Identification of protein kinase fibroblast growth factor receptor 1 (FGFR1) inhibitors among the derivatives of 5-(5,6-dimethoxybenzimidazol-1-yl)-3-hydroxythiophene-2-carboxylic acid

Article abstract of DOI:10.1007/s00706-019-02493-5

Abstract: Fibroblast growth factor receptor 1 (FGFR1) plays an important role in tumorigenesis, suggesting that inhibitors of this protein kinase may become important compounds for the development of anticancer agents. Using molecular docking approach, we have identified a novel class of FGFR1 inhibitors belonging to the derivatives of 5-(5,6-dimethoxybenzimidazol-1-yl)-3-hydroxythiophene-2-carboxylic acid. It was revealed that the most promising compound 5-(5,6-dimethoxybenzimidazol-1-yl)-3-[2-(methanesulfonyl)benzyloxy]thiophene-2-carboxylic acid methyl ester inhibits FGFR1 with an IC₅₀ value of 150?nM in in vitro kinase assay. The structure–activity relationships have been studied, and the binding mode of this chemical class has been proposed. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-019-02493-5

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-019-02493-5
ORIGINAL PAPER
Identifcation of protein kinase fbroblast growth factor receptor 1
(FGFR1) inhibitors among the derivatives of 5‑(5,6‑dimethoxybenzim
idazol‑1‑yl)‑3‑hydroxythiophene‑2‑carboxylic acid
Galyna Volynets1 · Sergiy Lukashov1 · Iryna Borysenko1 · Andrii Gryshchenko1 · Sergiy Starosyla1 ·
Volodymyr Bdzhola1 · Tetiana Ruban1 · Anna Iatsyshyna1 · Lyubov Lukash1 · Yaroslav Bilokin2 · Sergiy Yarmoluk1
Received: 7 June 2019 / Accepted: 5 August 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
Fibroblast growth factor receptor 1 (FGFR1) plays an important role in tumorigenesis, suggesting that inhibitors of this pro-
tein kinase may become important compounds for the development of anticancer agents. Using molecular docking approach,
we have identifed a novel class of FGFR1 inhibitors belonging to the derivatives of 5-(5,6-dimethoxybenzimidazol-1-yl)-
3-hydroxythiophene-2-carboxylic acid. It was revealed that the most promising compound 5-(5,6-dimethoxybenzimidazol-
1-yl)-3-[2-(methanesulfonyl)benzyloxy]thiophene-2-carboxylic acid methyl ester inhibits FGFR1 with an IC50 value of
150 nM in in vitro kinase assay. The structure–activity relationships have been studied, and the binding mode of this chemical
class has been proposed.
Graphic abstract
Keywords Drug research · Virtual screening · Kinase assay · Phosphorylations · Structure–activity relationships
Introduction
experimental results clearly show that this protein kinase is
involved in tumorigenesis. In particular, FGFR1 amplifca-
tions have been identifed in lung cancer [2, 3], oral tongue
squamous cell carcinoma [4], sinonasal cancer [5], gastric
cancer [6], pancreatic ductal adenocarcinoma [7], colorectal
cancer [8], etc. FGFR1 rearranged neoplasms lead to acute
myeloid leukemia, T or B acute lymphoblastic leukemia,
and mixed phenotypic acute leukemia [9, 10]. Therefore,
FGFR1 is an important therapeutic target for the treatment
of cancer diseases.
Fibroblast growth factor receptor 1 (FGFR1) is a transmem-
brane receptor tyrosine kinase which plays an important
role in the regulation of cell survival, proliferation, dif-
ferentiation, migration, and angiogenesis [1]. A number of
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-019-02493-5) contains
supplementary material, which is available to authorized users.
