Thienochromene derivatives inhibit pSTAT1 and pSTAT5 signaling induced by cytokines

Article abstract of DOI:10.1016/j.bmcl.2020.127415

Furanocoumarins, particularly furo[3,2-c]coumarins, are found in many natural products. However, coumarins annulated to a thiophene ring have received scarce attention to date in the literature. Therefore, we synthesized 4-oxo-4H-thieno[3,2-c]chromene derivatives and tested in vitro their anti-inflammatory activity. Anti-inflammatory potential of the synthesized compounds (1, 2, 6–8, 9a–e and 10a–c) has been evaluated by measuring various pSTAT (signal transducer and activator of transcription) inhibition within the JAK (Janus-activated family kinase)/STAT signaling pathway. Ethyl 7-hydroxy-4-oxo-4H-thieno[3,2-c]chromene-2-carboxylate (7) showed best inhibition properties on pSTAT5 in GM-CSF (Granulocyte-macrophage colony-stimulating factor)-triggered PBMC assay, with IC₅₀ value of 5.0 μM.

Full text of DOI:10.1016/j.bmcl.2020.127415

Bioorganic & Medicinal Chemistry Letters 30 (2020) 127415
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thienochromene derivatives inhibit pSTAT1 and pSTAT5 signaling induced
by cytokines
a,⁎
b
f
b
a
d
Karlo Wittine , Roberto Antolović , Dubravko Jelić , Sara Bracanović , Mario Cetina ,
a,e
a
c
Uroš Andjelkovic , Ozren Wittine , Sandra Kraljević Pavelić , Adrijana Vinter
a
b
c
d
e
f
University of Rijeka, Department of Biotechnology, Radmile Matejčić 2, 51000 Rijeka, Croatia
Catholic University of Croatia, Ilica 242, 10000 Zagreb, Croatia
Fidelta Ltd, Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia
University of Zagreb, Faculty of Textile Technology, Department of Applied Chemistry, Prilaz baruna Filipovića 28a, 10000 Zagreb, Croatia
University of Belgrade, Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, Studentski trg 12-16, 11000 Belgrade, Serbia
Pliva Croatia Ltd, Prilaz baruna Filipovića 25, 10000 Zagreb, Croatia
A R T I C L E I N F O
A B S T R A C T
InChIKeys:
Furanocoumarins, particularly furo[3,2-c]coumarins, are found in many natural products. However, coumarins
XRDYDKQPEUTKOZ-UHFFFAOYSA-N
JXWPVKTZTZYXNN-UHFFFAOYSA-N
WSGXYNCVIUSDGM-UHFFFAOYSA-N
UQXVYUODSBIAGJ-UHFFFAOYSA-N
ZLRYSLHVRPSJMO-UHFFFAOYSA-N
YJDFITHISLLEBT-UHFFFAOYSA-N
XUEJBMPAUSXSBT-UHFFFAOYSA-N
MKGZCNQVWIDXIT-UHFFFAOYSA-N
WRCVZCJJBWFEKM-UHFFFAOYSA-N
HAJLTRREQIWHTN-UHFFFAOYSA-N
STMSQPCRMHMFIW-UHFFFAOYSA-
NKeywords:
annulated to a thiophene ring have received scarce attention to date in the literature. Therefore, we synthesized
4
-oxo-4H-thieno[3,2-c]chromene derivatives and tested in vitro their anti-inflammatory activity. Anti-in-
flammatory potential of the synthesized compounds (1, 2, 6–8, 9a–e and 10a–c) has been evaluated by mea-
suring various pSTAT (signal transducer and activator of transcription) inhibition within the JAK (Janus-acti-
vated family kinase)/STAT signaling pathway. Ethyl 7-hydroxy-4-oxo-4H-thieno[3,2-c]chromene-2-carboxylate
(
7) showed best inhibition properties on pSTAT5 in GM-CSF (Granulocyte-macrophage colony-stimulating
factor)-triggered PBMC assay, with IC50 value of 5.0 µM.
Thieno[3,2-c]chromene
Coumarin
Anti-inflammatory
Interleukin
Cytokine
JAK/STAT
JAK/STAT is one of the major pathways through which many cy-
tokines exert and integrate their function. JAK is a family of non-
receptor cytoplasmic tyrosine kinases, consisting of four members in
process of target genes.2 Although JAK family has only four members,
different cytokines may act through the same JAK family member.
Additionally, each cytokine may engage with more than one JAK family
member to a varying degree facilitating different duration and signal
1
mammals: JAK1, JAK2, JAK3 and TYK2. When JAK kinases are acti-
3
vated by their respective effector molecules (ILs, IFNs, colony-stimu-
lating factors, growth factors and hormones) they phosphorylate
themselves (auto-phosphorylation) and/or adjacent molecules (trans-
phosphorylation) including STAT which consists of seven members in
mammals (STAT 1–4, 5a, 5b and 6), facilitating their translocation to
the nucleus, direct binding to DNA and activation of the transcription
intensity of STAT. Exaggerated or protracted cytokine signaling has
been implicated in many inflammatory and autoimmune diseases. Im-
paired delicate balance comes with a steep price of inflicting great
bodily harm. Therefore, targeting cytokines or their receptors presents a
favorable approach to treat diseases related to disturbed cytokine bal-
ance.
Abbreviations: DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GM-CSF, granulocyte-macrophage colony-sti-
mulating factor; IFNα, inerferon alpha; IL-2, interleukine-2; JAK, Janus kinase; PathScan, Intracellular Signaling Array Kit; PBMC, peripheral blood mononuclear cell;
PBS, phosphate buffered saline; STAT, signal transducer and activator of transcription
⁎
Corresponding author.
E-mail address: karlo.wittine@biotech.uniri.hr (K. Wittine).
https://doi.org/10.1016/j.bmcl.2020.127415
Received 5 June 2020; Received in revised form 13 July 2020; Accepted 14 July 2020
Available online 18 July 2020
0960-894X/ © 2020 Elsevier Ltd. All rights reserved.

