Piperidinyl-embeded chalcones possessing anti PI3Kδ inhibitory properties exhibit anti-atopic properties in preclinical models

Article abstract of DOI:10.1016/j.ejmech.2018.09.033

Phosphatidylinositide 3-kinases (PI3Ks) are widely expressed enzymes involved in membrane signalization pathways. Attempts to administer inhibitors with broad activity against different isoforms have failed due to toxicity. Conversely the PI3Kδ isoform is

Full text of DOI:10.1016/j.ejmech.2018.09.033

Accepted Manuscript
Piperidinyl-embeded chalcones possessing anti PI3Kδ inhibitory properties exhibit
anti-atopic properties in preclinical models
Charles Dumontet, Guillaume Beck, Fabrice Gardebien, Romain Haudecoeur,
Doriane Mathé, Eva-Laure Matera, Anne Tourette, Eve Mattei, Justine Esmenjaud,
Cédric Boyere, Alessandra Nurisso, Marine Peuchmaur, Basile Peres, Grégory
Bouchaud, Antoine Magnan, Guillaume Monneret, Ahcène Boumendjel
PII:
S0223-5234(18)30805-5
10.1016/j.ejmech.2018.09.033
EJMECH 10744
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 May 2018
Revised Date: 9 September 2018
Accepted Date: 11 September 2018
Please cite this article as: C. Dumontet, G. Beck, F. Gardebien, R. Haudecoeur, D. Mathé, E.-L. Matera,
A. Tourette, E. Mattei, J. Esmenjaud, Cé. Boyere, A. Nurisso, M. Peuchmaur, B. Peres, Gré. Bouchaud,
A. Magnan, G. Monneret, Ahcè. Boumendjel, Piperidinyl-embeded chalcones possessing anti PI3Kδ
inhibitory properties exhibit anti-atopic properties in preclinical models, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.09.033.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Piperidinyl-Embeded Chalcones Possessing anti PI3Kδ Inhibitory
Properties Exhibit Anti-atopic Properties in Preclinical Models
Charles Dumontet,a,b Guillaume Beck, Fabrice Gardebien, Romain Haudecoeur, Doriane
c
c
d
a
a
a
Mathé,a Eva-Laure Matera,a Anne Tourette, Eve Mattei, Justine Esmenjaud, Cédric Boyere,
d
e
,
d
d
Alessandra Nurisso, Marine Peuchmaur, Basile Peres, Grégory Bouchaud,f g Antoine
Magnan,f,g Guillaume Monneret,b and Ahcène Boumendjeld,*
aINSERM 1052/CNRS 5286/University of Lyon – Cancer Research Center of Lyon
bHospices Civils de Lyon
c
Biologie Intégrée du Globule Rouge UMR_S1134, Inserm, Univ. Paris Diderot, Sorbonne
Paris Cité, Univ. de la Réunion, Univ. des Antilles; Laboratoire d’Excellence GR-Ex,
Paris, France; Faculté des Sciences et Technologies, Saint Denis Messag, F-97715 La
Réunion, France
d
Univ. Grenoble Alpes, DPM UMR 5063, F-38041 Grenoble, France; CNRS, DPM UMR
5063, F-38041 Grenoble, France
eSchool of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CH-1211
Geneva 4, Switzerland
fINSERM, CNRS, UNIV Nantes, l'institut du thorax, Nantes, France; CHU Nantes, France.
gINRA, UR1268, BIA, Nantes, France
1

ACCEPTED MANUSCRIPT
ABSTRACT: Phosphatidylinositide 3-kinases (PI3Ks) are widely expressed enzymes
involved in membrane signalization pathways. Attempts to administer inhibitors with broad
activity against different isoforms have failed due to toxicity. Conversely the PI3Kδ isoform
is much more selectively expressed, enabling therapeutic targeting of this isoform. Of
particular interest PI3Kδ is expressed in human basophils and its inhibition has been shown to
reduce anti-IgE induced basophil degranulation, suggesting that PI3Kδ inhibitors could be
useful as anti-allergy drugs. Herein, we report for the first time the activity of compounds
derived from chalcone scaffolds as inhibitors of normal human basophil degranulation and
identified the most active compound with anti-PI3Kδ properties that was investigated in
preclinical
models.
Compound
18,
namely
1-[2-hydroxy-4,6-dimethoxy-3-(N-
methylpiperidin-4-yl)phenyl]-3-(2,4,6-trimethoxyphenyl)-prop-2-en-1-one, was found to
inhibit normal human basophil degranulation in a dose-dependent manner. In a murine model
of ovalbumin-induced asthma, compound 18 was shown to reduce expiratory pressure while
its impact on the inflammatory infiltrate in alveolar lavage and total lung was dependent on
the route of administration. In a DNFB-induced model of atopic dermatitis compound 18
administered systemically proved to be as potent as topical betamethasone. These results
support the anti-atopic and allergic properties of the title compound and warrant further
clinical development.
Key words: PI3Kδ, Chalcones, Basophil Degranulation, Allergic Properties, Anti-atopic
Activity
2

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1. Introduction
Phosphatidylinositide 3-kinases (PI3Ks) constitute a family of enzymes involved in several
functions such as cell growth, proliferation, differentiation, motility, survival and intracellular
trafficking. PI3Ks are involved in signal transduction through the phosphorylation of the
hydroxyl group at position 3 of the inositol ring of phosphatidylinositol (PtdIns), which may
be reduced by the phosphatase enzyme PTEN [1,2].
The PI3K family is divided into four different classes, based on the primary structure,
regulation, and in vitro lipid substrate specificity [2]. Class I PI3Ks are activated by G
protein-coupled receptors and tyrosine kinase receptors. They are heterodimeric molecules
composed of a regulatory (p85) and a catalytic (p110) subunit. There are three variants of the
p110 catalytic subunit designated p110α, β, or δ catalytic subunit, expressed by separate
genes. While the first two catalytic isoforms are ubiquitously expressed in all cell types, the
p110δ is expressed primarily in white blood cells [3]. PI3Kδ is particularly expressed in B
cells, leading to its targeting in B cell malignancies. PI3Kδ has presently essentially been
targeted in onco-hematology indications [4]. Idelalisib (CAL-101, GS-1101), an oral agent, is
the first inhibitor of PI3Kδ that has recently been approved for the treatment of lymphoid
malignancies such as chronic lymphocytic leukemia. Since then, a number of other PI3Kδ
inhibitors have been reported to be active in preclinical models against B cell malignancies
and/or in auto-immune diseases [5].
However PI3K has also been considered as a potential target in asthma [6,7] and the PI3Kδ
isoform has been found to be specifically involved in allergic reactions [8]. A phase 1 study
of idelalisib (NCT00836914) has been performed, demonstrating good tolerance, increased
activity against allergic responses in patients with allergic rhinitis in comparison to placebo
and a reduced percentage of ex vivo stimulated basophils [9]. A clinical trial with the related
compound CAL-263 (NCT01066611) has been initiated in patients with allergic rhinitis.
