluted to twice the desired final
maximum test concentration with complete medium containing
4.12. Synthesis of compounds 9j,k
Synthesis of compounds 9j,k was achieved by the same proce-
dure as described for compound 9a.
50
tions are made to provide a total of five drug concentrations plus
control. Aliquots of 100 L of these different drug dilutions are added
to the appropriate microtiter wells already containing 100 L of
medium, resulting in the required final drug concentrations.
Following drug addition, the plates are incubated for an additional
48 h at 37 ꢀC, 5% CO2, 95% air, and 100% relative humidity. For
adherent cells, the assay is terminated by the addition of cold TCA.
mg/mL gentamicin. Additional four, 10-fold or 1/2 log serial dilu-
4.12.1. N-(4-(4-Amino-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-4-
chloro-3-trifluoromethylbenzamide (9j)
It was purified by column chromatography (silica gel,
hexaneeethyl acetate 3:1 v/v then switching to hexaneeethyl
acetate 1:1 v/v); Yield 12%; mp > 280 ꢀC; 1H NMR (DMSO-d6):
m
m
d
(ppm) 8.07 (d, 1H, J ¼ 2.3 Hz), 7.94 (d, 1H, J ¼ 2.2 Hz), 7.70e7.63
(m, 4H), 7.59e7.50 (m, 3H), 7.44 (d, 2H, J ¼ 3.7 Hz), 7.40e7.33 (m,
2H), 7.00 (d, 1H, J ¼ 3.0 Hz), 6.88 (brd, 1H, J ¼ 6.9 Hz); MS m/z:
432.87 (Mþ þ 2, 40%), 431.89 (Mþ þ 1, 100%), 430.91 (Mþ, 58%).
Cells are fixed in situ by the gentle addition of 50 mL of cold 50% (w/v)
TCA (final concentration, 10% TCA) and incubated for 60 min at 4 ꢀC.
The supernatant is discarded, and the plates are washed five times
with tap water and air dried. Sulforhodamine B (SRB) solution
4.12.2. N-(4-(4-Amino-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-3-
trifluoromethyl-4-morpholinobenzamide (9k)
(100 mL) at 0.4% (w/v) in 1% acetic acid is added to each well, and
It was purified by column chromatography (silica gel,
hexaneeethyl acetate 3:1 v/v then switching to ethyl acetate); Yield
plates are kept for 10 min at room temperature. After staining,
unbound dye is removed by washing five times with 1% acetic acid
and the plates are air dried. Bound stain is subsequently solubilized
with 10 mM trizma base, and the absorbance is read on an auto-
mated plate reader at a wavelength of 515 nm. For suspension cells,
the methodology is the same except that the assay is terminated by
37%; mp: 253e256 ꢀC (dec.); 1H NMR (DMSO-d6):
d (ppm) 8.38(d,1H,
J ¼ 2.6 Hz), 8.29e8.26 (m, 2H), 8.02e7.99 (m, 2H), 7.75e7.51 (m, 5H),
7.20 (brd, 1H, J ¼ 8.0 Hz), 6.74 (brd,1H, J ¼ 8.2 Hz), 3.74e3.65 (m, 4H),
3.01e2.97(m,4H);MSm/z:482.50(Mþ þ 1,100%),481.46(Mþ,46.3%).
fixing settled cells at the bottom of the wells by gently adding 50 mL
4.13. Evaluation of the antiproliferative activity against A375P
human melanoma cell line and NIH3T3 fibroblasts
of 80% TCA (final concentration, 16% TCA). Using the seven absor-
bance measurements [time zero, (Tz), control growth, (C), and test
growth in the presence of drug at the five concentration levels (Ti)],
the percentage growth is calculated at each of the drug concentra-
tions levels. Percentage growth inhibition is calculated as:
A375P cells were purchased from AmericanType Culture Collection
(ATCC, Rockville, MD, USA) and maintained in Dulbecco’s modified
eagle medium (DMEM, Welgene, Daegu, Republic of Korea) supple-
mented with 10% fetal bovine serum (FBS, Welgene, Daegu, Republic of
Korea) and 1% penicillin/streptomycin (Welgene, Daegu, Republic of
Korea) in a humidified atmosphere with 5% CO2 at 37 ꢀC. A375P cells
were taken from culture substrate with 0.05% trypsine0.02% EDTA and
plated at a density of 5 ꢁ103 cells/well in 96 well plates and then
incubated at 37 ꢀC for 24 h in a humidified atmosphere with 5% CO2
prior to treatment with various concentrations (3-fold serial dilution,
12 points) of the tested compounds. The cells were incubated for 48 h
after treatment with the test compounds. The A357P cell viability was
assessed by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diph-
enyltetrazolium bromide (MTT) reduction assay. MTT assays were
carried out with CellTiter 96Ò (Promega) according to the manufac-
turer’s instructions. The absorbance at 590 nm was recorded using
EnVision 2103 (Perkin Elmer; Boston, MA, USA). The IC50 values were
calculated using GraphPad Prism 4.0 software. Triplicate testing was
performed for each test compound.
ꢃ [(Ti ꢂ Tz)/(C ꢂ Tz)] ꢁ 100 for concentrations for which Ti ꢄ Tz.
ꢃ [(Ti ꢂ Tz)/Tz] ꢁ 100 for concentrations for which Ti < Tz.
Growth inhibition of 50% (IC50) is calculated from [(Ti ꢂ Tz)/
(C ꢂ Tz)] ꢁ 100 ¼ 50, which is the drug concentration resulting in
a 50% reduction in the net protein increase (as measured by SRB
staining) in control cells during the drug incubation.
Acknowledgements
We would like to thank the National Cancer Institute (NCI),
Bethesda, Maryland, USA, for performing the anticancer testing
over nine melanoma cell lines. We are also grateful to Korea
Institute of Science and Technology (KIST) for financial support.
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