K. Wittine, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127415
1
9
through their ability to modulate inflammatory cells.
In contrast, coumarins annulated to a thiophene ring, known as
thieno[c]chromen-4-ones, have received scarce attention to date in the
literature (Fig. 1).
Thiophene and its derivatives represent a prevalent structural motif
in many natural products (Gronowitz et al., 1985) and pharmaceuti-
2
0–29
cally active agents
with some of the top selling marketed drugs
Fig. 1. Furano- and [2,3-c]thieno-coumarins.
including thiophene as part of their structure (including clopidogrel,
3
0
raloxifene, zileuton, and tiotropium bromide), as well as many others,
such as duloxetine (antidepressant), eprosartan (anti-hypertensive),
rivaroxaban (oral anticoagulant) and olanzapine (antipsyhotic). An
overview of the sulfur-containing FDA-approved drugs and their
3
1
structures was recently published.
Coumarins with additional rings condensed at the 3,4-position, have
3
2−33
long been reported to exhibit potent biological activity.
Most of
with only a small portion of
the literature concerning thieno[3,2-c]coumarin derivatives focusses on
3
4–48
different protocols for their synthesis
4
9–51
Fig. 2. Ethyl 4-oxo-4H-thieno[3,2-c]chromene-2-carboxylate (1) and 4-oxo-4H-
thieno[3,2-c]chromene-2-carboxamide (2).
publications describing biological activities such as anticancer,
52
53,54
anticancer and antimicrobial, antifungal activities
or those re-
5
5,56
lated to psoralen
.
A number of thieno[3,2-c][1]benzopyran-4-ones synthesized by
Derivatives of coumarin have attracted considerable attention due
thermal thio-Claisen rearrangement demonstrated anti-inflammatory,
4
57–61
to their diverse range of biological activities which include anti-in-
antipyretic, and antiallergic potential
.
5
6
7
8
flammatory, antimicrobial, antiangiogenic, antinociceptive, anti-
In searching for novel anti-inflammatory compounds, we synthe-
sized 4-oxo-4H-thieno[3,2-c]chromene derivatives with an ami-
noalkoxy chain linked at the benzene ring of the chromene scaffold.
To test the anti-inflammatory effect on various cytokine-triggered
production of pSTAT within the JAK/STAT signaling in cells, PBMCs
(peripheral blood mononuclear cells) stimulated with IFNα, IL-2 and
GM-CSF were utilized, with levels of pSTAT1 and pSTAT5 production
9
10−11
12
HIV, antitumour,
and anticoagulant. The last decade resulted
in over 5000 publications on coumarin derivatives in PubMed database
with a currently total of more than eleven thousand publications.
Furanocoumarins, particularly containing psorlen or angelicin
scaffold (Fig. 1), are found in many natural products and have been
seen to possess antifungicidal, insecticidal, insect antifeedant, anti-HIV
1
3–18
62
and anticancer activities,
as well as anti-inflammatory properties
used as a readout in ELISA-based method.
Scheme 1. Reagents and conditions: (a) NaH, Et
2
CO
3
, 100 °C, 3 h; (b) DMF, POCl
3
, 90 °C, 12 h; (c) ethyl-2-mercaptoacetate, pyridine, Et
3
N, 70 °C, 4 h; (d) 1 M BBr ,
3
CH Cl , rt, 5 h; (e) K CO , DMF, 100 °C, overnight; (f) NH
2
2
2
3
3
, methanol, r.t., overnight.
2