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Given potential applications in neoplasia, allergy, autoimmunity and inflammatory
diseases, there has been significant interest to identify novel PI3K inhibitors through
screening of chemical libraries or by optimization of clinically used drugs [10-13]. In this
regard, benzimidazole-based inhibitors were developed [14]. High throughput screening of
chemical libraries was also used to generate lead inhibitors [15,16]. More recently, Ferguson
and co-workers showed that a series of 5,11-Dihydro-6H-benzo[e]pyrimido[5,4-
b][1,4]diazepin-6-ones were selective PI3Kδ/gamma inhibitors [17]. Finally, Hoegenauer and
co-workers have developed novel pyrrolidineoxy-substituted heteroaromatics [18].
Interestingly xanthine-derived compounds, such as caffeine and theophylline, have also been
shown to have direct effects on PI3Kδ [19].
Being interested by the discovery of easily synthesizable scaffolds, acting as anti-allergy
agents with in vivo effectiveness, we screened a large series of compounds belonging to
flavonoids derivatives, including flavones, aurones and chalcones as inhibitors of the normal
human basophil degranulation. The choice of these chemical scaffolds was motivated by their
reported effect on human kinases and their therapeutic potential [20,21]. In addition, the
computational analysis of a collection of compounds derived from chalcones demonstrates
that the collection covers structural features for the inhibition of PI3K enzymes, especially
chalcones were the most promising compounds and that these scaffolds have never been
explored on this target. Herein, we report the activity of a selection of chalcones bearing
methoxyl and piperidinyl groups. The latter susbtituents were reported to contribute to the
biological activity of chalcones, allowed us to limit our investigation to chalcones bearing
these two groups [22-24]. The most active compounds revealed by the basophil degranulation
test were investigated for their inhibition effect on PI3Kδ, then the most active derivative was
tested in two preclinical models: a murine ovalbumin-induced asthma assay and a murine
DNFB-induced atopic dermatitis assay. The selected compound (18) displays selective anti-
4

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PI3Kδ activity and possesses therapeutic activity in preclinical murine models of allergy or
atopy.
R3
R2
R6'
R4
R5
MeO
OMe
OMe
A
R4'
R3'
MeO
N
Me
B
R6
OMe
R2'
O
OH
O
18
Figure 1. General structure of the studied compounds and the most active lead compound.
2. Results
2.1. Chemistry. The synthesis of chalcones (summarized in Table 1) were prepared according
to Scheme 1, through the condensation of acetophenone derivatives with substituted
benzaldehydes in the presence of KOH at 90 °C [22,23]. The benzaldehydes and
acetophenones required for the synthesis of derivatives 1-17 were all commercially available.
Scheme 1. General scheme for the synthesis of chalcones.a
R2
a
R1
R2
R1
+
O
O
O
acetophenones
benzaldehydes
aReagents and conditions: (a) for compounds 1-19: KOH (50% in H2O), MeOH, 90 °C, 24 h.
For compound 20: NaOH in MeOH (3%, w/v), rt, 12 h.
The acetophenone derivative required for the synthesis of compounds 18-20 was
accomplished as shown in Scheme 2 [24]. The condensation of trimethoxybenzaldehyde and
1-methyl-4-piperidinone in the presence of gaseous HCl produced compound 21 with 90%
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yield. Compound 21 was reduced under catalytic hydrogenation with H2 in a mixture of
AcOH/H2O to provide the expected compound 22 with 94%. The acylation of 22 with acetic
anhydride in the presence of an excess of BF3.Et2O was achieved, giving the acetophenone
derivative 23 in 57% yield.
Scheme 2. Synthesis of acetophenone 23 required for the preparation of chalcones 18-20.
Me
MeO
OMe
+
N
MeO
OMe
MeO
OMe
O
MeO
OMe
a
c
b
OMe
O
N
OMe
N
OH
23
N
OMe
Me
Me
Me
22
21
aReagents and conditions: (a) HCl (gas), glacial AcOH, 24h at rt then 3h at 100 °C; (b) H2,
Pd/C, AcOH/H2O, rt, 16h; (c) Ac2O, BF3.Et2O, CH2Cl2, rt, 24 h.
In agreement with the report of Liu et al. [24], it was found that the acylation process led to a
partial demethylation of the methoxy group positioned between the N-methylpipeirine and the
carbonyl. The demethylation regioselectivity was assessed by NMR NOESY experiments and
confirmed by X-ray diffraction analysis, conducted on derivatives 18 and 19 (Figure 2).
18
19
Figure 2. ORTEP drawing of compounds 18 and 19
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2.2. Biological Evaluation.
2.2.1. Effect of chalcones on normal human basophil degranulation. The selected
chalcones were tested for their ability to reduce overexpression of the activation markers
CD63 and CD203c in normal human basophils induced by anti-IgE cross-linking. The results
are representative of three experiments with three different normal donors. As shown in Table
1, tested chalcones possessed very different inhibitory properties on the expression of CD63
and CD203c markers. Overall, the effect of the most potent compounds was more pronounced
on CD63 rather than on CD203c expression. As it could be expected, the substition pattern
played a crucial role. The first structural feature to be highlighted is the positive effect due to
the presence of at least three methoxy groups on either the A or B-ring (compounds 12, 13,
14, 16 and 17). The most marked effect was obtained with compounds bearing a N-
methylpiperidinyl moiety (18, 19 and 20). The importance of the N-methylpiperidinyl
substituent could be clearly seen by comparing the inhibitory effect of 11 and 19 that share
the same substitution pattern except for the presence of the N-methylpiperidinyl in 19. It
should be highlighted that the replacement of the A-ring of compounds (18, 19 and 20) by
heteroaryl groups led to inactive compounds (results not shown). Thus, it can be concluded
that the presence of the N-methylpiperidinyl is essential but not sufficient and the all
substitution pattern of the chalcone scaffold is important. Finally it should be noticed that the
chlorhydrate salts of the most active compounds (compound 18) was deleterious for the
activity.
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Table 1. Screening of chalcone derivatives (at 100 µM) for their ability to inhibit IgE-induced
degranulation of normal human basophils.
