Design, synthesis and biological evaluation of naphthostyril derivatives as novel protein kinase FGFR1 inhibitors

Article abstract of DOI:10.3109/14756366.2014.895718

New class of FGFR1 kinase inhibitors with naphthostyril heterocycle has been identified. A series of N-phenylnaphthostyril-1-sulfonamides has been synthesized and tested in vitro. It was revealed that the most active compound N-(4-hydroxyphenyl)naphthostyril-1- sulfonamide inhibited FGFR1 with IC₅₀ of 2 μM. In our preliminary studies, N-phenylnaphthos-tyril- 1-sulfonamides demonstrated selectivity of FGFR1 inhibition and antiproliferative activity on cancer cell line. N-phenylnaphthostyril-1-sulfonamides have a good potential for further development as anticancer agents.

Full text of DOI:10.3109/14756366.2014.895718

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J Enzyme Inhib Med Chem, Early Online: 1–7
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.895718
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ORIGINAL ARTICLE
Design, synthesis and biological evaluation of naphthostyril derivatives
as novel protein kinase FGFR1 inhibitors
Andrii Anatoliyovych Gryshchenko1, Kostiantyn Vasyliovych Levchenko1, Volodymyr Grygorovich Bdzhola1,
Tatiana Panasivna Ruban2, Lyubov Leonidovna Lukash2, and Sergiy Mikolayovych Yarmoluk1
2
1Medicinal Chemistry Department and Human Genetics Department, Institute of Molecular Biology and Genetics, Kiev, Ukraine
Abstract
Keywords
New class of FGFR1 kinase inhibitors with naphthostyril heterocycle has been identified.
A series of N-phenylnaphthostyril-1-sulfonamides has been synthesized and tested in vitro.
It was revealed that the most active compound N-(4-hydroxyphenyl)naphthostyril-1-
sulfonamide inhibited FGFR1 with IC50 of 2 mM. In our preliminary studies, N-phenylnaphthos-
tyril-1-sulfonamides demonstrated selectivity of FGFR1 inhibition and antiproliferative activity
on cancer cell line. N-phenylnaphthostyril-1-sulfonamides have a good potential for further
development as anticancer agents.
Drug design, FGFR1 inhibitor, naphthostyril,
screening
History
Received 14 October 2013
Revised 12 February 2014
Accepted 13 February 2014
Published online 17 June 2014
Introduction
binding site. Several FGFR1 inhibitors reveal high antitumor
potency in tests in vivo and now in clinical trials8.
The fibroblast growth factor receptors (FGFRs) are a family
of four similar in structure and function transmembrane proteins
that belong to receptor tyrosine kinases (RTKs). The FGFR
protein consists of extracellular immunoglobulin-like receptor
domain, transmembrane helix domain and intracellular catalytic
kinase domain. The natural ligands of FGFR are fibroblast
growth factors (FGFs). The binding of this small protein to the
extracellular domain induces receptor dimerization, autopho-
sphorylation of tyrosine residues in the intracellular kinase
domain and recruitment of signaling proteins. This process
activates downstream pathways Ras/MAPK, PI3K/Akt and PLCg
leading to proliferation stimulation and cell survival1.
FGFR signaling plays important role in embryonic develop-
ment of mesenchymal tissue, formation of nervous system, lung,
breast and other organs. In the adult organism, FGFR1 is involved
in reparative processes, inflammation, angiogenesis and differen-
tiation of muscle and fat cells. FGFR gene amplification,
activated mutation or aberrant overexpression described in a
variety of cancers such as squamous cell lung cancer2, glioblast-
oma3, breast cancer4, prostate cancer5, etc. Other FGFRs family
members also take part in oncogenic disorders such as bladder
cancer, myeloma, sarcoma, stomach cancer and melanoma6.
FGFR1 also plays an indirect role in the development of solid
tumors, participating in the process of oncogenic angiogenesis7.