K. Wittine, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127415
Synthetic route to 4-oxo-4H-thieno[3,2-c]chromene scaffold and its
aminoalkoxy derivatives of thiophene esters (9a-e) and thiophene
amides (10a-c) is depicted in Scheme 1.
The parent compound (3) (1-(2-hydroxy-4-methoxyphenyl)ethan-1-
one) is commercially available and prone to base-mediated cyclization
(
(
ring closure) with diethylcarbonate. Reaction of 4-hydroxycoumarin
4) under Vilsmeier–Haack reaction conditions using POCl /DMF af-
3
forded β-chlorovinyl aldehyde (4-chloro-3-formylcoumarin; 4-chloro-
coumarin carboxaldehyde) (5), that is essentially a gem-activated al-
kene with a good leaving group (chlorine). In the consecutive step
annulation of thiophene ring is achieved according to the well-estab-
6
3–67
lished process
by reaction of β-chlorovinyl aldeyde 5 with ethyl 2-
mercaptoacetate in the presence of triethylamine and pyridine (6).
Fiesselmann thiophene synthesis has been well studied and it is known
that the formation of thiophene to take place via a domino process
involving Michael addition, followed by intramolecular Knoevenagel
reaction in the presence of different bases. This mechanism has been
well studied on ethyl thioglyconate and β-halo-α,β-unsaturated alde-
6
8
Fig. 3. Molecular structure of 6, with the atom-numbering scheme.
Displacement ellipsoids for non-hydrogen atoms are drawn at the 50% prob-
ability level.
hydes.
Deprotection of methoxy group with 1 M BBr
3
in di-
chloromethane yielded 7 with a hydroxyl group in the 7-position. Five
different aminoalkoxy chains were introduced to thieno[3,2-c]chro-
mene-2-carboxylate (9a-e) by Williamson ether synthesis using K
2
CO
3
Previously synthesized derivatives (1 and 2)50 are shown in Fig 2.
as a base. An important beneficial feature of introducing an alkoxy-
amino side chain is improved solubility of compounds in polar solvents
Inhibitory potential of these compounds on pSTAT1 and pSTAT5 pro-
duction was also evaluated together with a newly synthesized com-
pounds 6–8, 9a-e and 10a-c.
(
methanol, ethanol, water). Amide derivatives 10a-c were readily ob-
tained by ammonolysis using gaseous ammonia in methanol.
Fig. 4. Inhibition of pSTAT5 production in PBMCs triggered with IL-2 or GM-CSF with tested compounds in one testing concentration (150 µM). Results are presented
as average values of duplicates in two separate experiments.
3