R3
R2
R6'
R4
R5
R2
R4
A
R4'
R3'
MeO
OMe
N
Me
B
R6
R6
R2'
O
OH
O
18: R2 = R4 = R6 = OMe
19: R2 = OMe, R4 = H, R6 = OMe
20: R2 = Cl, R4 = H, R6 = Cl
1 - 17
Entry
1
R2
R3
R4
R5
R6
R2’
R3’
R4’
R6’ CD63 CD203c
OMe
H
H
H
OMe OMe
OMe OH
H
H
OMe
H
94
95
92
100
78
67
91
64
63
76
81
40
53
48
66
49
55
2
96
91
93
82
81
68
95
81
75
86
85
54
51
65
70
62
65
7
OMe
H
H
OMe
H
H
H
H
H
H
OMe
OMe
H
2
OMe OMe OMe
H
H
OH
H
3
H
H
H
OMe
H
OMe
OMe
OMe
4
OMe
OMe
OMe
H
H
OMe
OMe
OMe
CF3
OMe OMe
OMe OMe
H
5
H
H
OMe OMe
6
H
H
OMe
H
H
OMe OMe
H
7
H
H
OMe
H
H
H
H
H
H
H
H
OMe
8
OMe
OMe
OMe
H
H
OMe
OMe
H
H
OMe OMe
OMe OMe
OMe OH
NH2 OMe
OMe
9
H
H
H
10
11
12
13
14
15
16
17
18
19
20
H
H
OMe OMe
OMe OMe
OMe OMe
OMe OMe
OH
H
OMe
H
H
H
H
H
H
H
OMe
OMe
OMe
H
OMe
OMe
OMe
H
OMe OMe
H
H
H
H
OMe OMe
OMe OMe OMe
OMe OMe OMe
OMe OMe OMe
OH
H
OMe
H
H
H
H
OMe
H
4
41
24
6
2.2.2. Inhibition of PI3K isoforms by compound 18. The inhibition of PI3K isoforms by
compound 18 and two reference inhibitors (idelalisib and duvelisib) was assessed (Table 2).
These results show that derivative 18 is a weaker inhibitor of PI3Kδ than idelalisib or
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duvelisib, with IC50 values in the micromolar range versus nanomolar ranges for idelalisib and
duvelisib. However 18 displayed only weak effect on the other isoforms with IC50 values
higher than 10 µM, while idelalisib and duvelisib remained quite potent against these
isoforms in the micromolar or submicromolar range.
Table 2. Inhibition activity of compound 18, Idelalisib and Duvelisib on PI3K isoforms
Compound
18
PIK3
IC50 (µM)
> 10
> 10
7.2
PI3KCα/PIK3R1
PI3KCβ/PIK3R1
PIK3Cδ/PIK3R1
PI3KCγ
18
18
10
18
2
PI3KCα/PIK3R1
PI3KCβ/PIK3R1
PI3KCδ/PIK3R1
PI3KCγ
Idelalisib
Idelalisib
Idelalisib
Idelalisib
Duvelisib
Duvelisib
Duvelisib
Duvelisib
0.6
0.017
0.25
0.4
PI3KCα/PIK3R1
PI3KCβ/PIK3R1
PI3KCδ/PIK3R1
PI3KCγ
0.049
0.0023
0.012
2.2.3. Activity of chalcone 18 in the ovalbumin-induced murine asthma model. In a first
series, compound 18 was administered intraperitoneally (90 mg/kg daily) and compared to
sodium cromoglycate (10 mg/kg) and in a second series, aerosolized 18 was compared to
buffer and aerosolized dexamethasone (10 mg/kg). Dexamethasone and DSCG are highly
potent drugs and the dose of 10 mg/kg is a standard dose which has been widely used in
preclinical models. We do not expect a higher dose to have a more pronounced effect in our
assay. For the experimental drug 18 which has never been used in the clinic, the dose of 90
mg/kg was chosen for reasons of solubility and thus may be a suboptimal dose for this
compound.
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As shown in Figure 3 (A and B), compound 18 reduced lung resistance following increasing
dose of metacholine whereas sodium cromoglycate did not, with an effect comparable to that
of dexamethasone in the aerosolized administration regimen.
Systemically administered 18 significantly reduced the total leukocytic infiltrate in BAL
(Figure 3C) and in lungs (data not shown). Analysis of subpopulations suggested that the
most striking effect of 18 was on the eosinophilic infiltrate (Figure 3E). Conversely in the
aerosolized regimens, the analysis of the cellular infiltrate showed important differences
between animals receiving 18 and those treated with dexamethasone. While mice receiving
steroids displayed a strong decrease in their cellular infiltrate, 18 did not modify the total
number of cells present in BAL (Fig 3D) while analysis of leukocyte subpopulations did not
show an impact of 18 on leukocytic subpopulations (Figure 3F).
Taken together, these results support a dual role of 18 both on bronchoconstriction and on the
inflammatory infiltrate observed in the ovalbumin-induced asthma model. It is likely that the
effect of 18 on inflammation is dose-related and that stronger concentrations could also be
active by aerosolization.
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A
B
C
D
E
F
6.0 106
4.0 106
2.0 106
0
150000
PBS
18
PBS
18
SC
DEXA
100000
50000
0
Cellular subtype
Cellular subtype
Figure 3. Effect of lead compound (18) and reference compounds in a murine model of
OVA-induced asthma. (A) Plethysmographic analysis in mice treated intraperitoneally with
18 or cromoglycate. (B) Flexivent analysis of lung resistance in mice treated with nebuilized
18 or dexamethasone. (C) Total cell infiltrate in lung in mice treated intraperitoneally with 18
or cromoglycate. (D) Total cell infiltrate in lung in mice treated with nebuilized 18 or
dexamethasone. (E) Plethysmographic analysis in mice treated intraperitoneally with 18 or
cromoglycate. (F) Flexivent analysis of lung resistance in mice treated with nebuilized 18 or
dexamethasone. CM: cromoglycate; DEXA: dexamethasone; LB: B lymphocytes; LT: T
lymphocytes.
2.2.4. Activity of 18 on murine atopic dermatitis model. As shown in Figure 4, 18 was
found to exert a significant protective effect when administered systemically in the DNFB-
induced atopic dermatitis model. This effect appeared to be prolonged (up to three days after
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exposure to the inducing agent) and was significantly different from that observed with a
reference agent, topical steroids.
18
Figure 4. Effect of systemically administered 18 and topical diprosone in a murine model of
atopic dermatitis. Control: mice unexposed to DNFB; DNFB sensitized: mice sensitized and
challenged with DNFB; Diprosone: mice sensitized and challenged with DNFB then treated
with diprosone; 18: mice sensitized and challenged with DNFB then treated with 18.
3. Discussion
With the aim of developing new chemical scaffolds active against allergy through the
inhibition of PI3Kδ requiring simple and straightforward synthesis, we investigated a library
of 20 chalcones, selected from a large series of derivatives. These compounds were selected
on the basis of their substitution features and due to their therapeutic potential [25,26].
In order to identify lead compounds as candidates for in vivo studies and development, we
performed a screening of chalcones on normal human basophil degranulation. In response to
FcεRI stimulation, basophil activation was assessed through increased CD63 or CD203c
expression. CD63 is anchored in the basophilic granule membrane (which contains histamine)
and its exposure on the outside of the cells reflects cell degranulation due to fusion between
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granules and plasma membranes [27]. CD203c is a basophil-specific surface antigen for
which the expression is rapidly upregulated after stimulation with the appropriate allergen or
after crosslinking of FcεRI with anti-IgE antibodies [27]. Both levels of expression were
investigated in whole blood as recommended by manufacturers. The three most active
chalcones on human basophil degranulation share a common chemical feature, the 6’-
hydroxy-2’,4’-dimethoxy-3”-(1-methylpiperidinyl) group. The 3 compounds induced high
basophil degranulation with a marked effect on CD63 expression.