Thus, inhibition of FGFR1 activity represents an attractive
therapeutic strategy for tumors with abnormal activity of this
kinase. Most of the known FGFR1 inhibitors target the ATP
This article is devoted to development of naphthostyril FGFR1
inhibitors. Naphthostyril core is a close structural analog of
oxindole heterocycle and differs by fused benzene ring. Oxindoles
are known class of RTK inhibitors, including FGFR19. The
oxindole multitargeted RTK inhibitor Sunitinib (Figure 1) is used
for treatment of several solid tumors10. We have compared
pharmacophore features of oxindole RTK inhibitors with
naphtostyriles. Similar to oxindole naphthostyril heterocycle
contains 2-pyrrolidone ring. This moiety is responsible for
formation of hydrogen bonds with kinase hinge region in
oxindole-based inhibitors. Flat aromatic nature of naphthostyril
core potentially permits formation of hydrophobic contacts with
amino acids surrounding adenine binding subsite of FGFR1 as
in case of oxindole inhibitors. Based on these assumptions,
we suggested that naphthostyrils potentially have high affinity to
FGFR1 ATP-binding site similarly to oxindole FGFR1 inhibitors.
Successful oxindole substitution for naphthostyril was reported
for CDK2 inhibitors (Figure 1)11. X-ray complex of naphthostyril
inhibitor with CDK2 described in Ref. [11] reveal hydrogen bonds
between carbonyl and amide groups of naphthostyril with kinase
hinge region, similarly to hydrogen bonds formed by oxindole
kinase inhibitors.
Thus, naphthostyril is a promising scaffold for development
of inhibitors for receptor tyrosine protein kinases, including
FGFR1. The aim of this article is identification and optimiza-
tion of naphthostyril FGFR1 inhibitors as potential anticancer
agents.
Address for correspondence: Sergiy Mikolayovych Yarmoluk, Medicinal
Chemistry Department, Institute of Molecular Biology and Genetics,
Zabolotnogo str. 150, 03680 Kiev, Ukraine. E-mail: sergiy@yarmoluk.
org.ua
Experimental
All appropriate anilines and 1,8-naphtalic anhydride and other
chemicals were commercially available (Acros, Fairlawn, NJ). 1H

2
A. A. Gryshchenko et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Figure 1. Chemical structures of kinase inhibitors from oxindole and naphthostyril classes.
NMR spectra were recorded with a Varian Mercury VXR400 6-Aminobenz[cd]indol-2(1H)-one (10)
spectrometer (Varian Inc., Palo Alto, CA) operating at 400 MHz
About 0.15 g of 10% Pd/C was added to the mixture of 25.4 g
in a DMSO-d6 solution, with tetramethylsilane (TMS) as an
internal standard. Chemical shifts are shown as d values (ppm).
Signal multiplicities are represented as s (singlet), d (doublet), t
(triplet), q (quartet) or m (multiplet).
compound 9 in 200 ml of ethanol. The reaction vessel was
degasificated until the solvent begins to bubble, then vessel was
carefully backfilled with hydrogen. This operation was repeated
twice and then the reaction vessel was allowed to stir overnight
with attached source of hydrogen. Then, hydrogen was evacuated,
reaction vessel was backfilled with argon and Pd/C was filtered
Benz[cd]indol-2(1H)-one (8)
About 0.3 mol of 1,8-naphthalic anhydride, 0.3 mol of hydro- off. Ethanolic solution was evaporated and obtained compound 10
xylamine hydrochloride and 330.64 g of pyridine were charged was used without further purification. Yield 20.5 g (94%). 1H
in the reactor and heated while stiffing. The reaction was NMR (DMSO-d6), d: 5.63 (s, 2H), 7.19 (d, 1H), 7.30 (d, 1H), 7.46
conducted under reflux for 1 h, followed by cooling to 80 ꢀC. (t, 1H), 7.76 (d, 1H), 8.42 (d, 1H), 10.64 (s, 1H).
Then 0.6 mol of p-toluenesulfonyl chloride was added to the
reaction system. After addition, the temperature was raised and 2-oxo-1,2-dihydrobenz[cd]indol-6-sulfonyl chloride (11)
the reaction was carried out under reflux for 1 h, followed by
About 20.5 g of compound 10, 82 ml of concentrated hydrochloric
cooling. The reaction mixture was poured into 1.25 l of water
acid and 40 ml water were mixed and heated to 40 ꢀC and stirred
and stirred to precipitate crystals, which were collected by
for 30 min keeping that temperature. Then the reaction mixture
filtration. The crystals were transferred to a beaker and washed
was cooled to 0 ꢀC and the solution of 7.83 g sodium nitrite in
successively with 1 L of a sodium hydrogencarbonate aqueous
15 ml water was slowly added dropwise maintaining temperature
solution and 1 l of water, followed by filtration. The crystals were
between 0 and 5 ꢀC. After the addition, reaction mixture was
washed with water and dried to give an intermediate in a yield
stirred for 30 min at 0 ꢀC.