* Galyna Volynets
To date, the small-molecular inhibitors of protein
kinase FGFR1 have been reported among the deriva-
tives of 2-benzamido-4-(6-oxy-N-methyl-1-naphtha-
mide)pyridine [11], 4-bromo-N-(3,5-dimethoxyphenyl)-
benzamide [12], pyrrolo[2,3-b]pyrazine [13, 14],
g.p.volynets@imbg.org.ua; galina.volinetc@gmail.com
1
Institute of Molecular Biology and Genetics, the NAS
of Ukraine, 150 Zabolotnogo St., Kiev 03143, Ukraine
2
OTAVA Ltd., 400 Applewood Crescent, Unit 100, Vaughan,
ON L4K 0C3, Canada
Vol.:(0123456789)
1 3

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
G. Volynets et al.
indazole [15–18], 3-(5′-substituted)-benzimidazolyl-
5-[1-(3,5-dichloropyridin-4-yl)ethoxy]-1H-indazole [19],
[5-amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-
4-yl](1H-indol-2-yl)methanone [20], thienopyrimidine
[21–24], pyridine-3-amine [25], 1H-pyrazolo[3,4-b]pyri-
dine [26], 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl
[27], N-phenyl-N′-[4-(5H-pyrrolo[3,2-d]pyrimidin-
4-yloxy)phenyl]urea [28], 5,7-dimethyloxazolo[5,4-d]-
pyrimidine-4,6(5H,7H)-dione [29], 6-aryl-pyrido[2,3-d]-
pyrimidine [30], 3-formylchromone [31], 3-vinylquinox-
alin-2(1H)-one [32], pyrazolylaminoquinazoline [33],
trichlorobenzene-substituted azaaryl [34], C-8 substituted
guanine derivatives [35], oxindole and benzotiophene
[36, 37], thiazol-2-amine [38], bisaryl-1,4-dien-3-one
[39], naphthostyril [40], acenaphtho[1,2-b]pyrrole [41],
3-(3,5-dimethoxyphenyl)-1,6-naphthyridin [42], 4-[2,4-dif-
luoro-5-(methoxycarbamoyl)phenylamino]pyrrolo[2,1-f]-
[1,2,4] triazine [43], 5-amino-4-(1H-benzimidazol-2-yl)-
phenyl-1,2-dihydropyrrol-3-ones [44].
Results and discussion
To discover the novel class of small molecule inhibitors for
protein kinase FGFR1, we used a combination of computer
modeling approaches and in vitro kinase assay. The virtual
screening experiments were performed targeting the ATP-
binding site of FGFR1 kinase by browsing commercially
available OTAVA drug-like compound library containing
about 100,000 ligands. According to docking results and
visual inspection of the complexes of the best-scored ligands
with amino acid residues of FGFR1 active site, 273 the most
promising compounds were selected for biochemical screen-
ing. Among them, we have identifed seven small-molecular
inhibitors of FGFR1 carried 5-(5,6-dimethoxybenzimidazol-
1-yl)-3-hydroxythiophene-2-carboxylic acid moiety close to
IKKε inhibitor GSK319347A [48] and PLK1 [49] inhibitor.
Using molecular docking, it turned out that all com-
pounds in this class display similar binding mode with the
ATP-binding site of FGFR1 (Fig. 1). In accordance with the
results of molecular modeling, benzimidazole is involved
in the hydrophobic interactions with amino acid residues
in the adenine-binding site. The nitrogen atom of this het-
erocycle forms a hydrogen bond with Ala564 located in
the hinge region. In addition, carboxylic acid methyl ester
forms a hydrogen bond with conservative Lys514, which is
an important feature for the most of highly active FGFR1
inhibitors.
Nowadays, several FGFR1 inhibitors are undergoing
clinical studies [45, 46], but resistance to some inhibitors
has already emerged. One of the mechanisms of acquired
drug resistance in FGFR1 occurs through mutation of the
hinge residue N546 and the gatekeeper residue V561 in the
ATP-binding site of the kinase domain. N546K mutation
can hinder drug binding by increasing afnity for ATP in
comparison with the wild type. In addition, it was found
that V561M mutation causes a signifcant reduction in afn-
ity for highly potent FGFR1–3 inhibitor—PD173074 [47].
