PdCl2-catalyzed synthesis of a new class of isocoumarin derivatives containing aminosulfonyl / aminocarboxamide moiety: First identification of a isocoumarin based PDE4 inhibitor

Article abstract of DOI:10.1016/j.ejmech.2021.113514

While anti-inflammatory properties of isocoumarins are known their PDE4 inhibitory potential was not explored previously. In our effort the non-PDE4 inhibitor isocoumarins were transformed into the promising inhibitors via introducing an aminosulfonyl/aminocarboxamide moiety to the C-3 benzene ring attached to the isocoumarin framework. This new class of isocoumarins were synthesized via a PdCl₂-catalyzed construction of the 4-allyl substituted 3-aryl isocoumarin ring starting from the appropriate 2-alkynyl benzamide derivative. Several compounds showed good inhibition of PDE4B in vitro and the SAR indicated superiority of aminosulfonamide moiety over aminocarboxamide in terms of PDE4B inhibition. Two compounds 3q and 3u with PDE4B IC₅₀ = 0.43 ± 0.11 and 0.54 ± 0.19 μM and ≥ 2-fold selectivity over PDE4D emerged as initial hits. The participation of aminosulfonamide moiety in PDE4B inhibition and the reason for selectivity though moderate shown by 3q and 3u was revealed by the in silico docking studies. In view of potential usefulness of moderately selective PDE4B inhibitors the compound 3u (that showed PDE4 selectivity over other PDEs) was further evaluated in adjuvant induced arthritic rats. At an intraperitoneal dose of 30 mg/kg the compound showed a significant reduction in paw swelling (in a dose dependent manner), inflammation and pannus formation (in the knee joints) as well as pro-inflammatory gene expression/mRNA levels and increase in body weight. Moreover, besides its TNF-α inhibition and no significant toxicity in an MTT assay the compound did not show any adverse effects in a thorough toxicity studies e.g. teratogenicity, hepatotoxicity, cardiotoxicity and apoptosis in zebrafish. Thus, the isocoumarin 3u emerged as a new, safe and moderately selective PDE4B inhibitor could be useful for inflammatory diseases possibly including COVID-19.

Full text of DOI:10.1016/j.ejmech.2021.113514

European Journal of Medicinal Chemistry 221 (2021) 113514
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
PdCl2-catalyzed synthesis of a new class of isocoumarin derivatives
containing aminosulfonyl / aminocarboxamide moiety: First
identification of a isocoumarin based PDE4 inhibitor
,
, b
B. Thirupataiah a b, Guntipally Mounika a, Gangireddy Sujeevan Reddy a
,
Jetta Sandeep Kumar a b, Kazi Amirul Hossain a, Raghavender Medishetti a
,
,
, b
Snigdha Samarpita c, Mahaboobkhan Rasool c, Jayesh Mudgal b, Jessy E. Mathew b,
Gautham G. Shenoy b, C. Mallikarjuna Rao b, Kiranam Chatti a, Kishore V.L. Parsa a,
,
*
Manojit Pal a
a Dr. Reddy's Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad, 500 046, India
b Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Madhav Nagar, Manipal, 576 104, Karnataka, India
c Immunopathology Lab, School of Biosciences and Technology, Vellore Institute of Technology (VIT), Vellore, India
a r t i c l e i n f o
a b s t r a c t
Article history:
While anti-inflammatory properties of isocoumarins are known their PDE4 inhibitory potential was not
explored previously. In our effort the non-PDE4 inhibitor isocoumarins were transformed into the
promising inhibitors via introducing an aminosulfonyl/aminocarboxamide moiety to the C-3 benzene
ring attached to the isocoumarin framework. This new class of isocoumarins were synthesized via a
PdCl2-catalyzed construction of the 4-allyl substituted 3-aryl isocoumarin ring starting from the
appropriate 2-alkynyl benzamide derivative. Several compounds showed good inhibition of PDE4B
in vitro and the SAR indicated superiority of aminosulfonamide moiety over aminocarboxamide in terms
Received 19 March 2021
Received in revised form
22 April 2021
Accepted 28 April 2021
Available online 6 May 2021
Keywords:
Isocoumarin
Pd-catalyst
PDE-4
Inflammation
Zebrafish
of PDE4B inhibition. Two compounds 3q and 3u with PDE4B IC50 ¼ 0.43 0.11 and 0.54 0.19 M and ꢀ
m
2-fold selectivity over PDE4D emerged as initial hits. The participation of aminosulfonamide moiety in
PDE4B inhibition and the reason for selectivity though moderate shown by 3q and 3u was revealed by
the in silico docking studies. In view of potential usefulness of moderately selective PDE4B inhibitors the
compound 3u (that showed PDE4 selectivity over other PDEs) was further evaluated in adjuvant induced
arthritic rats. At an intraperitoneal dose of 30 mg/kg the compound showed a significant reduction in
paw swelling (in a dose dependent manner), inflammation and pannus formation (in the knee joints) as
well as pro-inflammatory gene expression/mRNA levels and increase in body weight. Moreover, besides
its TNF-a inhibition and no significant toxicity in an MTT assay the compound did not show any adverse
effects in a thorough toxicity studies e.g. teratogenicity, hepatotoxicity, cardiotoxicity and apoptosis in
zebrafish. Thus, the isocoumarin 3u emerged as a new, safe and moderately selective PDE4B inhibitor
could be useful for inflammatory diseases possibly including COVID-19.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
Fig. 1) isolated from the capitula of paepalanthus bromelioides has
shown intestinal anti-inflammatory activity in the trini-
Isocoumarins represent one of the important class of naturally
occurring aromatic lactones possessing numerous biological
properties including anti-inflammatory activities [1]. For example,
a highly substituted isocoumarin derivative i.e. paepalantine (A,
trobenzenesulphonic acid model of rat colitis [2] whereas activities
of fluorinated isocoumarins as well as 3,4-dihydroisocoumarins
against TPA (12-O-tetradecanoylphorbol-13-acetate)-induced
inflammation in mice have been documented [3]. Besides, a dual
inhibitor of 5-LOX (5-lipoxygenase) and PGE2 (prostaglandin E2)
production was identified via screening of a series of 3-aryl iso-
coumarin derivatives in HeLa cells [4]. Indeed, a naturally occurring
* Corresponding author.