of 81.1%. The whole amount of the intermediate, 175 ml of
In total, 105 ml of 2.5 M solution of SO2 in acetic acid and
ethanol and 200 ml of water were put in a reactor and stirred.
solution of 3.9 g copper(II) chloride dehydrate in 10 ml water was
Then 325 ml of a 2.7 mol/L aqueous solution of sodium hydroxide
mixed in another flask and this mixture was cooled to 10 ꢀC and
was added dropwise to the mixture. Thereafter, the mixture was
solution of diazonium compound was added in one portion. The
heated to refluxing temperature, at which the reaction was carried
temperature was raised and gas was started bubbling. The reaction
out for 3 h while distilling off ethanol. After completion of the
mixture was stirred for 1 h and then mixture was poured to 250 ml
reaction, the reaction mixture was cooled to 75 ꢀC, and 105 ml
of ice-chilled water. Resulting mixture was stirred to precipitate
of concentrated hydrochloric acid was added dropwise. In the
crystals, which were collected by filtration and washed with water
meantime, crystals precipitated at 60 ꢀC. After completion of
the dropwise addition, the mixture was further cooled. The
precipitated crystals were collected by filtration, washed with
1
twice. Yield 19.7 g (66%); H NMR (DMSO-d6), d: 7.63 (d, 1H),
7.80 (t, 1H), 8.11 (d, 1H), 8.26 (d, 1H), 8.69 (d, 1H), 10.65 (s, 1H).
ion-exchanged water, and dried to give 38.67 g (yield: 94.0%)
General procedure of 2-oxo-1,2-dihydrobenz[cd]indol-6-
of the title compound. 1H NMR (DMSO-d6), d: 7.42 (d, 1H), 7.63
sulfanilanilides 12–28 synthesis
(t, 1H), 7.69 (t, 1H), 7.87 (d, 1H), 7.91 (d, 1H), 8.42 (d, 1H),
10.64 (s, 1H).
Two equivalent of pyridine was added to the solution of 1 eq. of
11 and 1,2 eq. corresponding aniline in THF (3 ml/g 11).
Resulting mixture was stirred for 20 h at room temperature and
then crystals of targeted compound were collected by filtration.
Precipitate was washed with 20% aqueous methanol, dried on
filter, washed with hot ethanol and dried in the open air. Yields
and NMR spectra data are presented in Table 1.
6-Nitrobenz[cd]indol-2(1H)-one (9)
Nitric acid (18.76 g, 0.297 mol) was added dropwise to the
solution of benz[cd]indol-2(1H)-one 8 (38.7 g, 0.229 mol) in
170 ml of glacial acetic acid. At first, there was a very minor
exotherm and then, over the course of 1 h, the reaction tempera-
ture raised to 50 ꢀC. The reaction mixture was gradually cooled to
room temperature with a cold water bath. A thick dark green paste
Kinase assays
resulted. This mixture was filtered, washed with 50% aqueous The FGFR1 kinase assays with recombinant cytoplasmic domain
acetic acid, and pulled as dry as possible. The resulting wet filter of the FGFR1 tyrosine kinase (Millipore, Billerica, MA, Cat. N.
cake was refluxed in 600 ml methanol and then cooled to 0 ꢀC. 14-582) were performed in a total volume of 30 ml containing
The mixture was filtered, washed with cold methanol and dried 10 mM MOPS (pH 7.2), 0.1 mM sodium orthovanadate, 0.2 mM
in vacuo to yield 25.4 g (51%) of 6-nitrobenz[cd]indol-2(1H)-one EDTA, 0.002% Brij 35, 0.2 mg/ml BSA, 0.02 % b-mercaptoetha-
9. 1H NMR (DMSO-d6), d: 7.82 (t, 1H), 7.94 (d, 1H), 8.55 nole, 250 mM of peptide substrate (KKKSPGEYVNIEFG,
(d, 1H), 8.59 (d, 1H), 8.93 (d, 1H), 10.65 (d, 1H).
