Design and synthesis of novel 6-aryl substituted 4-anilinequinazoline derivatives as potential PI3Kδ inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.03.020

In this study, a series of new 6-aryl substituted 4-anilinequinazolines was designed and synthesized as PI3Kδ inhibitors based on our reported chemical structures. The preliminary structure-activity relationship (SAR) was established, and compounds 13h an

Full text of DOI:10.1016/j.bmcl.2017.03.020

Accepted Manuscript
Design and synthesis of novel 6-aryl substituted 4-anilinequinazoline deriva-
tives as potential PI3Kδ inhibitors
Minhang Xin, Yuan-Yuan Hei, Hao Zhang, Ying Shen, San-Qi Zhang
PII:
S0960-894X(17)30258-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.020
BMCL 24772
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
23 January 2017
5 March 2017
9 March 2017
Please cite this article as: Xin, M., Hei, Y-Y., Zhang, H., Shen, Y., Zhang, S-Q., Design and synthesis of novel 6-
aryl substituted 4-anilinequinazoline derivatives as potential PI3Kδ inhibitors, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.03.020
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Design and synthesis of novel 6-aryl substituted 4-anilinequinazoline derivatives as
potential PI3Kδ inhibitors
Minhang Xin, Yuan-Yuan Hei, Hao Zhang, Ying Shen, San-Qi Zhang *
Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an
Jiaotong University, No 76, Yanta West Road, Xi’an 710061, P.R. China
Footnotes
*
Corresponding author: E-mail: sqzhang@xjtu.edu.cn.
1

Abstract: In this study, a series of new 6-aryl substituted 4-anilinequinazolines was
designed and synthesized as PI3Kδ inhibitors based on our reported chemical structures.
The preliminary structure-activity relationship (SAR) was established, and compounds
1
3h and 13k displayed most potent PI3Kδ inhibitory activities with the IC50 values of 9.3
nM and 9.7 nM, respectively. Compound 13h demonstrated similar anti-proliferative
profiles to idelalisib against three human B cell lines. Three key hydrogen bonding
interactions were found in the docking of 13h with PI3Kδ enzyme. These results suggest
that compound 13h possessed nanomolar PI3Kδ inhibitory activity and distinctive
chemical structure, deserving further structural optimization.
Keywords: PI3Kδ inhibitors; 6-aryl substituted 4-anilinequinazolines; structure-activity
relationship; drug design.
2

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that catalyze the
1
phosphorylation of the 3’-hydroxy of phosphatidylinositol . They mediate multiple
cellular signaling transductions and play essential roles in cell growth, development,
2
motility and survival . According to the primary structure, regulation and lipid substrate
specificity, PI3Ks are generally classified as three different classes: class I, class II and
class III. Class I PI3Ks have been mostly studied, and further delineated into class IA and
IB subclasses. Class IA PI3Ks are composed of PI3Kα, PI3Kβ and PI3Kδ, which are
primarily activated by receptor tyrosine kinase (RTK). Class IB comprises only one
component of PI3Kγ, which is activated by G-protein coupled receptors (GPCR). PI3Kα
and PI3Kβ are expressed in various cells, while PI3Kδ and PI3Kγ are restrictedly
3
, 4
expressed in immune and hematopoietic cells . In particular, PI3Kδ is identified to play
a critical role in the B-cell proliferation and differentiation. Furthermore,
pharmacological studies showed that abnormal expression of PI3Kδ was relevant to the
5
B-cell malignant transformation . Thus, PI3Kδ is regarded as promising therapeutic
6
targets for treating hematological malignancies .
Indeed, several small-molecule PI3Kδ inhibitors have been developed in recent
7
years . Considering PI3Kγ also playing an important role in hematopoietic cells, the
PI3Kδ inhibitors are permitted to possess PI3Kγ inhibitory activity. Idelalisib (CAL-101,
1
) was the first approved by US FDA in 2014 as a selective PI3Kδ inhibitor for use in the
treatment of chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and small
8
, 9
lymphocytic lymphoma (SLL) . Duvelisib (IPI-145, 2), a potent PI3Kδ/γ inhibitor, was
ever evaluated in phase III clinical trials. Other selective PI3Kδ inhibitors, such as
acalisib (GS-9820, 3), TGR-1202 (4), RV-1729 (5), RP-6530 (6) and AMG-319 (7), were
6
also progressed to multiple clinical stages (Fig. 1).
3