E-mail address: manojitpal@rediffmail.com (M. Pal).
https://doi.org/10.1016/j.ejmech.2021.113514
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
Fig. 1. Examples of isocoumarin derivatives possessing anti-inflammatory activities.
isocoumarin thunberginol (B, Fig. 1) showed inhibition of PGE2
production in the same study. Recently, a natural isocoumarin GDC
[3R-(40-hydroxyl-30-O-
b-D-glucopyranosyl phenyl)-dihydro iso-
coumarin] (C, Fig. 1) has shown remarkable anti-inflammatory ac-
tivities including the inhibition of production of tumor necrosis
factor alpha (TNF-a) and interleukin-6 (IL-6) induced by LPS [5].
Interestingly, exploration of isocoumarin derivatives as inhibitor of
PDE4 (phosphodiesterase 4) is rather uncommon though inhibition
of calmodulin-sensitive cyclic guanosine 30,50-monophosphate
phosphodiesterase by three natural isocoumarins (D, Fig. 1) was
studied way back in 1989 [6]. Notably, the inhibitors of PDE4 are not
only known to be useful for the treatment of inflammatory diseases
[7e9] like COPD/asthma, psoriasis, psoriatic arthritis etc but also
have been suggested to attenuate the cytokine storm in COVID-19
especially TNF-a [10e12]. The currently available inhibitors such
as roflumilast and apremilast though found to be potent and
effective but their emetic and other side effects emphasize the
necessity for the discovery and development of alternative agents.
In our effort [13,14] for the identification of new inhibitors of PDE4
we explored benzo[c]phenanthridine derivatives that were syn-
thesized from the isocoumarin-based key intermediates (E, Fig. 2)
[15]. Prompted by the reported usefulness of isocoumarins as anti-
inflammatory agents we examined PDE4 inhibitory potential of E
when none of these derivatives showed significant inhibition of
PDE4 in vitro. However, introduction of an aminosulfonyl moiety to
the benzene ring at C-3 position enhanced the inhibitory properties
of the resultant analogue (F, Fig. 2) remarkably in some cases.
Indeed, the role of aminosulfonyl moiety in PDE4B inhibition was
further supported by the docking study of a representative mole-
cule F-1 (Fig. 3, see also Fig. S-6 in suppl info) where one of the
oxygen atom of the eNHSO2-group formed a H-bond with the
HIS450 residue. Overall, with a high binding affinity (ꢁ11.9 com-
parable to rolipram's ꢁ10.4) F-1 interacted well with the PDE4B in
silico (ID: 4MYQ) via (i) two H-bonds with HIS450 and TYR405, (ii)
Fig. 3. 2D and 3D interaction diagram between PDE4B (ID: 4MYQ) and F-1 (or 3q,
Table 1) prepared by using Maestro visualizer (Schrodinger, LLC) (the H-bond and pi-pi
stacking are shown in magenta and green colour in 2D diagram, and yellow and cyan
dashed lines in 3D diagram).
one aromatic pi interaction with PHE586 and (iii) a number of van
der Waals/hydrophobic contacts with hydrophobic residues like
TYR575, PHE618 etc. and hydrophobic regions of polar or charged
Scheme 1. Synthesis of isocoumarin derivatives containing aminosulfonyl/amino-
Fig. 2. Exploring isocoumarin derivatives as PDE-4 inhibitor.
carboxamide moiety.
2

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
Table 1
List of isocoumarin derivatives (3) synthesized and their in vitro evaluation against PDE4B.a,b
3c (84%)
3b (80%)
PDE4B: 54.85%
3a (82%)
PDE4B: 55.80%
PDE4B: 82.89% (IC50 ¼ 1.24 0.76)
3f (82%)
PDE4B: 63.49%
3e (86%)
3d (81%)
PDE4B: 77.83% (IC50 ¼ 2.07 1.16)
PDE4B: 87.57% (IC50 ¼ 3.91 0.31)
3h (80%)
3g (85%)
3i (78%)
PDE4B: 55.34%
PDE4B: 80.02% (IC50 ¼ 3.33 0.25)
PDE4B: 85.89% (IC50 ¼ 1.76 0.35)
3j (82%)
PDE4B: 72.58%
3k (79%)
PDE4B: 46.45%
3l (83%)
PDE4B: 62.25%
3n (84%)
3m (80%)
PDE4B: 55.97%
3o (82%)
PDE4B: 81.21% (IC50 ¼ 4.78 0.01)
PDE4B: 75.65% (IC50 ¼ 4.01 0.73)
3p (85%)
3r (79%)
3q (86%)
PDE4B: 90.86% (IC50 ¼ 1.94 0.67)
PDE4B: 94.39% (IC50 ¼ 1.91 0.50)
PDE4B: 82.25% (IC50 ¼ 0.43 0.11)
3s (83%)
3t (80%)
3u (87%)
PDE4B: 96.02 (IC50 ¼ 0.54 0.19)
PDE4B: 79.68% (IC50 ¼ 3.04 1.17)
PDE4B: 93.96 (IC50 ¼ 2.36 0.17)
3v (80%)
PDE4B: 74.63%
a
Figure in the bracket indicates isolated % yield.