GenScript, Piscataway, NJ), various concentrations of inhibitor

DOI: 10.3109/14756366.2014.895718
Naphthostyril derivatives as novel protein kinase FGFR1 inhibitors
3
Table 1. Chemical characteristics of synthesized compound.
Compound No.
1H NMR data
Yield (%)
84
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1H NMR (DMSO-d6), d: 7.15 (d, 2H), 7.22 (d, 2H), 7.85 (d, 1H), 8.13 (t, 1H), 8.36 (d, 1H), 8.56 (d, 1H), 8.61
(d, 1H), 10.50 (s, 1H), 10.66 (s, 1H)
1H NMR (DMSO-d6), d: 7.13 (d, 1H), 7.26 (d, 1H), 7.43 (s, 1H), 7.83 (d, 1H), 8.14 (t, 1H), 8.38 (d, 1H), 8.54
(d, 1H), 8.59 (d, 1H), 10.47 (s, 1H), 10.63 (s, 1H)
81
80
86
79
77
74
88
82
78
73
86
84
82
83
73
68
1H NMR (DMSO-d6), d: 7.05 (t, 1H), 7.17 (d, 1H), 7.23 (d, 1H), 7.83 (d, 1H), 8.12 (t, 1H), 8.36 (d, 1H), 8.56
(d, 1H), 8.61 (d, 1H), 10.46 (s, 1H), 10.65 (s, 1H)
1H NMR (DMSO-d6), d: 7.09 (d, 2H), 7.37 (d, 2H), 7.85 (d, 1H), 8.15 (t, 1H), 8.38 (d, 1H), 8.57 (d, 1H), 8.62
(d, 1H), 10.53 (s, 1H), 10.68 (s, 1H)
1H NMR (DMSO-d6), d: 6.94 (d, 2H), 7.09 (d, 2H), 7.80 (d, 1H), 8.11 (t, 1H), 8.35 (d, 1H), 8.54 (d, 1H), 8.59
(d, 1H), 10.47 (s, 1H), 10.60 (s, 1H)
1H NMR (DMSO-d6), d: 6.96 (m, 3H), 7.08 (d, 1H), 7.81 (d, 1H), 8.12 (t, 1H), 8.35 (d, 1H), 8.54 (d, 1H),
8.59 (d, 1H), 10.47 (s, 1H), 10.60 (s, 1H)
1H NMR (DMSO-d6), d: 7.22 (d, 1H), 7.33 (m, 2H), 7.53 (s, 1H), 7.81 (d, 1H), 8.13 (t, 1H), 8.37 (d, 1H),
8.55 (d, 1H), 8.60 (d, 1H), 10.50 (s, 1H), 10.63 (s, 1H)
1H NMR (DMSO-d6), d: 7.01 (t, 1H), 7.12 (d, 2H), 7.18 (d, 2H), 7.80 (d, 1H), 8.14 (t, 1H), 8.34 (d, 1H), 8.55
(d, 1H), 8.57 (d, 1H), 10.48 (s, 1H), 10.60 (s, 1H)
1H NMR (DMSO-d6), d: 6.67 (d, 2H), 7.05 (d, 2H), 7.80 (d, 1H), 8.10 (t, 1H), 8.32 (d, 1H), 8.53 (d, 1H), 8.60
(d, 1H), 10.46 (s, 1H), 10.61 (s, 1H)
1H NMR (DMSO-d6), d: 6.78 (d, 1H), 7.17 (d, 1H), 7.23 (s, 1H), 7.81 (d, 1H), 8.13 (t, 1H), 8.35 (d, 1H), 8.57
(d, 1H), 8.61 (d, 1H), 10.47 (s, 1H), 10.62 (s, 1H)
1H NMR (DMSO-d6), d: 2.00 (s, 3H), 6.54 (d, 1H), 6.62 (s, 1H), 7.03 (d, 1H), 7.80 (d, 1H), 8.12 (t, 1H), 8.35
(d, 1H), 8.55 (d, 1H), 8.60 (d, 1H), 10.45 (s, 1H), 10.60 (s, 1H)
1H NMR (DMSO-d6), d: 3.60 (s, 3H), 6.73 (d, 2H), 7.02 (d, 2H), 7.82 (d, 1H), 8.14 (t, 1H), 8.36 (d, 1H), 8.57
(d, 1H), 8.62 (d, 1H), 10.47 (s, 1H), 10.62 (s, 1H)
1H NMR (DMSO-d6), d: 3.71 (s, 6H), 5.80 (s, 1H), 6.71 (s, 2H), 7.81 (d, 1H), 8.12 (t, 1H), 8.30 (d, 1H), 8.52
(d, 1H), 8.58 (d, 1H), 10.43 (s, 1H), 10.57 (s, 1H)
1H NMR (DMSO-d6), d: 3.72 (s, 3H), 3.80 (s, 3H), 6.55 (d, 1H), 6.59 (s, 1H), 7.24 (d, 1H), 7.82 (d, 1H), 8.13
(t, 1H), 8.32 (d, 1H), 8.54 (d, 1H), 8.59 (d, 1H), 10.47 (s, 1H), 10.61 (s, 1H)
1H NMR (DMSO-d6), d: 3.66 (s, 3H), 3.75 (s, 3H), 6.67 (d, 1H), 6.93 (d, 1H), 6.98 (s, 1H), 7.85 (d, 1H), 8.15