This fnding provides the stimulus to search novel chemical
classes of FGFR1 inhibitors.
Several important structural features of 5-(5,6-dimeth-
oxybenzimidazol-1-yl)-3-hydroxythiophene-2-carboxylic
Fig. 1 Complex of compound
methyl 5-(5,6-dimethoxy-
1H-1,3-benzodiazol-1-yl)-
3-[[2-(methanesulfonyl)-
phenyl]methoxy]thiophene-2-
carboxylate (7b) with amino
acid residues in the active site
of the FGFR1 catalytic subunit,
obtained with molecular dock-
ing. Hydrogen bonds are shown
by the green dotted lines and
hydrophobic interactions are
presented by the magenta dotted
lines (color fgure online)
1 3

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Identifcation of protein kinase fbroblast growth factor receptor 1 (FGFR1) inhibitors among…
Table 1 Chemical structure of 5-(5,6-dimethoxybenzimidazol-1-yl)-
acid derivatives were identifed from a qualitative analysis
of their activity on FGFR1 (Table 1).
3-hydroxythiophene-2-carboxylic acid derivatives and in vitro inhibi-
tory activity towards protein kinase FGFR1
The FGFR1 inhibitory activity of the studied deriva-
tives depends on the structure of R1 substituents in this
heterocyclic system. We observed that compound 7b
with R1 = methoxy group is more active (IC50 = 0.15 μM)
than compound 5-(5,6-dimethoxy-1H-1,3-benzodiazol-
1-yl)-3-[[2-(methanesulfonyl)phenyl]methoxy]thiophene-
2-carboxamide (8b) with amino group (IC50 =0.50 μM) or
compound 5-(5,6-dimethoxy-1H-1,3-benzodiazol-1-yl)-
3-[[2-(methanesulfonyl)phenyl]methoxy]thiophene-2-car-
boxylic acid (8a) with hydroxy group (IC50 = 0.50 μM)
in this position, while the replacement of methoxy group
(compound methyl 5-(5,6-dimethoxy-1H-1,3-benzodi-
azol-1-yl)-3-[[4-(methanesulfonyl)phenyl]methoxy]-
thiophene-2-carboxylate (7c)) to dimethyl amine
(compound 5-(5,6-dimethoxy-1H-1,3-benzodiazol-1-yl)-
3-[[4-(methanesulfonyl)phenyl]methoxy]-N,N-dimethylthio-
phene-2-carboxamide (8c)) positively impacts the inhibitory
activity (IC50 values are 5 and 3.7 μM, respectively). From
the obtained results, the order of potency for the substituent
R1 could be proposed as follows: dimethyl amine>methoxy
group>amino group>hydroxy group.
R2
O
O
R1
S
O
O
N
N
IC50 /μM
Compound
R1
R2
7b
OMe
0.15
O
S
S
S
O
8b
8a
NH2
OH
0.5
O
O
O
0.66
The nature of R2 substituent also signifcantly afects
the inhibitory activity towards FGFR1. We observed that
compounds with R2 =2-(methanesulfonyl)benzyl are more
active than those with 4-(methanesulfonyl)benzyl in this
position. To compare this efect, one can refer to compound
pairs such as 7b, 7c; 8a, 5-(5,6-dimethoxy-1H-1,3-ben-
zodiazol-1-yl)-3-[[4-(methanesulfonyl)phenyl]methoxy]-
thiophene-2-carboxylic acid (8d) (corresponding IC50 values
are 0.15 μM, 5 μM; 0.66 μM, > 30 μM). The compounds
with hydrogen (methyl 5-(5,6-dimethoxy-1H-1,3-benzo-
diazol-1-yl)-3-hydroxythiophene-2-carboxylate (6)) and
3-hydroxythiophene-2-carboxylic acid methyl ester (methyl-
5-(5,6-dimethoxy-1H-1,3-benzodiazol-1-yl)-3-[[4-hydroxy-
5-(methoxycarbonyl)thiophen-2-yl]oxy]thiophene-2-car-
boxylate (7a)) have IC50 values equal to 0.56 and 23 μM,
respectively. Therefore, based on the obtained results, the
order of potency for the substituent R2 could be proposed as
follows: 2-(methanesulfonyl)benzyl>hydrogen>4-(meth-
anesulfonyl)benzyl>3-hydroxythiophene-2-carboxylic acid
methyl ester.