K. Wittine, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127415
Fig. 5. Inhibition of pSTAT1 production in PBMCs triggered with IFNα with tested compounds in one testing concentration (150 µM). Results are presented as
average values of duplicates in two separate experiments.
In 669, ethyl carboxylate and methoxy groups are bonded to the
thienyl and phenyl rings, respectively, of thieno[3,2-c]chromene
moiety (Fig. 3).
assay (Fig. 5).
Since compound 7 showed more significant inhibition in all above
mentioned tests, its activity was additionally analysed in secondary
screening (10-concentration dose–response inhibition profile, 300 µM
final starting concentration, 1:3 dilution factor), in order to calculate
IC value for every assay and to estimate its full inhibitory profile and
The whole molecule is essentially planar. The biggest deviation one
of non-hydrogen atoms from mean plane is 0.215(2) Å, for the terminal
C16 atom of the methoxy group. Only one similar structure has been
published until now, in which methyl carboxylate and trifluoromethyl
5
0
potency. Compound 7 showed best activity properties on inhibition of
4
4
groups are bonded to the thienyl ring carbon atoms. Bond lengths and
angles in 6 show no unexpected features, and in common parts of the
molecule agree well with equivalent ones in structurally related com-
pSTAT5 production in GM-CSF-triggered assay, with IC value of
5
0
5.0 µM. Inhibition of pSTAT5 in IL-2-triggered assay showed IC value
5
0
of 12.1 µM, while inhibition of pSTAT1 in IFNα-triggered assay showed
4
4
pound. The sulphur S1 atom of the thienyl ring adopts an anti-
periplanar conformation with respect to the oxygen O3 atom of the ethyl
carboxylate group, as defined by the S1–C12–C13–O3 torsion angle of
IC value of 11.9 µM, with lower top values in both of those assays
5
0
(Fig. 6).
In summary, a series of 4-oxo-4H-thieno[3,2-c]chromene deriva-
−
179.86(14)°.
tives with an aminoalkoxy side chain have been synthesized and their
anti-inflammatory potential on JAK/STAT signaling pathway eval-
uated. Among the three different JAK/STAT cellular assays in PBMCs
(pSTAT5 in GM-CSF-triggered PBMCs, pSTAT5 in IL-2-triggered
PBMCs, and pSTAT1 in IFNα-triggered PBMCs, it is observed that tested
compounds inhibit much stronger production of pSTAT5 induced by
GM-CSF, in comparison to the production of pSTAT5 triggered by IL-2
in PBMCs, which could point to the conclusion that compounds are
more selective toward signaling pathways which goes over JAK2, in
comparison to pathways which are more selective to JAK1/3. Although
most of the tested compounds showed some level of activity on the
production of the pSTAT5 and pSTAT1 in PBMCs, only compound 7
showed more significant inhibition of pSTAT5 production in both, IL-2
and GM-CSF triggered assay, as well as inhibition of pSTAT1 induced by
7
0
All compounds were tested on IFNα-triggered measurement of
pSTAT1 production, and IL-2 or GM-CSF-triggered measurement of
7
1
pSTAT5 production in PBMCs in one single concentration (150 µM) in
7
2
two separate experiments for each of the assay. Results are presented
in Figs. 5 and 6. Tofacitinib and Baricitinib, marketed potent anti-in-
flammatory drugs (low nanomolar JAK inhibitors which interferes with
the JAK/STAT signaling pathways), were used as control compounds
for inhibition of JAK/STAT signaling, and their obtained activities were
in correspondence with internal historical data values in all cell-based
assays (Fig. S2, Supporting information).
Taken all together, only compound 7 showed more significant in-
hibition of pSTAT5 production in both IL-2 or GM-CSF triggered assay
in PBMCs (Fig. 4), as well as inhibition of pSTAT1 production in IFNα
Fig. 6. Inhibition of pSTAT5 production in PBMCs
triggered with IL-2 or GM-CSF, or pSTAT1 produc-
tion in PBMCs triggered with IFNα with Compound
7
(10-concentration dose–response inhibition pro-
file, 300 µM final starting concentration, 1:3 dilu-
tion factor). Results are presented as average values
of duplicates. Top values were fixed for IL-2 and
IFNα-triggered assay, for more precise calculation
of IC50 values.
4

K. Wittine, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127415
IFNα in PBMCs. Compared to compound 1, pSTAT inhibitory activity of
thienochroemene derivative 7 having OH-7 group was increased.
Hydroxyl group is generally known to bring favorable features to many
biologically-active coumarins. Therefore, compound 7 is a good can-
didate for further structural modifications to improve detected anti-
inflammatory potential. Nevertheless, it should be noted that the rules
linking the structural features of coumarin derivatives and their me-
chanisms of action are unfortunately still lacking.
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