(t, 1H), 8.34 (d, 1H), 8.56 (d, 1H), 8.61 (d, 1H), 10.48 (s, 1H), 10.63 (s, 1H)
1H NMR (DMSO-d6), d: 3.72 (s, 6H), 6.52 (d, 2H), 6.84 (t, 1H), 7.84 (d, 1H), 8.13 (t, 1H), 8.32 (d, 1H), 8.55
(d, 1H), 8.59 (d, 1H), 10.45 (s, 1H), 10.61 (s, 1H)
1H NMR (DMSO-d6), d: 7.18 (t, 1H), 7.27 (d, 1H), 7.42 (t, 1H), 7.73 (d, 1H), 7.85 (d, 1H), 8.16 (t, 1H), 8.40
(d, 1H), 8.59 (d, 1H), 8.65 (d, 1H), 10.64 (s, 1H), 11.36 (s, 1H), 11.50 (s, 1H)
dissolved in DMSO (final DMSO concentration in probe less than into 96 ell tissue plate at 2 ꢁ 105 cells/mL pre-incubated for 24 h
1%) and 7 ng of enzyme. The reaction was initiated by the and treated with the synthesized compounds (compounds in
addition of ATP (50 mM ATP, 25 mM MgAc containing 0.1 mCi DMSO solution, final DMSO concentration less than 0.5%) at
of [g-32 P]ATP per incubation) and samples incubated at 30 ꢀC various concentrations. After 72 h treatment, the cells were
for 25 min. The reaction was terminated by the addition of 5% incubated with 15 ml MTT (3 -(4,5-dimethyl-thiazol-2-yl)-2,5-
phosphoric acid and the precipitation of material onto phospho- diphenyl-tetrazolium bromide from Sigma, St. Louis, MO)
cellulose filter mats ‘‘Whatman P81’’. Filters were washed thrice solution (5 mg/mL) for 4 h at 37 ꢀC, 5% CO2. The formazan
with 0.75% phosphoric acid and the incorporation of [32P] into the precipitates were dissolved in 100 ml DMSO and the absorbance at
peptide substrate was determined by counting the radioactivity 495 nm of each well was measured by spectrofluorometer MR 700
retained on the filters in a PerkinElmer (Waltham, MA) scintil- (Dynatech, Chantilly, VA). The cell viability was expressed as a
lation counter. Kinase residual activity was expressed in percent- percentage relative to the untreated control cells.
ages with respect to DMSO control. The concentration of
compound that inhibited enzymatic activity by 50% (IC50) was Results and discussion
determined graphically.
Screening of naphthostiriles
Inhibition of selectivity panel kinases was performed accord-
ing to enzyme provider protocols (Millipore, Billerica, MA). ATP Search of compounds with naphthostyril scaffold was per-
concentration in reaction mixture was 100 mM.
formed in our in-house collection. In total, 397 compounds with
naphthostyril core were found by substructure search. This set
of compounds was clustered using ECFP_4 fingerprints and
subjected to diversity sorting, resulting in selection of 18
compounds for FGFR1 inhibition tests. For compounds reducing
the kinase activity more than 50% at concentration 33 mM IC50
was determined and their close structural analogs were tested.