Our study shows that the selected chalcone, 18 possesses anti- PI3Kδ inhibitory activity.
While the IC50 values are significantly higher than those of reference compounds such as
idelalisib or duvelisib, an interesting feature of our compound is that it has little effect on
other PI3K isoforms such as the α and β isoenzymes, a property which could prove to be
important in terms of tolerance. This is of particular importance since idelalisib has been
shown to be associated with some severe side effects in some cases [28].
Aiming to rationalize the binding of chalcone 18 to the active site of PIK3δ, we performed a
prediction of the orientation (or pose) of 18 in the binding site of PI3Kδ. Among the 11 poses
generated by the GOLD and rDOCK docking softwares [29,30], one of these, shown in
Figure 5, has the top rank when rescoring by using each of the methods XSCORE [31],
ITScore [32], and ID-Score [33].
In this predicted geometry, ligand 18 interacts with the core of the six residues that are usually
found in interaction with known PI3K inhibitors (W760, E826, V827, V828, M900 and I910).
The central ring along with the enone moiety fit into the adenine pocket; the carbonyl group
forming a first hydrogen bond with V828. A second hydrogen bond is found between the
protonated nitrogen of the 1-methylpiperidinyl moiety and D911; this ligand moiety, which is
critical for the activity as revealed by our experimental assays, occupies the affinity pocket
and participates in hydrophobic contacts with I910 [34]. The 1-methylpiperidinyl moiety,
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which is critical for the activity as revealed by experimental assays, occupies the affinity
pocket making hydrophobic contacts with I910 and could potentially form another hydrogen
bond with D911 through its protonated nitrogen.
From the chalcone orientation shown in Figure 5, we can also understand the structure-
activity relationships for compounds 19 and 20. Indeed, the methoxy group in para position of
the trimethoxyphenyl moiety in 18 points outside the binding site of the protein and forms no
contacts in the predicted orientation. Hence, the removal of this group in 19 is not expected to
have a large impact on the activity measured for this chalcone in comparison to that for 18. In
chalcone 20, the two chlorine atoms can form similar van der Waals contacts with the protein
as for the two methoxy groups found as counterparts in 18, again accounting for the similar
activities measured for these two chalcones.
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Figure 5. Prediction of the orientation of lead compound 18 in the binding site of PI3Kδ. The
carbon atoms of the ligand are displayed in gray. For the protein, the residues that form at
least one contact (measured by atomic distances less than 4 Å) with the ligand are displayed
in salmon; the other residues are in orange. The hydrogen bonds that the ligand forms with the
residues V828 and D911 are represented as dashed lines.
Importantly, the in vivo studies show that systemic administration of 18 is associated with
anti-allergic effects and that therapeutic concentrations can therefore be reached. Of interest,
chalcone 18 was administered intraperitoneally and intravenously up to doses of 90 mg/kg, 3
to 5 five times weekly, up to 4 weeks with no observed effect on animal well-being or weight
(data not shown). Compound 18 displayed potent activity in both the murine atopic dermatitis
model and in the asthma models, with effects close to or similar to reference steroids. In the
atopic dermatitis model intraperitoneal administration of 18 displayed a protective effect
comparable to topical betamethasone. In the asthma model, the lead compound proved to
reduce bronchial airway resistance both after intraperitoneal administration and after
aerosolization. Analysis of the cellular infiltrate in the asthma model suggests that the
compound had a strong effect on total leukocytic infiltrate, in particular on eosinophilic cells.
Interestingly Doukas et al. found that aerosolized administration of the PIK3δ inhibitor,
PI3KTG100-115, proved to be beneficial in murine asthma model and induced a decrease of
the eosinophilic infiltrate [35]. When 18 was administered by aerosolization, we did not
observe a strong effect on the inflammatory infiltrate while steroids induced a decrease of all
leukocytic subpopulations. In spite of this aerosolized-administration mode, 18 was associated
with a decrease in lung hyperresponsiveness to metacholine. Whether this is due to reduced
release of cytokines by basophils or other leukocytic cells or to an alternate mechanism
remains to be determined. However this observation suggests that the compound might not
induce immunosuppression, as it is commonly observed with steroids. It is likely that the
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aerosolization conditions were not optimized in our study, preventing us from observing the
same BAL modifications as those observed after systemic administration of 18.
The potential consequences of chronic PI3Kδ inhibition constitute an important aspect since
anti allergic agents are likely to be administered over prolonged periods of time. As PI3Kδ
has been shown to be an important actor in the differentiation and/or function of various types
of leucocytes, it is possible that chronic inhibition may be deleterious. Guo et al. reported that
PI3Kδ plays a critical role in Natural Killer (NK) cell maturation and cytokine release [36].
PI3Kδ has also been shown to affect neutrophil function either directly or via its expression in
endothelial cells [37-39]. Okeke et al. have recently shown that deficiency of PI3Kδ signaling
could lead to diminished number of regulatory T cells as well as increased neutrophil activity
in the context of sepsis [40]. While the clinical relevance of these observations in patients
receiving PI3Kδ inhibitors is unclear, it will be important to determine whether chronic
administration of these agents has to be modulated in the context of sepsis.
4. Conclusions
Our results show that judiciously functionalized chalcone derivatives administered
systemically (or by inhalation in the asthma model) possess protective activity in preclinical
allergy or atopy models. The observed activity is tightly linked to the inhibition of PI3Kδ.
The docking studies reveals that the scaffold of chalcone 18 fits well within the active site of
the PI3Kδ structure. The relevant in vivo activity of the lead compound, its safety at the active
doses, the easy synthesis and scale-up (4 steps and 38% overall yield) warrant further
optimization and development of the title compound in allergy and asthma management.
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5. Experimental Section
5.1. Chemistry. All reagents were purchased from commercial sources and were used directly
without further purification. Synthetic grade solvents were purchased and were used without
further distillation. NMR spectra were recorded on Bruker Advance-400 instrument (400
MHz) using CDCl3 as the solvent. The chemical shifts (δ) were reported in parts per million
(ppm). ESI-MS spectra were recorded at the mass spectrometry facility of the Institut de
Chimie Moléculaire de Grenoble (ICMG, FR 2607) using an Esquire 300 Plus Bruker
Daltonis instrument with a nano spray inlet. Thin-layer chromatography (TLC) were
performed on Merck silica gel F-254 plates (thickness 0.25 mm).