Other figures present % inhibition of PDE4B at 10
b
m
M and IC50
(
m
M). Rolipram (PDE4B: 77.22%; IC50 ¼ 0.94 0.2) was used as a reference compound.
3

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
Fig. 4. Partial representation of 1H as well as 13C NMR and IR spectral data of com-
pound 3u.
residues like; SER454, ASN455, GLN456, THR579, HIS406, ASN567,
GLN615 etc. Notably, in addition to participating in the hydrophobic
interactions with PDE4B the allyl group enhances lipophilicity that
may cause a rapid onset. Herein we present a detailed account of
this study. We also examined the PDE4 inhibitory potential of the
corresponding aminocarboxamide analogues designed via replac-
ing the eNHSO2R2 of F by eNHCOR2. While PDE4 inhibition of
compounds containing sulphonamide moiety has been studied
earlier [16,17] the synthesis and PDE4 evaluation of isocoumarin
derivatives containing aminosulfonyl/aminocarboxamide moiety
was not explored previously.
2. Results and discussion
2.1. Chemistry
The targeted new class of isocoumarin derivatives were syn-
thesized via a Pd-catalyzed method similar to that developed by us
recently (Scheme 1) [15]. This interesting approach involved the
construction of a 4-allyl substituted 3-aryl isocoumarin ring start-
ing from the appropriate 2-alkynyl benzamide derivative. Thus the
commercially available terminal alkynes containing an aniline
moiety were converted to the corresponding sulfonamides or am-
ides (1a-k) (step-1 of Scheme 1) that on Sonogashira coupling with
2-iodobenzamides afforded a range of 2-alkynyl benzamides (2a-v)
in good yields (step 2 of Scheme 1, see also Tables Se3 in Supp info).
Treating the compound 2 with allyl bromide in the presence of
PdCl2 in aqueous DMF (10% H2O in DMF) afforded the desired iso-
coumarins (3a-v) (step 3 of Scheme 1 and Table 1). Notably in
compared to Pd(OAc)2 used in our earlier approach [15] the rela-
tively cheaper PdCl2 was found to be more effective in the current
study as the reaction was completed within 1e1.5 h (for optimi-
zation study see Tables Se5 in suppl info). Nevertheless, the
starting 2-alkynyl benzamides (2a-v) may contain various NHX
group (when X ¼ SO2R2 or COR2) and the reaction proceeded
smoothly via CeO/CeC bond formation to give the desired products
with desired regioselectivity. Indeed, the formation of no isomeric
5-membered ring product i.e. the corresponding phthalide as a side
product was observed during the reaction. All the isocoumarins
synthesized were characterized by spectral (NMR, MS) data. The
allylic protons could be detected for most of the compounds in their
Fig. 5. Concentration dependent study of (A) compound 3q and (B) compound 3u
against PDE4B and PDE4D.
fluorine atom appeared near
d 10.6 (broad singlet) and 162 ppm in
the 1H and 13C NMR spectra, respectively. Mechanistically [15], the
reaction involved a PdCl2 catalyzed 6-endo-dig ring closure in the
presence of allyl bromide to give the isochromen-1-imine species
that afforded 3 in acidic media (see Supp info).
1HNMR spectra generally as (i) a doublet near
in the range 6.0-5.9 and (iii) two double doublets near
d
3.3, (ii) a multiplet
5.1 and
2.2. Biology
d
d
5.0 due to the CH2, CH and ¼ CH2 moiety, respectively. The presence
of lactone C]O group was indicated by the IR absorptions near
1720 cmꢁ1 and the 13CNMR signal near 160 ppm, respectively. The
partial 1H and 13C NMR data along with IR absorptions of a repre-
sentative compound 3u is shown in Fig. 4. The NH and C-7 bearing a
Since PDE4B (one of the four major subtypes of PDE4 e.g. PDE4A,
B, C and D) is known to be responsible for mediating LPS-induced
inflammation [7e9] hence the synthesized isocoumarin de-
rivatives were evaluated in vitro initially at 10
mM for their PDE4B
inhibitory properties using an enzyme based assay (Table 1) [18].
4

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
nature of the aminosulfonyl or aminocarboxamide substituent i.e.
NHX attached to the benzene ring at C-3 position of the iso-
coumarin ring appeared to play a key role in the PDE4 inhibitory
activities (Fig. 6). In general, (i) an aminosulfonyl group was more
favorable than the aminocarboxamide moiety, (ii) the NHX sub-
stituent preferred the m-position over the p-position of the C-3
benzene ring, (iii) the NHX group was preferred when X ¼ aryl or
heteroarylsulfonyl group rather than methylsulfonyl moiety (with
the exception of compound 3p), and (iv) a fluorine at the C-7 po-
sition of the isocoumarin ring was beneficial though a chlorine at C-
6 position was less effective. Notably, the presence of NHX sub-
stituent at the o-position of the C-3 benzene ring was not assessed
because this position might not be optimum for the NHX substit-
uent to interact effectively with the target protein (due to the po-
tential steric crowding or hindrance caused by the adjacent and
proximate group or ring present within the molecule). Moreover,
substituents e.g. Cl or F at C-6/C-7 but not at C-5/C-8 position of the
isocoumarin ring were explored to block the potential metabolism
if any under in vivo conditions.