Totally, it was found six active FGFR1 inhibitors (1–6). Their
chemical structures and activity are shown in Table 2.
Molecular modeling
Ligands were prepared for docking with conformation minimiza-
tion and converted in pdbqt format with Vega ZZ (Milano,
Italy)12. Docking was carried out in crystal structure of FGFR1
(pdb bank code 2FGI) by AutoDock 4.2 (La Jolla, CA) with
default parameters. Docking results were analyzed with
AutoDockTools-1.5.6 (La Jolla, CA)13.
The most active compound (1) identified during screening
has N-(3-chlorophenyl)sulfonamide substituent at the position 6
of naphthostyril ring and IC50 value of 4.2 mM. Substituted in
Cellular proliferation assay
KG1 cell viability was examined using a standard MTT assay14. 6 and 8 positions naphthostyriles 2 and 3 effectively reduce
Cells were grown in DMEM containing 10% fetal bovine serum kinase activity at screening concentration 33 mM but have
(FBS), 100 mg/mL penicillin and 100 mg/mL streptomycin in unexpectedly high IC50 values. A possible reason for this effect
humidified air at 37 ꢀC with 5% CO2. Viable cell were seeded is the low solubility of these compounds. Compound 4 with

4
A. A. Gryshchenko et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Table 2. Chemical structure and in vitro activities of the naphthostyril derivatives.
Compounds
Structure
Kinase activity (% at 33 mM*)
Log Sy
ꢂ4.64
IC50 (mM)
1
15
4.2
2
6
ꢂ5.46
48.9
3
11
ꢂ5.32
34
4
42
ꢂ3.5
22
5
35
ꢂ2.72
12.1
6
17
ꢂ2.79
4.4
*Residual kinase activity is percent of kinase activity at inhibitor concentration 33 mM related to control with DMSO. yCalculated logarithm of
solubility.

DOI: 10.3109/14756366.2014.895718
Naphthostyril derivatives as novel protein kinase FGFR1 inhibitors
5
6-methylacetamide substituent has IC50 of 22 mM. Several struc- chosen for chemical optimization of N-phenylnaphthostyril-1-
tural analogs of this compound were inactive for kinase inhibition sulfonamides as FGFR1 inhibitors.
(data not shown). Compound 5 has lower FGFR1 inhibition
activity than structurally close compound 1 implying the signifi- Chemistry
cant role of phenyl group in the structure of compound 1 for the
Taking into consideration, the prospects of search of FGFR1
interaction with kinase. Compound 6, 6-Methyl-1H-indolo[5,4,3-
kinase inhibitors among N-phenylnaphthostyril-1-sulfonamide
def]isoquinoline-2,5,7-trione with IC50 of 4.4 mM was slightly less
derivatives, a number of compound 1 structural analogs with
active than compound 1. But analogs of compound 6 with bulkier
different substituted phenyl were synthesized according to
pyridine or butyl substituents instead of methyl at position 6 were
Scheme 1. First, 2- hydroxybenzo[de]isoquinolin-1,3-dione was
inactive for FGFR1 inhibition (data not shown).
obtained from 1,8-naphtalic anhydride through the reaction with
Thus, the most promising inhibitor from screening of
hydroxylamine hydrochloride. Then the oxygen of hydroxyl of 2-
naphthostyriles was compound 1 with a single digit micromolar
hydroxybenzo[de]isoquinolin-1,3-dione was acylated one-pot by
activity, therefore N-phenylnaphthostyril sulfonamide was chosen
p-toluenesulfonyl chloride and benz[cd]indol-2(1H)-one (8)
as scaffold for further optimization.
was obtained through rearrangement15. Then compound 8 was
nitrated by concentrated nitric acid in glacial acetic acid and
Molecular modeling
obtained compound 9 was hydrogenated on 10% Pd/C. Sulfonyl
Docking of compound 1 in the FGFR1 catalytic site was chloride 11 was obtained through diazotation of obtained on
carried out to clarify the relationship between chemical structure previous stage aniline 10 and following reaction resulting
and inhibition activity of kinase by N-phenylnaphthostyril diazonium compound with solution of SO2 in acetic acid and
sulfonamide compounds (Figure 2). The naphthostyril core of copper(I) as a catalyst. Targeted compounds 12–28 were obtained
compound 1 occupies the adenine binding portion of the ATP by reaction of sulfonyl chloride 11 with a number of correspond-
binding site and forms two hydrogen bonds with kinase hinge ing anilines.