5.2. General procedure for the synthesis of intermediates 21 - 23
1-(N-methylpiperidin-3,4-en-4-yl)-2,4,6-trimethoxybenzene (21) [24]. N-methylpiperidin-
4-one (10.0 g, 59.5 mmol) was added to a solution of trimethoxybenzene (6.72 g, 59.5 mmol)
in glacial acetic acid (80 mL). Gaseous HCl was generated by the dropwise addition of HCl
(37%, 50 mL) to concentrated H2SO4 (50 mL). The flux of HCl was bubbled into the solution
containing the reagents, under stirring and for 1 h. The reaction mixture was stirred for 24 h
at room temperature then for 3 h à 100 °C. The solvent was removed under reduced pressure
and the residue was dissolved in H2O. The obtained solution was alkalinized by adding NaOH
(1 M), then extracted 3 times with ethyl acetate. The organic solution was dried over MgSO4
and the solvent was evaporated to provide compound 21 as a white solid (14.12 g). Yield
90%. The compound was used without purification for the next step.
1-(N-methylpiperidin-4-yl)-2,4,6-trimethoxybenzene (22). Compound 21 (14.12 g, 53.7
mmol) was dissolved in a mixture of acetic acid (400 mL) and water (40 mL). Palladium on
charcoal (10%, 1.4 g, 0.1 eq) was added (without stirring during the addition of Pd/C). The
mixture was stirred under hydrogen atmosphere for 16 h, then filtered over Celite. After
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filtration, the solvent was removed under reduced pressure to provide 13.37 g of compound
22 with satisfactory purity (94% yield).
2,4-Dimethoxy-6-hydroxy-3-(N-methylpiperidin-4-yl)acetophenone (23). Boron trifluoride
diethyl etherate (BF3.Et2O, 71.67 g, 505 mmol, 10 eq) was dropwise added to a solution of
compound 22 (13 g, 49 mmol) in dichloromethane (210 mL) at 0 °C. Acetic anhydride (50
mL) was then added dropwise and the mixture was stirred at rt for 24 h. The mixture was
diluted with H2O, alkalinized with Na2CO3 then extracted with dichloromethane. The organic
layer was dried over MgSO4 then evaporated under reduced pressure. The obtained solid was
dissolved in a mixture of ethyl acetate/cyclohexane, heated up to 90 °C then immediately
filtered. The residue was dissolved in small amount of methanol then a small volume of H2O
was added. The solid formed was filtered on filter paper. The solid was dried under reduced
pressure to provide compound 23 as a light green powder (8.45 g). Yield 57%.
5.3. General procedure for the synthesis of chalcones 1-19. The acetophenone (10 mmol)
and the benzaldehyde derivatives (1 eq.) were solubilized in methanol (200 mL). Potassium
hydroxide (10 mL, 50%) was added and the solution was stirred at 90 °C for 24 h. The
solution was evaporated to dryness and the residue was suspended in H2O. The solid formed
was filtered, washed with H2O then dried in oven at 40 °C and reduced pressure to give the
desired chalcone as yellow pale solids which were fully characterized by NMR and mass
spectrometry analyses.
3-(2,6-Dichlorophenyl)-1-[2-hydroxy-4,6-dimethoxy-3-(N-methylpiperidin-4-yl)phenyl]-
prop-2-en-1-one (20) was prepared as previously reported [24].
Synthesis and characterization of compounds 1-6, 8-11 and 14 were reported in our previous
papers.22,23 Compound 7 was described in references [41,42]. Compound 16 was described in
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reference [43]. Compound 12 is commercially available (available from Sigma-Aldrich
company). Compound 13, 15 and 17 were previously reported [44,45].
1-[2-Hydroxy-4,6-dimethoxy-3-(N-methylpiperidin-4-yl)phenyl]-3-(2,4,6-
1
trimethoxyphenyl)-prop-2-en-1-one (18). Yield 60%; H NMR (CDCl3) δ 14.51 (s, 1H),
8.27 (d, 1H, J = 15.8 Hz), 8.18 (d, 1H, J = 15.8 Hz), 6.12 (s, 2H), 5.97 (s, 1H), 3.91 (s, 3H),
3.89 (s, 6H), 3.86 (s, 3H), 3.85 (s, 3H), 3.15 (dt, 1H, J = 12.3, 3.6 Hz), 2.92 (m, 2H), 2.43 (qd,
13
2H, J = 12.6, 3.6 Hz), 2.29 (s, 3H), 2.02 (m, 2H), 1.49 (m, 2H). C NMR (CDCl3) δ 194.7,
164.6, 163.8, 162.9, 161.7, 161.2, 133.9, 127.7, 113.3, 107.3, 107.1, 90.7, 86.9, 57.3, 55.9,
55.7, 55.5, 55.3, 47.0, 32.1, 29.3. MS (ESI) m/z 472 (M+H)+.
3-(2,6-Dimethoxyphenyl)-1-[2-hydroxy-4,6-dimethoxy-3-(N-methylpiperidin-4-
yl)phenyl]-prop-2-en-1-one (19). Yield 35%; 1H NMR (CDCl3) δ 14.49 (s, 1H), 8.28 (s, 2H),
7.27 (t, 1H, J = 8.4 Hz), 6.58 (d, 2H, J = 8.4 Hz), 5.98 (s, 1H), 3.93 (s, 3H), 3.91 (s, 9H), 3.24
13
(m, 1H), 3.10 (m, 2H), 2.61 (m, 2H), 2.45 (br s, 3H), 2.29 (s, 2H), 1.55 (m, 2H). C NMR
(CDCl3) δ 194.8, 164.5, 164.1, 161.5, 160.4, 133.4, 131.2, 130.6, 113.6, 112.7, 107.0, 103.9,
86.9, 57.0, 55.9, 55.7, 55.4, 46.4, 31.7, 28.7. MS (ESI) m/z 442 (M+H)+.
3-(2,6-Dichlorophenyl)-1-[2-hydroxy-4,6-dimethoxy-3-(N-methylpiperidin-4-yl)phenyl]-
prop-2-en-1-one (20). Yield 30%; 1H NMR (CDCl3) δ 14.20 (s, 1H), 8.00 (d, 1H, J = 16.0
Hz), 7.85 (d, 1H, J = 16.0 Hz), 7.37 (d, 2H, J = 8.0 Hz), 7.17 (t, 1H, J = 8.0 Hz), 5.97 (s, 1H),
3.93 (s, 3H), 3.90 (s, 3H), 3.27 (dt, 1H, J = 12.2, 3.6 Hz), 3.19 (m, 2H), 2.70 (m, 2H), 2.53 (s,
3H), 2.42 (m, 2H), 1.57 (m, 2H). 13C NMR (CDCl3) δ 192.8, 165.1, 164.6, 162.0, 135.8,
135.4, 135.3, 133.2, 129.6, 129.0, 111.9, 106.4, 86.9, 56.5, 56.0, 55.6, 45.4, 31.0, 27.6. MS
(ESI) m/z 450 (M+H)+.
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5.3. Biology.