O
O
F
O
N
Cl
O
O
F
F
F
N
N
N
N
N
N
F
HN
N
HN
N
HN
N
N
N
N
N
N
N
N
H N
2
NH
NH
NH
O
Idelalisib (CAL-101, 1 )
Duvelisib (IPI-145,
2
)
Acalisib (GS-9820, 3)
TG-1202 (4)
Gilead
Launched
Infinity/Abbvie
Phase III
Gilead
Phase II
TG Therapeutics
Phase II
O
O
N
N
O
O
N
Cl
O
O
N
N
N
F
F
N
N
HN
N
HN
N
N
N
N
N
N
HO
H N
2
NH
NH
RP-6530 (6)
AMG-319 (7)
RV-1729 (5)
Amgen
Phase I
RespiVert Ltd
Phase I
Rhizen Pharmaceutics
Phase I
Fig. (1). Representative clinical agents of PI3Kδ inhibitors
Although PI3Kδ inhibitors have been investigated abundantly, most of their
chemical structures are similar to idelalisib. In fact, idelalisib was identified to cause
serious side effects including fatal hepatotoxicity, severe diarrhea or colitis, pneumonitis
and intestinal perforation thereby being marked by black box warnings. And due to its
continuously adverse events and deaths, idelalisib was stopped in six ongoing clinical
1
0
trials in March 2016 . These side-effect profiles associated with idelalisib might be
mostly relative to its chemical structure due to the fact that duvelisib did not suffer from
these severe adverse events. However, unfortunately, duvelisib was recently withdrawn
1
1
from the phase III studies because of the disappointing clinical efficacy . Based on these
scientific events, the discovery of new chemotype of PI3Kδ inhibitors which are different
from idelalisib (or duvelisib) may bring unexpected therapeutic index and clinical benefit.
4

Recently, a series of small molecules with distinct structural characteristics were
reported as potent PI3Kδ inhibitors by Novartis which were structurally featured by
1
2, 13, 14
4
-aryl quinazolines
. Among these, the potent compound 8 was disclosed showing
very potent and selective PI3Kδ inhibition (PI3Kδ: IC50 = 4 nM, δ/α selectivity: > 400).
Based on this pioneer structure, we recently developed a series of 6-benzamide
substituted 4-phenylquinazoline derivatives showing potent PI3Kδ inhibitors, in which
the corresponding 6-benzamide group afforded the desired hydrogen-bond interaction as
1
5
the pyridyl group did in the PI3Kδ binding pocket . As a continuation of our study
towards development of new PI3Kδ inhibitors, based on our previous structures, we
intend to insert an amino group between the 4-aryl moiety and quinazoline scaffold
which could reduce the molecular planarity and rigidity thereby improving drug-like
properties. In particular, this strategy was found to be frequently used in the kinase-based
drug design such as epidermal growth factor receptor (EGFR) inhibitors and bruton’s
tyrosine kinase (BTK) inhibitors. Herein, we describe the syntheses, biochemical and
cellular evaluation of these novel 6-aryl substituted 4-anilinequinazoline derivatives as
PI3Kδ inhibitors (Fig. 2).
Fig. (2). Design of novel 6-aryl substituted 4-anilinequinazolines as PI3Kδ inhibitors
The new 6-aryl substituted 4-anilinequinazoline derivatives (13a-13k and 16a-16b)
were synthesized as depicted in Scheme 1. Following the literature procedures, the key
intermediate 6-bromo-4-chloroquinazoline (11) was synthesized by two steps in an
overall yield of 94% using the commercially available 2-amino-5-bromobenzoic acid (9)
1
5
as the starting material . Treatment of 11 with several readily prepared anilines in
refluxed isopropanol produced multiple 4-anilinequinazolines (12a-12d). Compound 12
was then reacted with either appropriate aryl boronate which was prepared under the
5

Pd-catalyzed Miyaura borylation coupling condition by our previously reported
procedures or the commercially available 2-methoxy-5-pyridineboronic acid in the
presence of PdCl
2
(dppf) and Na
2
CO
. The similar synthetic routes were employed to prepare the 6-aryl
substituted 4-anilinequinolines 16a-16b. Beginning with the commercially available
3
generated the targeted compound 13a-13k in
1
6, 17
satisfactory yields
6
3
-bromo-4-chloroquinoline
(14)
and
the
readily
prepared
-(4-methylpiperazine)carbonylphenylamine, compound 15 was obtained by nucleophilic
substitution, followed by Suzuki coupling with appropriate arylboric substrates to give
targeted compounds 16a-16b (Scheme 1).
o
Scheme 1. Reagents and conditions: (a) Formamidine acetate, 150-160 C, 50 min, 96%; (b) POCl
3
,
1
-2 drops DMF, reflux, 4 h, 98%; (c) substituted phenylamine, i-PrOH, reflux 4 h, 62-86%; (d)
arylboric esters, Na CO Pd(dppf)Cl DME/H O, reflux, h, 11-91%; (e)
-(4-methylpiperazine)carbonylphenylamine, i-PrOH, 1-2 drops of concentrated HCl, reflux 5 h, 26%;
f) for 16a: (i) bis(pinacolato)diboron, 5-bromo-3-methoxybenzamide, Pd(dppf)Cl , KOAc, dioxane,
reflux, 2 h; (ii) 15, Na CO , Pd(dppf)Cl , DME/H O, reflux, 4 h, 95%; for 16b:
-methoxy-5-pyridineboronic acid, 15, Na , DME/H O, reflux, 4 h, 76%.
2
3
,
2
,
2
4
3
(
2
2
3
2
2
2
2
CO
3
, Pd(dppf)Cl
2
2
6