region. The NH moiety of naphthostyril is involved in hydrogen
bond formation with Glu562 residue while the carbonyl group Structural activity relationship (SAR) of
forms a hydrogen bond with the backbone of Ala564. The N-phenylnaphthostyril-1-sulfonamides
hydrophobic interactions of the naphthostyril core with Leu484,
A series of newly synthesized N-phenylnaphthostyril-1-sulfona-
Leu630, Val492, Ala512 (not labeled) stabilize the position
mides (12–28) was tested for inhibition of FGFR1 using P32
of inhibitor in binding site. Obtained docking binding pose well
radioassay. The chemical structures and biological activity of
correlates with X-ray position of CDK2 naphthostyril inhibitors
these compounds are shown in Table 3.
from Ref. [11].
Relationship between substituents in phenyl group of
The N-(3-chlorophenyl)sulfonamide substituent at the position
N-phenylnaphthostyril-1-sulfonamid and FGFR1 inhibitory activ-
6 of compound 1 extends in depth of ATP binding site. The amide
ity was investigated. Compound 19 with unsubstituted phenyl
group of sulfonamide linker forms a hydrogen bond with carboxyl
group was a weak inhibitor of FGFR1. Compound 28 with ortho-
group of Asp641 side chain. The chlorophenyl group of N-(3-
carboxyl group and compounds with methoxy groups located in
chlorophenyl)sulfonamide substituent is involved in hydrophobic
different positions of phenyl (23–27) also displayed poor inhibi-
interactions with amino acid residues Lys514, Met535, Val559,
tory activity and their IC50 values were not determined. But
Ile545, Ala640 and Val561 in hydrophobic region I. Meta chlorine
compounds with methoxy group in ortho position (25, 27) were
on the phenyl group of N-phenylnaphthostyril-1-sulfonamide
slightly more active than N-phenylnaphthostyril-1-sulfonamides
interact with amino acid residues in the hydrophobic pocket
with meta and para methoxy groups in kinase residual activity
depth, and possibly have a significant impact on binding affinity
test. Compounds with halogen substituents in phenyl group
in our interaction model of N-phenylnaphthostyril-1-sulfonamides
demonstrated higher activity than the methoxy derivatives and
with FGFR1. Based on these considerations, phenyl group was
have IC50 values below 10 mM. Compounds with halogen
substituent in the para position of the phenyl were generally
more active inhibitors than compounds with halogen substituent
in the ortho position (16 versus 17, 13 versus 14). The most active
halogenated compound 13 has two chlorine atoms in the para and
meta positions (IC50 ¼ 2.3 mM). Activity of this compound was
significantly higher in comparison with compounds 1 and 12
containing only para and meta chloro phenyl and having twofold
and threefold lower activity, respectively. From investigated N-
phenylnaphthostyril-1-sulfonamide derivatives the highest inhibi-
tor activity was observed in compounds containing hydroxyl
group positioned at the para position (20, 21, 22). Compound 20
with hydroxyl group in para position of phenyl and without any
others substituents in phenyl was the most active inhibitor in
examined compound series. The introduction of chlorine in the
meta position (21) reduced activity compared to compound 20;
presence of methyl in the ortho position (22) also slightly
decreased activity. These results indicated that hydrogen bonding
donor group introduced to the phenyl group might results in an
increased activity. According to molecular modeling studies,
hydroxyl group in the para position of the phenyl can form a
hydrogen bond with the carbonyl group of Ile545 deeply in
hydrophobic pocket I. This fact can explain increasing of
compound 20 activity.
Figure 2. Docking model of compound 1 bound to the ATP-binding site
of the FGFR1. Hydrogen bonds marked as dashed lines.

6
A. A. Gryshchenko et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
O
O
Scheme 1. Synthesis of N-phenylnaphthos-
tyril-1-sulfonamides.