5.3.1. Effect on normal human basophil degranulation. Cells were incubated with a
crosslinking anti-FcεRI antibody in addition with various potential PI3Kδ inhibitors. Flow2
CAST assay (Buhlmann, Schönenbuch, Switzerland) was used for assessing CD63 expression
(basophils were identified on the basis of CCR3 expression) whereas Allergenicity kit
(Beckman Coulter, Hialeah, FL) was used for CD203c expression measurement (basophils
were identified through CRTH2+/CD3- expressions). Results, recorded as means of
fluorescence intensities (MFI), were expressed as percentages of maximal Ig-E mediated
effect (i.e., that obtained without kinase inhibitors).
5.3.2. Murine asthma model. Analyses of the activity of chalcone derivatives in preclinical
models of asthma were performed by the Therassay platform in Nantes, France. In this
ovalbumin-based model, mice are sensitized by intraperitoneal administration of OVA
together with alum as adjuvant on days 1 to 21 then intra-nasally exposed to OVA on days 27
to 29 andanalyzed the day after. Two series of tests were performed. In a first series, groups
of 10 mice received intraperitoneal injections of PBS, compound 18 or cromoglycate
(considered to be an inhibitor of basophil degranulation). Non-invasive whole body
plethysmography was performed in response to metacholine, a bronchoconstrictor. Broncho-
alveolar lavage was obtained from 5 mice in each group.
In a second series, groups of 15 mice received nebulization of PBS, of compound 18 or of
dexamethasone. 7 to 8 mice per group were then analyzed by Flexivent®, an invasive forced
oscillation method to determine respiratory function while broncho alveolar lavage was
performed on the remaining animals.
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5.3.3. Murine atopic dermatitis model. The murine atopic dermatitis model was obtained as
previously described using DNFB (1-fluoro-2,4-dinitrobenzène, Sigma: D1529), diluted in a
mixture of acetone and olive oil (4:1) [46]. Briefly mice were sensitized by application of
DNFB on their abdomen then sensitized by topical administration of DNFB on their ears. To
evaluate the protective effect of compounds, each mouse was used as its own control with
groups receiving excipient as a negative control, topical steroid (Diprosone™) or chalcone 18
(at 90 mg/kg intraperitoneally). Ear thickness was then measured daily with a caliper in
groups of 5 mice.
5.4. Molecular Modeling Calculations
The PI3Kδ structure taken from the Protein Data Bank [43] (PDB code 2WXF, resolution
1.90 Å) [34] was used for the search of the pose of the compound 18. This search was
performed with docking calculations using GOLD and rDOCK softwares [29,30] followed by
geometry optimization of the poses generated by using CHARMM program [48] and binding
energy evaluations for all of these relaxed poses by three scoring methods. The protein was
prepared using Chimera (original ligand and water molecules were removed) [49]. During the
docking experiments, a hydrogen bond with the backbone NH bond of residue V828 was used
as a pharmacophore restraint since all known ligands of this protein exhibit such a hydrogen
bond. Among the generated poses by GOLD and rDOCK, those with the best scores were
compared and 14 of the non-redundant poses were chosen for the next step of geometry
optimization. Prior to this optimization, each of the protein-ligand predicted structures were
immersed in a sphere of water molecules of 20-Å radius. This sphere size is sufficient to
provide a hydration shell around the binding site of the complex. A potential was imposed at
the water-vacuum boundary to avoid evaporation. The force field CHARMM36 was used for
the protein and the CGenFF parameters were used for the ligand [50]. First, only the water
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molecules were subjected to energy minimization (gradient tolerance of 0.5 kcal/mol/Å) while
the protein and ligand atoms were kept fixed. Second, the protein and the solvent were
harmonically restrained while the ligand was energy-minimized (gradient tolerance of 0.1
kcal/mol/Å). Third, only the harmonical restraints on the ligand and on the solvent were
applied while the protein was energy-minimized (gradient tolerance of 0.1 kcal/mol/Å).
Fourth, all the atoms of the system were subjected to energy minimization (gradient tolerance
of 0.1 kcal/mol/Å).
The binding energy of interaction of each of the poses we generated were then rescored by
three methods: XSCORE [31], ITScore [32], and ID-Score [33]. Only one pose, which is
discussed in the text, was ranked as number one in every of these rescoring methods.
ACKNOWLEDGMENTS
The authors would like to thank the Cytocell platform of Nantes for assistance with the flow
cytometry analysis. Also, we would like to thank the Therassay platform for the respiratory
equipment (Plethysmography and Flexivent systems). We are also grateful to the members of
the UTE IRS-UN animal facility for the support they provide us with the mice. Authors would
like to thank Dr. Christian Philouze from the Département de Chimie Moléculaire, UMR
5250 for the X-ray structures acquisition. This work has been partially supported by Labex
ARCANE and CBH-EUR-GS (ANR-17-EURE-0003).
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6. References
[1]
S.J. Leevers, B. Vanhaesebroeck, M.D. Waterfield, Signalling through
phosphoinositide 3-kinases: the lipids take centre stage, Curr. Opin. Cell. Biol. 11
(1999) 219-225.
[2]
[3]
B. Vanhaesebroeck, L. Stephens, P. Hawkins, PI3K signalling: the path to discovery
and understanding. Nat. Rev. Mol. Cell. Biol. 13 (2012) 195-203.
B. Vanhaesebroeck, M.J. Welham, K. Kotani, R. Stein, P.H. Warne, M.J. Zvelebil, K.
Higashi, S. Volinia, J. Downward, M.D, Waterfield, P110delta, a novel
phosphoinositide 3-kinase in leukocytes. Proc. Natl. Acad. Sci. U S A. 94 (1997)
4330-4335.
[4]
[5]
E. Jabbour, O.G. Ottmann, M. Deininger, A. Hochhaus, Targeting the
phosphoinositide 3-kinase pathway in hematologic malignancies. Haematologica 99
(2014) 7-18.
H. Chiu, S. Mallya, P. Nguyen, A. Mai, L.V. Jackson, D.G. Winkler, J.P. DiNitto, E.E.
Brophy, K. McGovern ,J.L. Kutok, D.A. Fruman, The Selective Phosphoinoside-3-
Kinase p110delta Inhibitor IPI-3063 Potently Suppresses B Cell Survival,
Proliferation, and Differentiation. Front. Immunol. 8 (2017) 747. doi:
10.3389/fimmu.2017.00747
[6]
[7]
S.J. Park, K.H. Min, Y.C. Lee, Phosphoinositide 3-kinase delta inhibitor as a novel
therapeutic agent in asthma. Respirology 13 (2008) 764-771.
K.S. Lee, S.J. Park, S.R. Kim, K.H. Min, S.M. Jin, K.D. Puri, Y.C. Lee,
Phosphoinositide 3-kinase-delta inhibitor reduces vascular permeability in a murine
model of asthma. J. Allergy Clin. Immunol. 118 (2006) 403-409.