All the newly synthesized 6-aryl substituted 4-anilinequinazolines were initially
evaluated for their PI3Kδ inhibitory activities under a concentration of 100 nM and the
reference compound idelalisib (CAL-101) was used as the positive control. As
summarized in Table 1, most of the targeted compounds displayed significant PI3Kδ
inhibitory activities at the concentration, suggesting that our design of this series of 6-aryl
substituted 4-anilinequinazoline derivatives is reasonable. It was found that the position
of the side chains on the 4-aniline group was closely related to the PI3Kδ inhibitory
activity of title compounds. The mara-position of side chains on the 4-aniline exhibited
greater
inhibitory
’-(4-methylpiperazine)carbonyl (13a: 12%) vs 3’-(4-methylpiperazine)carbonyl (13b:
3%) and 4’-(morpholine)carbonyl (13d: 0% and 13f: 61%) vs 3’-(morpholine)carbonyl
activity
than
that
of
para-position,
such
as
4
3
(
13e: 21% and 13g: 87%). Moreover, the 6-aryl moiety also played significant impact on
the activity. Considering the previous SAR finding that a presence of a hydrogen-bond
1
5
accepting group was beneficial to the PI3Kδ inhibitory activities . Several 6-aryl
moieties containing hydrogen-bond accepting groups such as substituted benzamides,
substituted pyridin-3-yl groups were investigated. It was found that
6
3
’-methoxylpyridin-3-yl (13c: 71%) afforded more potent activity than
’-aminocarbonyl-4’-methoxylphenyl (13b: 33%), and this clear SAR trend was also
obviously observed between compounds 13g (87%) and 13e (21%). In particular,
replacement of 6’-methoxylpyridin-3-yl group with 5’-cyano-6’-methoxylpyridin-3-yl
group led to afford the most potent activity (13h: 93%). Additionally, several different
substituted benzamides were also studied, such as 3’-aminocarbonyl-4’-methoxylphenyl
(
13e),
’-aminocarbonyl-5’-methoxylphenyl (13j), 3’-aminocarbonyl-5’-trifluoromethylphenyl
13k). It was found that introduction of an extra nitro group at 5′-position resulted in
increased activity (13i: 49% vs 13e: 21%). When the methoxy group was moved from
3’-aminocarbonyl-4’-methoxyl-5-nitrophenyl
(13i),
3
(
4
2
′-position to 5′-position led to improved PI3Kδ inhibitory activity (13j: 61% vs 13e:
1%), and especially, the methoxy group at 5′-position was changed into trifluoromethyl
induced the potency greatly enhanced (13j: 61% vs 13k: 91%). In addition, the quinoline
as a bioisosteric scaffold was also investigated and two representative compounds (16a
7

and 16b) were synthesized, which showed very weak PI3Kδ inhibitory activity. In this
PI3Kδ screening condition, idelalisib showed 96% inhibition at 100 nM.
After the preliminary PI3Kδ inhibition results, two title compounds 13h and 13k,
showing more than 90% inhibition rate, were subsequently evaluated for their IC50 values
against PI3Kδ. To our delight, compounds 13h and 13k exhibited significantly potent
PI3Kδ inhibitory activities, with the IC50 values of 9.3 nM and 9.7 nM, respectively,
although they were still not comparable to the positive drug idelalisib showing an IC50
value of 2.7 nM. It is noted that this series of 6-aryl substituted 4-anilinequinazolines
possessed novel chemical structures, which were different from idelalisib or its analogues,
as well as showed nanomolar PI3Kδ inhibitory activities, deserving further structural
optimization (Table 1).
Table 1. PI3Kδ inhibitory activity of 6-aryl substituted 4-anilinequinazolines
PI3Kδ
PI3Kδ
IC50 b
(nM)
compds
Ar
X
Inhibition
a
(
%)
1
3a
3b
N
12
ND
1
N
N
33
71
ND
ND
1
3c
1
3d
N
0
ND
8