Fig. 6. Summary of SAR for PDE4B inhibitory activities of isocoumarins (3).
Rolipram, a well-known inhibitor of PDE4 was used as a reference
compound. While most of the compounds showed mediocre to
good activities the analogues that showed >75% inhibition i.e. 3c,
3d, 3e, 3g, 3h, 3n, 3o, 3p, 3q, 3r, 3s, 3t and 3u were identified as
initial hits and taken for concentration dependent studies. The IC50
values of these compounds are presented in Table 1 and the con-
centration response curves of the most active compounds 3q and
3u are shown in Fig. 5. Notably, while some compounds e.g. 3d, 3r,
While significant inhibition of PDE4D was shown by both the
compound 3q (IC50
(IC50 ¼ 1.34 0.09
¼
0.90
0.06
mM) and 3u
m
M) (Fig. 5) the compound 3u showed
marginally better selectivity i.e. ~2.5 fold over 3q possessing ~2 fold
selectivity. Notably, while the inhibition of PDE4D was thought to
be responsible for undesired side effects especially emesis later it
was demonstrated that inhibition of PDE4D by allosteric inhibitors
(Maximum inhibition, Imax 80e90%) did not cause emetic side ef-
fects [19] raising an intriguing possibility that PDE4B inhibitors
with partial but not complete inhibition of PDE4D (Imax of
~60e80%) could be developed to treat inflammatory diseases
3t etc showed good inhibition initially at 10
to be higher due to their inferior inhibition at low concentrations
particularly below 1 M. Though a precise Structure-Activity-
mM their IC50 was found
m
Relationship (SAR) within the current series of isocoumarin de-
rivatives was not clear it was evident that the position as well as
Fig. 7. (A) The 2D (H-bond in magenta and pi-pi in green colour) and 3D interaction diagram (H-bond in yellow and pi-pi stacking in cyan dashed lines) between PDE4B (ID: 4MYQ)
and compound 3u; (B) 2D and 3D interaction diagram between PDE4D (ID: 5K32) and 3u.
5

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
Fig. 8. Effect of compound 3u on (A) paw swelling and (B) body weight changes in adjuvant induced arthritic (AIA rats). All error bars represent mean SEM. ***P < 0.001 vs
control. #P < 0.05, ##P < 0.01, ###P < 0.001 vs AIA rats. (C) Macroscopic images depicting the severity of paw swelling in experimental animals on day 21.
Fig. 9. Representative radiographic images of rat joints in experimental animals on day 21 (red arrow shows joint deformity).
Fig. 10. Radiographic scoring graded on a semi-quantitative four-point scale of 0e4: grade 0: (absent); grade 1: (weak); grade 2: (moderate); grade 3: (high); grade 4: (very high).
Radiographic scores were confirmed by independent observers on the basis of joint structural changes such as joint space, and joint deformity and averaged.
without causing emetic side effects. Thus these moderately selec-
tive isocoumarins especially 3u seemed to be of further interest. To
understand its interaction with PDE4B as well as PDE4D in silico the
compound 3u was docked into both the proteins (Fig. 7). The
molecule participated in two H-bond interactions with HIS450 and
TYR405, and one aromatic pi interaction with PHE586. Additionally,
3u was also involved in a number of non-bonded contacts (e.g.
hydrophobic/van der Waals) with hydrophobic residues ILE582,
TYR575, LEU674, MET519, PRO568, PHE678 and hydrophobic re-
gions of polar or charged residues such as ASN567, SER406 and
THR579. On the other hand, 3u showed lower number of in-
teractions with PDE4D indicating its moderate selectivity towards
6

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
Fig. 11. Experimental rat knee joint sections were H & E stained for examining histological changes like joint space, cellular infiltration and pannus formation. Black arrow: pannus
formation; yellow box: cartilage degradation (magnification: 100ꢂ).
Fig. 12. Representative H & E staining scoring graded on a semi-quantitative four-point scale in a blinded manner; grade 0: (absent); grade 1: (weak); grade 2: (moderate); grade 3:
(high); grade 4: (very high).
Fig. 13. Immunohistochemistry staining analysis was performed to examine for the expression of TNF-
a
and IL-6. (magnification: 100ꢂ).
PDE4B over D. A similar trend was observed in case of compound
3q (or compound F-1) also when the molecule was docked into
PDE4B (Fig. 3) as well as PDE4D (see Fig S-7, suppl info).
In a separate study the PDE4 selectivity of compound 3u over
other families of PDEs (e.g. PDE1-PDE11) was assessed using an
Next, the potential for anti-inflammatory effects of compound
3u was evaluated when the compound showed significant inhibi-
tion of TNF-
10, 3,1 and 0.3
inhibition at 0.1
a
i.e. 65.5 5.1, 45.6 6.4, 40.3 4.8 and 30.9 8.3% at
M, respectively (when rolipram showed 50.3 1.2%
M). Subsequently, the therapeutic efficacy of
m
m
in vitro enzymatic assay [14]. At 10
m
M the % inhibition shown by 3u
compound 3u was evaluated at an intraperitoneal dose of 10 and
30 mg/kg in an adjuvant induced arthritic (AIA) rat model using
methotrexate (1 mg/kg) as a positive control [20]. The progression
of disease severity was monitored periodically by assessing
changes in body weight and paw swelling post complete Freund's
adjuvant injection. As shown in Fig. 8, substantial increase in paw
was either low or poor, e.g. 21% (PDE1A1), 18% (PDE1B), 24%
(PDE1C), 9% (PDE2A1), 11% (PDE3A), 7% (PDE3B), 8% (PDE5A1), 10%
(PDE6C), 6% (PDE7A1), 3% (PDE7B), 2% (PDE8A1), 8% (PDE9A2), 12%
(PDE10A1), 19% (PDE10A2) and 11% (PDE11A4) indicating its PDE4
selectivity over other PDEs.