K. Ali, M. Camps, W.P. Pearce, H. Ji, T. Rückle, N. Kuehn, C. Pasquali, C. Chabert,
C. Rommel, B. Vanhaesebroeck, Isoform-specific functions of phosphoinositide 3-
kinases: p110 delta but not p110 gamma promotes optimal allergic responses in vivo.
J. Immunol. 180 (2008) 2538-2544.
[8]
23

ACCEPTED MANUSCRIPT
[9]
F. Horak, K.D. Puri, B.H. Steiner, L. Holes, G. Xing, Randomized phase 1 study of
the phosphatidylinositol 3-kinase delta inhibitor idelalisib in patients with allergic
rhinitis. J. Allergy Clin. Immunol. 137 (2016) 1733-1741.
[10] K. Ellard, M. Sunose, K. Bell, N. Ramsden, G. Bergamini, Discovery of novel
PI3Kgamma/delta inhibitors as potential agents for inflammation. Bioorg. Med. Chem.
Lett. 22 (2012) 4546-4549.
[11] P.T. Hawkins, L.R. Stephens, PI3K signalling in inflammation. Biochim. Biophys.
Acta, 1851 (2015) 882-897.
[12] D.G. Winkler, K.L. Faia, J.P. DiNitto, J.A. Ali, K.F. White, PI3K-delta and PI3K-
gamma inhibition by IPI-145 abrogates immune responses and suppresses activity in
autoimmune and inflammatory disease models. Chem. Biol. 20 (2013) 1364-1374.
[13] B.S. Safina, S. Baker, M. Baumgardner, P.M. Blaney, B.K. Chan, Discovery of novel
PI3-kinase delta specific inhibitors for the treatment of rheumatoid arthritis: taming
CYP3A4 time-dependent inhibition. J. Med. Chem. 55 (2012) 5887-5900.
[14] J.M. Murray, Z.K. Sweeney, B.K. Chan, M. Balazs, E. Bradley, G. Castanedo, C.
Chabot, D. Chantry, M. Flagella, D.M. Goldstein, R. Kondru, J. Lesnick, J. Li, M.C.
Lucas, J. Nonomiya, J. Pang, S. Price, L. Salphati, B. Safina, P.P. Savy, E.M. Seward,
M. Ultsch, D.P. Sutherlin, Potent and highly selective benzimidazole inhibitors of PI3-
kinase delta. J. Med. Chem. 55 (2012) 7686-7695.
[15] E. Malek, J.J. Driscoll, High throughput chemical library screening identifies a novel
p110-delta inhibitor that potentiates the anti-myeloma effect of bortezomib.
Oncotarget 7 (2016) 38523-38538.
[16] D. Kong, K. Yamazaki, T. Yamori, Discovery of phosphatidylinositol 3-kinase
inhibitory compounds from the Screening Committee of Anticancer Drugs (SCADS)
library. Biol. Pharm. Bull. 33 (2010) 1600-1604.
[17] F.M. Ferguson, J. Ni, T. Zhang, B. Tesar, T. Sim, N.D. Kim, X. Deng, J.R. Brown, J.J.
Zhao, N.S. Gray, Discovery of a Series of 5,11-Dihydro-6H-benzo[e]pyrimido[5,4-
b][1,4]diazepin-6-ones as Selective PI3K-delta/gamma Inhibitors. ACS. Med. Chem.
Lett. 7 (2016) 908-912.
[18] K. Hoegenauer, N. Soldermann, C. Hebach, G.J. Hollingworth, I. Lewis, A. von Matt,
A.B. Smith, R.M. Wolf, R. Wilcken, D. Haasen, C. Burkhart, F. Zécri, Discovery of
novel pyrrolidineoxy-substituted heteroaromatics as potent and selective PI3K delta
inhibitors with improved physicochemical properties. Bioorg. Med. Chem. Lett. 26
(2016) 5657-5662.
24

ACCEPTED MANUSCRIPT
[19] L.C. Foukas, N. Daniele, C. Ktori, K.E. Anderson, J. Jensen, P.R. Shepherd, Direct
effects of caffeine and theophylline on p110 delta and other phosphoinositide 3-
kinases. Differential effects on lipid kinase and protein kinase activities. J. Biol. Chem.
277 (2002) 37124-37130.
[20] R. Haudecoeur, A. Boumendjel, Recent advances in the medicinal chemistry of
aurones. Curr. Med. Chem. 19 (2012) 2861-2875.
[21] A. Boumendjel, X. Ronot, J. Boutonnat, Chalcones Derivatives Acting as Cell Cycle
Blockers: Potential Anti-Cancer Drugs? Curr. Drug Targets 10 (2009) 363-371.
[22] A. Boumendjel, J. Boccard, P.-A. Carrupt, E. Nicolle, M. Blanc, A. Geze, L.
Choisnard , D. Wouessidjewe, E.L. Matera, C. Dumontet, Antimitotic and
antiproliferative activities of chalcones: forward structure-activity relationship. J. Med.
Chem. 51 (2008) 2307-2310.
[23] G. Valdameri, C. Gauthier, R. Terreux, R. Kachadourian, B.J. Day, S.M.
Winnischofer, M.E. Rocha , V. Frachet, X. Ronot, A. Di Pietro,A. Boumendjel,
Investigation of chalcones as selective inhibitors of the breast cancer resistance
protein: critical role of methoxylation in both inhibition potency and cytotoxicity. J.
Med. Chem. 55 (2012) 3193-3200.
[24] X. Liu, M.L. Go, Antiproliferative properties of piperidinylchalcones. Bioorg. Med.
Chem. 14 (2006) 153-163.
[25] M.N. Gomes, E.N. Muratov, M. Pereira, J.C. Peixoto, L.P. Rosseto, P.V.L. Cravo,
C.H. Andrade, B.J. Neves, Chalcone Derivatives: Promising Starting Points for Drug
Design. Molecules 22 (2017) doi: 10.3390/molecules22081210.
[26] S. Ducki, The development of chalcones as promising anticancer agents. IDrugs 10
(2007) 42-46.
[27] R. Boumiza, A.L. Debard, G. Monneret, The basophil activation test by flow
cytometry: recent developments in clinical studies, standardization and emerging
perspectives. Clin. Mol. Allergy 3 (2005) 9. doi:10.1186/1476-7961-3-9.
[28] I. Greenwell, A. Ip, J. Cohen, PI3K inhibitors: understanding toxicity mechanisms and
management. Oncology 31 (2017) 821-828.
[29] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and validation of
a genetic algorithm for flexible docking. J. Mol. Biol. 267 (1997) 727-748.
[30] S. Ruiz-Carmona, D. Alvarez-Garcia, N. Foloppe, A.B. Garmendia-Doval, S. Juhos, P.
Schmidtke, X. Barril, R.E. Hubbard, S.D. Morley, rDock: a fast, versatile and open
25

ACCEPTED MANUSCRIPT
source program for docking ligands to proteins and nucleic acids. PLoS Comput Biol.