O
S
O
O
O
7c
8c
8d
OMe
5
O
O
O
N(Me)2
OH
3.7
S
S
> 30
6
OMe
OMe
Н
0.56
23
7a
Therefore, substituents R1 and R2 in thiophene ring of
5-(5,6-dimethoxybenzimidazol-1-yl)-3-hydroxythiophene-
2-carboxylic acid derivatives play a significant role for
inhibitory activity towards FGFR1.
OH
S
O
O
We have tested two the most active compounds 7b and 8b
for antiproliferative activity against human T-cell lympho-
blast-like cell line Jurkat. This cell line is characterized by
high expression of FGFR1, FGFR4, and FGFR3 (FGFR1
expression is highest) [50]. The EC50 values are presented
in Table 2. According to the results of in cellulo testing, the
antiproliferative activity of compound 8b was higher than in
7b despite lower enzymatic potency, suggesting that com-
pound 8b has better membrane permeability. In addition,
we have tested compounds 7b and 8b for cytotoxicity in
1 3

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
G. Volynets et al.
Table 2 Antiproliferative activities of compounds 7b and 8b against
General synthetic procedure for 5‑(5,6‑dimethoxy‑
benzimidazol‑1‑yl)‑3‑hydroxythiophene‑2‑carboxylic
acid derivatives
Jurkat and HEK293 cell lines
Compound
Jurkat cell proliferation
HEK293 cell
proliferation
EC50/ µMa
EC50/ µMa
The addition of methyl thioglycolate (1) to methyl pro-
piolate (2) in the presence of sodium methylate was fol-
lowed with simultaneous cyclization into thiophene ring
[51]. Mild chlorination of 3 with sulfuryl chloride gave
chlorinated compound 4 [52]. Slow addition of 4 to ben-
zimidazole 5 in homogeneous DMF solution induced an
exothermic reaction that gave a complex mixture of prod-
ucts [49]. Therefore, 6 was isolated after chromatographic
separation only with the yield of less than 30%. Among
by-products was isolated compound 7a bearing two thio-
phene moieties. Alkylation of 6 with corresponding ben-
zylhalogenides in DMF using K2CO3 as a base gave almost
quantitative yields of 7b and 7c that were converted into
acids, and amides 8a–8d under conditions, as indicated in
Scheme 1. All synthesized compounds have ≥ 95% purity.
7b
8b
215
125
>250
>250
aValues are the mean of three independent experiments
“normal” non-tumorous cell line—HEK293. In this case, the
compounds did not have efect on cell viability.
Conclusion
Therefore, the derivatives of 5,6-dimethoxy-1-thiophen-
2-yl-1H-benzimidazole represent a novel class of FGFR1
inhibitors. The most active compound 5-(5,6-dimethoxy-
benzimidazol-1-yl)-3-[2-(methanesulfonyl)benzyloxy]thio-
phene-2-carboxylic acid methyl ester (7b) inhibits FGFR1
with an IC50 value of 150 nM. According to the molecular
docking results, the inhibitors from this class bind FGFR1
in such a way to form hydrogen bonds simultaneously with
hinge region and conservative Lys514. The identifed com-
pounds can be used for further optimization of FGFR1 inhib-
itory activity and membrane permeability.