7

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
Fig. 14. Effect of 3u on the expression of pro-inflammatory molecules. (A) The expression level of TNF-
a
and IL-6 are examined in the serum of experimental animals. (B) The
expression level of TNF-
a and IL-6 are examined at the mRNA level in experimental animals. All error bars represent mean SEM. ***P < 0.001 vs control. #P < 0.05, ##P < 0.01,
###P < 0.001 vs AIA rats.
swelling and body weight loss was observed in AIA rats as
compared to control rats. However, 3u was found to be effective in
reducing paw swelling in a dose dependent manner. Indeed, at
30 mg/kg the compound 3u significantly reduced paw volume of
AIA rats similar to that of 1 mg/kg of methotrexate (Fig. 8A).
Similarly, 3u (30 mg/kg) improved intestinal absorption of nutri-
ents as observed from significant increase in body weight in AIA
rats (Fig. 8B).
Histopathological examination of synovial tissues displayed
joint space narrowing, abundant cellular infiltration and pannus
formation in AIA rats. In addition, radiographic assessment [21]
showed increased joint deformity in AIA rats (Figs. 9e13). While
the methotrexate treatment was not so effective in reducing
changes in joint structure deformity (perhaps due to its significant
role in inflammation inhibition rather than controlling bone dam-
age) the treatment with 3u (30 mg/kg) decreased pathological
changes and significantly reduced joint degenerative changes
(Figs. 9e13). Furthermore, the expression of TNF-
evaluated in the synovial tissue and serum of experimental ani-
mals. As shown in Fig. 14, an increased expression level of TNF-
and IL-6 was observed in AIA rats. However, a significant decrease
in the expression level of TNF- was observed in 3u and metho-
a and IL-6 were
a
a
trexate treated AIA rats as compared to AIA rats. Interestingly, not
much significant changes were observed in the expression level of
IL-6 upon treatment with 3u (Fig. 14). Notably, being considered as
a
first line disease-modifying anti-rheumatic drug (DMARD)
methotrexate is the first choice of clinicians for the treatment of
rheumatoid arthritis (RA) though the drug has been shown to
possess adverse effects on bone metabolism in RA patients [22].
Overall, based on the strong evidences of the anti-arthritic potential
of 3u the molecule appeared to be a promising hit and may be
investigated further for its preclinical development.
In order to gain an initial assessment about potential toxicity the
compound 3u was tested against Raw 264.7 cells via an MTT assay
[23] when the compound did not show any significant toxicity
(IC50 > 100 mM). However, due to the presence of an allyl moiety as
well 5-unsubstituted thiophene ring it was necessary to evaluate
3u for potential toxicities in vivo. It is known that the allyl group not
only enhance the lipophilicity but also could be the reason for
potential metabolic instability to generate toxic metabolites in vivo.
Being a toxicophore the 5-unsubstituted thiophene on the other
hand is known to undergo oxidation affording hepatotoxic me-
tabolites. Hence the compound was further evaluated for its
probable toxicity specifically its potential ability to induce
Fig. 15. Compound 3u did not induce teratogenicity in Zebrafish embryos. Top panel:
Teratogenicity scores of compound 3u and positive control phenobarbitol. Bottom
panel: Representative images of teratogenicity assay. Data are represented as
mean þSEM. Statistical analysis was performed by graphpad prism software using
two-way ANOVA post-hoc test. *, p < 0.05 was considered as significant.
8

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
Fig. 16. Compound 3u did not induce hepatotoxicity in Zebrafish embryos. Top panel: the graph represents the qualitative data of % liver size and % liver degeneration in compound
3u treated embryos at different concentrations when compared to positive control amiodarone. Bottom panel: Representative images of hepatotoxicity assay. Data are represented
as mean þSEM. Statistical analysis was performed by graphpad prism software using two-way ANOVA post-hoc test. ***, p < 0.001 was considered as significant.
teratogenicity, hepatotoxicity, cardiotoxicity and apoptosis in
zebrafish. The well-known agents such as methotrexate, pheno-
barbitol, amiodarone, terfenadine and atrazine were used individ-
ually as a positive control in these assays respectively. Across the
ADME (absorption, distribution, metabolism, and excretion) or
pharmacokinetic properties of compound 3u in silico. The compu-
tational ADME prediction was carried out using Swiss ADME web-
tool [24] and results are summarized in Table 2 (among the various
descriptors only notable one are listed in the table). While the low
GI absorption was predicted for 3u an acceptable bioavailability
(score 0.55) and no P-gp substrate potential was also predicted for
range of concentration tested e.g. 1, 3, 10, 30 and 100
mM the
compound 3u was found to be safe in all these toxicity assays
(Figs. 15e18). Finally, we conducted some initial assessment about
9

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
Fig. 17. Compound 3u did not induce cardiac toxicity in Zebrafish embryos. The above graph represents the heart rate of compound 3u treated embryos at different concentrations
when compared to positive control terfenadine. Data are represented as mean SEM. Statistical analysis was performed by graphpad prism software using two-way ANOVA post-
hoc test. ***, p < 0.001 was considered as significant.
this molecule. Moreover, no BBB permeation predicted for this
molecule indicated its less likely interaction with the emetic center
in the brain thereby avoiding the possible emetic side effects.