10 (2014) e1003571.
[31] R. Wang, L. Lai, S. Wang, Further development and validation of empirical scoring
functions for structure-based binding affinity prediction. J. Comput. Aided Mol. Des.
16 (2002) 11-26.
[32] S.Y. Huang, X. Zou, Inclusion of solvation and entropy in the knowledge-based
scoring function for protein-ligand interactions. J. Chem. Inf. Model 50 (2010) 262-
273.
[33] G.B. Li, L.L. Yang, W.J Wang, L.L. Li, S.Y. Yang, ID-Score: a new empirical scoring
function based on a comprehensive set of descriptors related to protein-ligand
interactions. J. Chem. Inf. Model 53 (2013) 592-600.
[34] A. Berndt, S. Miller, O. Williams, D.D. Le, B.T. Houseman, J.L. Pacold, F. Gorrec,
W.C. Hon, Y. Liu, C. Rommel, P. Gaillard, T. Rückle, M.K. Schwarz, K.M. Shokat,
J.P. Shaw, R.L. Williams, The p110 delta structure: mechanisms for selectivity and
potency of new PI(3)K inhibitors. Nat. Chem. Biol. 6 (2010) 117-124.
[35] J. Doukas, L. Eide, K. Stebbins, A. Racanelli-Layton, L. Dellamary, et al. Aerosolized
phosphoinositide 3-kinase gamma/delta inhibitor TG100-115 [3-[2,4-diamino-6-(3-
hydroxyphenyl)pteridin-7-yl]phenol] as a therapeutic candidate for asthma and chronic
obstructive pulmonary disease. J. Pharmacol. Exp. Ther. 328 (2009) 758-765.
[36] H. Guo, A. Samarakoon, B. Vanhaesebroeck, S. Malarkannan, The p110 delta of PI3K
plays a critical role in NK cell terminal maturation and cytokine/chemokine
generation. J. Exp. Med. 205 (2008) 2419-2435.
[37] K.D. Puri, T.A. Doggett, J. Douangpanya, Y. Hou, W.T. Tino, T. Wilson, T. Graf, E.
Clayton, M. Turner, J.S. Hayflick, T.G. Diacovo, Mechanisms and implications of
phosphoinositide 3-kinase delta in promoting neutrophil trafficking into inflamed
tissue. Blood 103 (2004) 3448-3456.
[38] C. Sadhu, B. Masinovsky, K. Dick, C.G. Sowell, D.E. Staunton, Essential role of
phosphoinositide 3-kinase delta in neutrophil directional movement. J. Immunol. 170
(2003) 2647-2654.
[39] M.N. Duong, E.L. Matera, D. Mathe, A. Evesque, S. Valsesia-Wittmann, B.
Clémenceau, C. Dumontet, Effect of kinase inhibitors on the therapeutic properties of
monoclonal antibodies. MAbs 7 (2015) 192-198.
26

ACCEPTED MANUSCRIPT
[40] E.B. Okeke, Z. Mou, N. Onyilagha, P. Jia, A.S. Gounni, J.E. Uzonna, Deficiency of
Phosphatidylinositol 3-Kinase delta Signaling Leads to Diminished Numbers of
Regulatory T Cells and Increased Neutrophil Activity Resulting in Mortality Due to
Endotoxic Shock. J. Immunol. 199 (2017) 1086-1095.
[41] N. Mateeva, R. Kode, K. Redda, Synthesis of novel flavonoid derivatives as potential
HIV-integrase inhibitors. J. Heterocycl. Chem. 39 (2002) 1251-1258.
[42] C. Mills, N. Mateeva, K. Redda, Synthesis of novel substituted flavonoids. J.
Heterocycl. Chem. 43 (2006) 59-64.
[43] B. Srinivasan, T.E. Johnson, R. Lad, C. Xing, Structure-activity relationship studies of
chalcone leading to 3-hydroxy-4,3',4',5'-tetramethoxychalcone and its analogues as
potent nuclear factor kappaB inhibitors and their anticancer activities. J. Med. Chem.
52 (2009) 7228-7235.
[44] S. Shenvi, K. Kumar, K.S. Hatti, K. Rijesh. L. Diwakar, G.C. Reddy, Synthesis,
anticancer and antioxidant activities of 2,4,5-trimethoxy chalcones and analogues from
asaronaldehyde: structure-activity relationship. Eur. J. Med. Chem. 62 (2013) 435-42.
[45] B. Balsera, J. Mulet, A. Fernández-Carvajal, R. de la Torre-Martínez, A. Ferrer-
Montiel, J.G.. Hernández-Jiménez, J. Estévez-Herrera, A.E. Borges, A.E. Freitas,
M.G. López, M.T García-López, R. González-Muñiz, M.J. Pérez de Vega, L.M. Valor,
L. Svobodová, S. Sala, F. Sala, M. Criado, Chalcones as positive allosteric
modulators of α7 nicotinic acetylcholine receptors: a new target for a privileged
structure. Eur. J. Med. Chem. 86 (2014) 724-39.
[46] M. Bonneville, C. Chavagnac, M. Vocanson, A. Rozieres, J. Benetiere, I. Pernet, A.
Denis, J.F. Nicolas, A. Hennino, Skin contact irritation conditions the development
and severity of allergic contact dermatitis. J. Invest. Dermatol. 127 (2007) 1430-1435.
[47] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, et al, The Protein Data
Bank. Nucleic Acids Res. 28 (2000) 235-242.
[48] B.R. Brooks, C.L., 3rd. Brooks, A.D.Jr. Mackerell, L. Nilsson, R.J. Petrella, B. Roux,
Y. Won, G. Archontis, C. Bartels, S. Boresch, A. Caflisch, L. Caves, Q. Cui, A.R.
Dinner, M. Feig, S. Fischer, J. Gao, M. Hodoscek, W. Im, K. Kuczera, T. Lazaridis, J.
Ma, V. Ovchinnikov, E. Paci, R.W. Pastor, C.B. Post, J.Z. Pu, M. Schaefer, B. Tidor,
R.M. Venable, H.L. Woodcock, X. Wu, W. Yang, D.M. York, M. Karplus,
CHARMM: the biomolecular simulation program. J. Comput. Chem. 30 (2009) 1545-
1614.
27

ACCEPTED MANUSCRIPT
[49] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng,
T.E. Ferrin, UCSF Chimera--a visualization system for exploratory research and
analysis. J. Comput. Chem. 25 (2004) 1605-1612.
[50] K. Vanommeslaeghe, E. Hatcher, C. Acharya, S. Kundu, S. Zhong, J. Shim, E. Darian,
O. Guvench, P. Lopes, I. Vorobyov, A.D. Jr. Mackerell, CHARMM general force
field: A force field for drug-like molecules compatible with the CHARMM all-atom
additive biological force fields. J. Comput. Chem. 31 (2010) 671-690.
28