Methyl 5‑(5,6‑dimethoxy‑1H‑1,3‑benzodiazol‑1‑yl)‑3‑hy‑
droxythiophene‑2‑carboxylate (6, C15H14N2O5S) 18 g of
5,6-dimethoxybenzimidazole (5, 0.10 mol) and 17 cm3 of tri-
ethylamine (0.12 mol) were dissolved in 150 cm3 of dimeth-
ylformamide. Under stirring and cooling with cold water
bath, 19.5 g of 4 (0.10 mol) were added portionwise. Stirring
continued overnight, and then, 300 cm3 of water were added
and crude 6 was extracted with 3× 100 cm3 of methylene
chloride. Combined organic layers were evaporated with
50 g of silica gel. Solid residue transferred into the top of
70 cm-long chromatographic column over 50 cm of SiO2 and
eluted with CH2Cl2/MeOH 95:5. After fractions contained
rests of 3 and traces of 7a fractions with product 6 were
collected and evaporated to dryness giving 9.15 g (27%)
Experimental
Starting materials and solvents were purchased from com-
mercial suppliers and were used without further purifca-
tion. H NMR and 13C NMR spectra were recorded on a
1
1
Varian VXR 400 instrument at 400 MHz. Chemical shifts
are described as parts per million (δ) downfeld from an
internal standard of tetramethylsilane, and spin multiplici-
ties are given as s (singlet), d (doublet), dd (double doublet),
t (triplet), q (quartet), or m (multiplet). HPLC–MS analysis
was performed using the Agilent 1100 LC/MSD SL separa-
tions module and Mass Quad G1956B mass detector with
electrospray ionization (positive or negative ion mode as
indicated) and with HPLC performed using Zorbax SB-C18,
Rapid Resolution HT Cartridge 4.6 30 mm 1.8-Micron (Agi-
lent P/N:823975-902) i.d. column, at a temperature of 40 °C
with gradient elution of 0–100% CH3CN (with 1 cm3/dm3
HCO2H): H2O (with 1 cm3/dm3 HCO2H) at a fow rate of
3 cm3/min and a run time of 2.8 min. Compounds were
detected at 215 nm using a Diode Array G1315B detector.
All purifed synthetic intermediates and tested compounds
gave 95% purity as determined by this method.
of white powder. M.p.: 157–158 °C; H NMR (400 MHz,
DMSO-d6): δ=10.40 (s, 1H), 8.28 (s, 1H), 7.24 (s, 1H), 7.21
(s, 1H), 7.08 (s, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H)
ppm; 13C NMR (75 MHz, DMSO-d6): δ=162.30, 159.76,
147.84, 147.06, 141.28, 140.74, 136.93, 134.02, 125.92,
112.52, 102.55, 93.97, 56.00, 55.92, 51.62 ppm; LC–MS
(ESI): tr =0.845, m/z=335.0 ([M+H]+).
Methyl 5‑(5,6‑dimethoxy‑1H‑1,3‑benzodiazol‑1‑yl)‑3‑
[[4‑hydroxy‑5‑(methoxycarbonyl)thiophen‑2‑yl]oxy]thio‑
phene‑2‑carboxylate (7a, C21H18N2O8S2) was isolated as
a byproduct of compound 6 synthesis as golden powder.
Yield 1.35 g (4%); m.p.: 204–205 °C; 1H NMR (400 MHz,
DMSO-d6): δ=10.75 (br.s, 1H), 9.98 (br.s, 1H), 8.00 (s, 1H),
7.27 (s, 1H,), 6.69 (s, 1H), 6.59 (s, 1H), 3.91 (s, 3H), 3.85
(s, 3H), 3.72 (s, 6H) ppm; 13C NMR (75 MHz, DMSO-d6):
δ=162.63, 162.18, 159.62, 156.82, 147.85, 146.90, 141.88,
136.25, 135.57, 134.48, 127.50, 122.25, 119.87, 105.22,
1 3