Further, the compound 3u did not show the violation of Lipinski or
Veber rule. Overall, based on the outcome of all in vitro and in vivo
studies the isocoumarin 3u has been identified as a potential
candidate that may be taken forward for further in vivo studies.
no significant toxicity in an MTT assay the compound did not show
any adverse effects in a thorough toxicity (e.g. teratogenicity,
hepatotoxicity, cardiotoxicity and apoptosis) studies in zebrafish.
Overall, based on the outcome of all in vitro and in vivo studies the
isocoumarin 3u was identified as a new, safe and moderately se-
lective inhibitor of PDE4B that can progress further into the pre-
clinical development. Moreover, in view of the potential utility of
PDE-4/TNF-a inhibitors against COVID-19 in addition to their well-
known effectiveness against inflammatory diseases the current
efforts on PdCl2-catalyzed synthesis of a new class of isocoumarins
containing aminosulfonyl/aminocarboxamide moiety towards the
identification of PDE4 inhibitors may attract considerable interest.
3. Conclusion
In view of anti-inflammatory activities of isocoumarin de-
rivatives a new class of PDE4B inhibitors was designed via intro-
ducing an aminosulfonamide/aminocarboxamide moiety into the
C-3 benzene ring attached to an isocoumarin framework followed
by docking of a representative compound into the PDE4B in silico.
These compounds were synthesized via a PdCl2-catalyzed method
involving the construction of a 4-allyl substituted 3-aryl iso-
coumarin ring starting from the appropriate 2-alkynyl benzamide
derivative. The current method has some advantages over the
previously reported Pd(OAc)2 catalyzed method because PdCl2 was
cheaper and decreased the reaction time. A variety of isocoumarins
were prepared in good yields and several of them showed good
inhibition when evaluated initially against PDE4B in vitro. The SAR
within the series indicated superiority of aminosulfonamide over
aminocarboxamide moiety in terms of PDE4B inhibition. Two
4. Experimental section
4.1. General procedure for the preparation of compound 1
Preparation of compound 1a-i: Appropriate Sulfonyl chloride
(1.2 mmol) was added to a solution containing 3-ethynylaniline or
4-ethynylaniline (1.0 mmol), and pyridine (3.0 mmol) in DCM
(10 mL) under a nitrogen atmosphere at 0 ꢃC for 10 min. The
resulting mixture was stirred at room temperature for overnight.
After completion of the reaction (indicated by TLC) DCM was
evaporated. The residue was treated with 2 N aqueous HCl (10 mL)
and the mixture was diluted with ice-water (60 mL) and extracted
with ethyl acetate (3 x 15 mL). The organic layers were collected,
combined, dried over anhydrous Na2SO4, filtered and concentrated
under low vacuum. The residue was purified by column chroma-
tography using hexane and EtOAc (9:1) as eluent to afford the title
compound.
compounds 3q and 3u with PDE4B IC50 ¼ 0.43
0.11 and
0.54 0.19 and ꢀ 2-fold selectivity over PDE4D emerged as initial
hits. The participation of aminosulfonamide moiety in PDE4B in-
hibition and the reason for selectivity though moderate shown by
3q and 3u was analyzed by the in silico docking studies. In view of
potential usefulness of moderately selective PDE4B inhibitors the
compound 3u (that showed PDE4 selectivity over other PDEs) was
further evaluated in adjuvant induced arthritic rats. At an intra-
peritoneal dose of 30 mg/kg the compound showed a significant
reduction in paw swelling (in a dose dependent manner), inflam-
mation and pannus formation (in the knee joints) as well as pro-
inflammatory gene expression/mRNA levels and increase in body
weight along with a drastic reduction in joint degenerative
4.1.1. Preparation of compound 1j-k
Appropriate benzoyl chloride (1.2 mmol) was added to a solu-
tion containing 3-ethynylaniline [or 4-ethynylaniline (1.0 mmol)],
and triethylamine (3.0 mmol) in DCM (10 mL) under a nitrogen
atmosphere at 0 ꢃC for a duration of 10 min. The resulting mixture
was stirred at room temperature for overnight. After completion of
the reaction (indicated by TLC) DCM was evaporated. The mixture
was diluted with cold water (60 mL) and extracted with ethyl
changes. Moreover, besides its inhibitory activity against TNF-a and
10

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
Table 2
Computational ADME prediction of 3u.
Properties
Molecule 3u
(i) Physicochemical
Molecular Weight (g/mol)
Consensus Log Pa
Log S (ESOL)b
441.50
4.74
ꢁ5.72 (moderately soluble)
(ii) Pharmacokinetics
GIc absorption
Low
No
No
P-gpd substrate
BBBe permeation
(iii) Druglikenss
Lipinski rule
No violation
No violation
0.55
Veber rule
Bioavailability score
a
Log P: Lipophilicity.
b
Log S (ESOL): water solubility, calculated by ESOL method which is a Quanti-
tative Structure-Property Relationship (QSPR) based model.
c
GI: Gastrointestinal.
P-gp: permeability glycoprotein.