N
HNO
H2/Pd/C
EtOH
HCI*H N–OH
2
3
AcOH
Py, PTSC
NH
NH
9
O
O
7
O
O
O
O
O
8
Cl
O S
O S NH
NH
2
O
R
1
HNO2, HCl
H N
2
R
SO2/AcOH
CuCl2*2H2O
Py, THF
NH
NH
10
NH
11
O
O
12-28
Table 3. Chemical structure and in vitro activities of the
N-phenylnaphthostyril-1-sulfonamide derivatives.
Table 4. Kinase selectivity of 20 and 22.
Kinase
Inhibitor 20
Inhibitor 22
FGFR1
Tie-2
c-MET
Aurora A
JNK3
11 4.1
117.7 11.6
99.7 4.0
92 8.4
115.3 13.2
83.4 3.0
89.6 6.1
15 3.9
67 9.0
75 7.9
63.4 3.5
108.6 11.3
69 4.7
CK2
ASK1
103.8 7.4
Kinase residual activity is percent of kinase activity at
inhibitor concentration 33 mM related to control with
DMSO, ATP concentration was 100 mM for all kinases.
Kinase residual
activity (%)*
Compounds
R1
IC50 (mM)y
Table 5. Antiproliferative activity of compounds 13, 20 and 22 at KG1
cancer cell line.
1
30-Cl
40-Cl
15
21.5
5.5
4.22
6.3
2.3
9.5
7.4
5.9
8.2
3.4
ND
2
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
30,40-diCl
20,30-diCl
40-Br
Compounds
13
20
22
8.3
17.5
18.7
15.3
11.6
32.7
7.2
Cell viability, % at 100 mM
Cell viability, % at 10 mM
58.5 2.0
98.9 3.5
69.8 2.4
90.8 3.8
103.8 2.4
108.6 6.4
40-F
20-F
30-CF3
H
40-OH
The percentage of cell viability was presented as mean of three replicates
with standard deviation.
30-Cl, 40-OH
40-OH, 20-CH3
40-OCH3
30,50-diOCH3
20,40-diOCH3
30,40-diOCH3
20,50-diOCH3
20-COOH
8.8
3.5
41.5
68
33.1
76.3
31.7
47.5
3.55
2.25
ND
ND
ND
ND
ND
ND
Constitutive activity of FGFR1 was shown in this cancer cell
line16 that used to investigate FGFR1 inhibitors cytotoxicity17.
Results of proliferation inhibition tests of N-phenylnaphthostyril-
1-sulfonamides are shown in Table 5. The antiproliferative
activity of compounds 13 and 20 was weak but dose dependent,
compound 22 was inactive. The lack of compound 22 activity
was possibly established with its low membrane permeability
caused by the presence of methyl group in the ortho position of
phenyl ring.
*Kinase residual activity is percent of kinase activity at inhibitor
concentration 33 mM related to control with DMSO. yND, not
determined.
Conclusions
Selectivity study
A new series of N-phenylnaphthostyril-1-sulfonamide compounds
with low micromolar FGFR1 inhibition activity was identified.
The SAR indicated that introducing of hydroxyl group in para
position of phenyl group contributed much to the inhibition
activity. In parallel, molecular docking analysis helped to
rationalize SAR observations, led to a proposal of the preferred
binding conformation. The most potent inhibitor in the series (20)
with IC50 of 2 mM showed good selectivity and some antiproli-
ferative effect. N-phenylnaphthostyril-1-sulfonamides are per-
spective for further development of FGFR1 inhibitors.
Two active compounds 20 and 22 were taken for kinase selectivity
analysis with in-house panel of 6 kinases. The results, presented in
Table 4, show that studied N-phenylnaphthostyril-1-sulfonamides
have good selectivity profile in the panel of tested kinases,
including receptor tyrosine protein kinases Tie-2 and c-MET.
Thereby N-phenylnaphthostyril-1-sulfonamides are potentially
selective class of FGFR1 inhibitors.
Inhibition of proliferation in cancer cell lines
Three the most active compounds (compounds 13, 20 and 22)
with IC50 below 3 mM were tested for antiproliferative activity
Declarations of interest
against KG1 myeloma cell line at concentrations 10 and 100 mM. The authors report no declarations of interest.

DOI: 10.3109/14756366.2014.895718
Naphthostyril derivatives as novel protein kinase FGFR1 inhibitors
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