BBB: Blood Brain Barrier.
d
e
(1.0 mmol)], (PPh3)2PdCl2 (5 mol%), CuI (5 mol%) and N,N-Diiso-
propylethylamine (3.0 mmol) in DMF (10 mL) under a nitrogen
atmosphere. The mixture was stirred at 70 ꢃC for 1e2 h. After
completion of the reaction (confirmed by TLC), the mixture was
diluted with cold water (60 mL) and extracted with ethyl acetate (3
x 15 mL). The organic layers were collected, combined, dried over
anhydrous Na2SO4, filtered, and concentrated under low vacuum.
The residue was purified by column chromatography using hexane
and EtOAc (7:3) as eluent to afford the title compound.
4.3. General procedure for the preparation of compound 3
To a solution of compound 2 (1.0 mmol) and PdCl2 (5 mol%) in
9:1 DMF: H2O (5 mL) was added allyl bromide (1.5 mmol) under a
nitrogen atmosphere. The mixture was stirred at 50 ꢃC for 1e1.5 h.
After completion of the reaction (indicated by TLC), the mixture
was diluted with ice-water (60 mL) and extracted with ethyl ace-
tate (3 x 15 mL). The organic layers were collected, combined, dried
over anhydrous Na2SO4, filtered, and concentrated under low vac-
uum. The residue was purified by column chromatography using
9:1 hexane-EtOAc as eluent to afford the title compound 3.
Physical and spectroscopic characterization data of all the syn-
thesized compounds are given in the supplementary data.
Declaration of competing interest
Fig. 18. Compound 3u did not induce apoptosis in Zebrafish embryos. Top panel: the
graph represents the apoptosis intensity (mean grey value intensity) of compound 3u
treated embryos at different concentrations and compared to the positive control
atrazine. Bottom panel: Representative images of apoptosis induction assay. Data are
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
represented as mean
SEM. Statistical analysis was performed by graphpad prism
software using two-way ANOVA post-hoc test. ***, p < 0.001 was considered as
significant.
Acknowledgements
Authors acknowledge the financial support received from
Department of Biotechnology, New Delhi, India (Grant No. BT/
PR22126/BRB/10/1585/2017). Authors thank the Management of
DRILS, Hyderabad, India, and Manipal University, Manipal, India for
encouragement and support.
acetate (3 x 15 mL). The organic layers were collected, combined,
dried over anhydrous Na2SO4, filtered and concentrated under low
vacuum. The residue was purified by column chromatography us-
ing 9:1 hexane-EtOAc as eluent to afford the title compound.
4.2. General procedure for the preparation of compound 2
Appendix A. Supplementary data
Aryl acetylenes (1) (1.2 mmol) was added to a solution of 2-iodo-
N-methylbenzamide [or substituted 2-iodo-N-methylbenzamide
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113514.
11

B. Thirupataiah, G. Mounika, G.S. Reddy et al.
European Journal of Medicinal Chemistry 221 (2021) 113514
Biomol. Chem. 11 (2013) 2075e2079.
[14] R. Sunke, R. Bankala, B. Thirupataiah, E.V.V.S. Ramarao, J.S. Kumar, H.M. Doss,
R. Medishetti, P. Kulkarni, R.K. Kapavarapu, M. Rasool, J. Mudgal, J.E. Mathew,
G.G. Shenoy, K.V.L. Parsa, M. Pal, InCl3 mediated heteroarylation of indoles and
their derivatization via C-H activation strategy: discovery of 2-(1Hindol-3-yl)-
References
[1] S. Pal, M. Pal, Isocoumarin, Thiaisocoumarin and Phosphaisocoumarin: Natural
Occurrences, Synthetic Approaches and Pharmaceutical Applications, Elsevier,
Paperback, 2018, ISBN 9780128154113.
[2] L.C. Di Stasi, D. Camuesco, A. Nieto, W. Vilegas, A. Zarzuelo, J. Galvez, Intestinal
anti-inflammatory activity of paepalantine, an isocoumarin isolated from the
capitula of Paepalanthus bromelioides, in the trinitrobenzenesulphonic acid
model of rat colitis, Planta Med. 70 (2004) 315e320.
[3] G. Qadeer, N.H. Rama, M. Gardu~no-Ramírez, Synthesis and anti-inflammatory
activity of fluorinated isocoumarins and 3, 4-dihydroisocoumarins, J. Fluor.
Chem. 128 (2007) 641e646.
[4] M. Ramanan, S. Sinha, K. Sudarshan, I.S. Aidhen, M. Doble, Inhibition of the
enzymes in the leukotriene and prostaglandin pathways in inflammation by
3-aryl isocoumarins, Eur. J. Med. Chem. 124 (2016) 428e434.
[5] Y. Zhang, G. Yan, C. Sun, L. Nan, X. Wang, W. Xu, K. Chu, Compound GDC, an
isocoumarin glycoside, protects against LPS-induced inflammation and po-
tential mechanisms in vitro, Inflammation 42 (2019) 506e515, https://doi.org/
10.1007/s10753-018-0908-2.
[6] V.R. Hegde, H. Wittreich, M.G. Pate, A.C. Horan, R.F. Hart, J.J. Troyanovich,
M.S. Puar, V.P. Gullo, Naturally produced isocoumarins: inhibitors of calm-
odulinsensitive cyclic guanosine 3',5'-monophosphate phosphodiesterase,
J. Ind. Microbiol. 4 (1989) 209e214.
quinoxaline derivatives as
a new class of PDE4B selective inhibitors for
arthritis and/or multiple sclerosis, Eur. J. Med. Chem. 174 (2019) 198e215.
[15] B. Thirupataiaha, G.S. Reddy, S.S. Ghule, J.S. Kumar, G. Mounika, K.A. Hossain,
J. Mudgal, J.E. Mathew, G.G. Shenoy, K.V.L. Parsaa, M. Pal, Synthesis of 11,12-
dihydro benzo[c]phenanthridines via a Pd-catalyzed unusual construction of
isocoumarin ring/FeCl3-mediated intramolecular arene-allyl cyclization: first
identification of
a benzo[c]phenanthridine based PDE4 inhibitor, Bioorg.
Chem. 97 (2020), 103691.
[16] S.V. Reddy, G.M. Rao, B.V. Kumar, C.L.T. Meda, G.S. Deora, K.S. Kumar,
K.V.L. Parsa, M. Pal, Novel imidazophenoxazine-4-sulfonamides: their syn-
thesis and evaluation as potential inhibitors of PDE4, Bioorg. Med. Chem. 21
(2013) 1952e1963, https://doi.org/10.1016/j.bmc.2013.01.023.
[17] J.G. Montana, G.M. Buckley, N. Cooper, H.J. Dyke, L. Gowers, J.P. Gregory,
P.G. Hellewell, H.J. Kendall, C. Lowe, R. Maxey, J. Miotla, R.J. Naylor,
K.A. Runcie, B. Tuladhar, J.B. Warneck, Aryl sulfonamides as selective PDE4
inhibitors, Bioorg. Med. Chem. Lett 8 (1998) 2635e2640, https://doi.org/
10.1016/s0960-894x(98)00491-0.
[18] P. Wang, J.G. Myers, P. Wu, B. Cheewatrakoolpong, R.W. Egan, M.M. Billah,
Expression, purification, and characterization of human cAMP-specific phos-
phodiesterase (PDE4) sub-types A, B, C, and D, Biochem. Biophys. Res. Com-
mun. 234 (1997) 320e324.
[19] A.B. Burgin, O.T. Magnusson, J. Singh, P. Witte, B.L. Staker, J.M. Bjornsson,
M. Thorsteinsdottir, S. Hrafnsdottir, T. Hagen, A.S. Kiselyov, L.J. Stewart,
M.E. Gurney, Design of phosphodiesterase 4D (PDE4D) allosteric modulators
for enhancing cognition with improved safety, Nat. Biotechnol. 28 (2010)
63e70, https://doi.org/10.1038/nbt.1598.
[20] A. Koyama, A. Tanaka, H. To, Daily oral administration of low-dose metho-
trexate has greater antirheumatic effects in collagen-induced arthritis rats,
J. Pharm. Pharmacol. 69 (2017) 1145e1154.
[7] H. Li, J. Zuo, W. Tang, Phosphodiesterase-4 inhibitors for the treatment of
inflammatory diseases, Front. Pharmacol.
10.3389/fphar.2018.01048.
9 (2018) 1048, https://doi.org/
[8] C.H. Lin, S.H. Chang, J.Y. Fang, Recent advances using phosphodiesterase 4
(PDE4) inhibitors to treat inflammatory disorders: animal and clinical studies,
Curr. Drug Ther. 11 (2016) 21e40.
[9] A. Kodimuthali, S.S.L. Jabaris, M. Pal, Recent advances of phosphodiesterase 4
inhibitors for the treatment of asthma and chronic obstructive pulmonary
disease, J. Med. Chem. 18 (2008) 5471e5489.
[10] C. Bridgewood, G. Damiani, K. Sharif, A. Watad, N.L. Bragazzi, L. Quartuccio,
S. Savic, D. McGonagle, Rationale for evaluating PDE4 inhibition for mitigating
against severe inflammation in COVID-19 pneumonia and beyond, Isr. Med.
Assoc. J. 22 (2020) 335e339.
[11] M. Dalamaga, I. Karampela, C.S. Mantzoros, Commentary: phosphodiesterase
4 inhibitors as potential adjunct treatment targeting the cytokine storm in
COVID-19, Metab. Clin. Exp. 109 (2020), 154282.
[12] R. Chemboli, R. Kapavarapu, K. Deepti, K.R.S. Prasad, A.G. Reddy,
A.V.D.N. Kumar, M.V.B. Rao, M. Pal, Pyrrolo[2,3 -b ]quinoxalines in attenuating
cytokine storm in COVID-19: their sonochemical synthesis and in silico/in vitro
assessment, J. Mol. Struct. 1230 (2021), 129868.
[13] D.R. Gorja, S. Mukherjee, C.L.T. Meda, G.S. Deora, K.L. Kumar, A. Jain,
G.H. Chaudhari, K.S. Chennubhotla, R.K. Banote, P. Kulkarni, K.V.L. Parsa,
K. Mukkanti, M. Pal, Novel N-indolylmethyl substituted olanzapine de-
rivatives: their design, synthesis and evaluation as PDE4B inhibitors, Org.
[21] R. Ganesan, H.M. Doss, M. Rasool, Majoon ushba, a polyherbal compound
ameliorates rheumatoid arthritis via regulating inflammatory and bone
remodeling markers in rats, Cytokine 77 (2016) 115e126.
ꢀ
[22] J. Swierkot, K. Gruszecka, A. Matuszewska, P. Wiland, Assessment of the effect
of methotrexate therapy on bone metabolism in patients with rheumatoid
arthritis, Arch. Immunol. Ther. Exp. 63 (2015) 397e404.
[23] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays, J. Immunol. 65 (1983)
55e63.
[24] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of
small molecules, Sci. Rep. 7 (2017) 1e13.
12