1
3e
N
N
21
61
ND
ND
1
3f
1
3g
3h
N
N
87
93
ND
9.3
1
1
3i
N
N
49
61
ND
ND
1
3j
1
3k
N
91
0
9.7
1
6a
6b
CH
ND
1
CH
-
23
96
ND
2.7
idelalisib
-
-
a
b
PI3Kδ inhibition at the concentration of 100 nM; The IC50 value for PI3Kδ inhibition; ND means
“
Not Detected”. All the data are shown as the mean for two experiments.
Based on the PI3Kδ inhibitory activities, compound 13h and 13k were further
evaluated for their anti-proliferative activities against three human B cell lines including
RPMI-8226, Ramos and Raji via 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
1
8, 19
bromide (MTT) cell proliferation assay
. The idelalisib and SAHA were selected as
reference compounds. The results were listed in Table 2. The reference compound
9

idelalisib displayed moderate anti-proliferative activity against RPMI-8226 with an IC50
value of 5.49 µM, and exhibited weak anti-proliferative activity against Ramos and Raji
with the IC50 values of >10 and 9.95 µM, respectively, whereas another reference
compound SAHA showed significantly potent anti-proliferative activities against
RPMI-8226, Ramos and Raji with the IC50 values of 0.66, 0.52 and 0.97 µM, respectively.
Noticeably, compound 13h also exhibited moderate potency against RPMI-8226 with an
IC50 value of 6.61 µM, and weak anti-proliferative activities against Ramos and Raji with
IC50 values both >10 µM, while compound 13k showed significantly potent
anti-proliferative activities against RPMI-8226, Ramos and Raji with the IC50 values of
1
.10, 1.06 and 0.95 µM, respectively. In a word, compound 13h displayed similar
anti-proliferative profiles to idelalisib in given three human B cell lines, which suggested
compound 13h enabled to share the same enzyme-based mechanism to idelalisib (Table
2
).
a
Table 2. Anti-proliferative activities for compounds 13h and 13k in vitro
IC50 (µM)
Ramos
Compounds
Chemical Structures
RPMI-8226
6.61
Raji
> 10
1
1
3h
3k
> 10
CF3
O
N
HN
H N
2
O
1.10
5.49
0.66
1.06
> 10
0.52
0.95
9.95
0.97
N
O
N
idelalisib
SAHA
a
the data are shown as the mean for at least two experiments.
To further explain the activities of the title compounds, a molecular docking of
compound 13h with human PI3Kδ enzyme was conducted by C-DOCKER program
10

within Discovery Studio 3.0 software package. The docking result was shown in Figure 3.
It showed three key hydrogen bonds were formed within the PI3Kδ enzyme pocket. The
1
-nitrogen atom of quinazoline core interacted within the hinge region by a hydrogen
bonding with the Val828. The 6-pyridyl moiety extended into the affinity pocket, and its
3
4
′-nitrogen
generated
another
hydrogen
bond
with
Lys779.
The
-(3-morpholinylcarbonyl)aniline moiety made close to the PI3Kδ isoform specificity
pocket, where the morpholine group formed a hydrogen bond with Lys708 which was
1
8, 20
key to generate specific PI3Kδ inhibition
. These specific hydrogen bonding
interactions with PI3Kδ conferred compound 13h to a potent PI3Kδ inhibitor (Fig. 3).
Fig. (3). Compound 13h docked into the site of PI3Kδ (PDB code: 2wxp). Compound 13h is shown
as stick modes. Hydrogen bonds within 2.5 Å are shown as yellow dashed lines.
In conclusion, a series of new 6-aryl substituted 4-anilinequinazolines was designed
as PI3Kδ inhibitors based on our previous chemical structures by reduction of molecular
planarity and rigidity. These targeted derivatives were synthesized delicately and
evaluated for their inhibitory activity against PI3Kδ enzyme. The preliminary
structure-activity relationship (SAR) for this series of 6-aryl substituted
4
-anilinequinazolines was established. Compounds 13h and 13k afforded low nanomolar
PI3Kδ inhibitory activities with IC50 values of 9.3 nM and 9.7 nM, respectively, and
compound 13h showed similar anti-proliferative profiles to idelalisib in the
anti-proliferative assay of three human B cell lines. Molecular docking study indicated
key hydrogen bonding formation is essential for generating potent PI3Kδ inhibition for
11

compound 13h. These results suggest that compound 13h possessed nanomolar PI3Kδ
inhibitory activity and distinctive chemical structure, deserving further structural
optimization to develop highly potent and specific PI3Kδ inhibitors.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
(
81402792) and China Postdoctoral Science Foundation (2014M560793 and
015T81038). The authors also thank the New Teachers Supporting Program of Xi’an
2
Jiaotong University.
Supplementary data
Supplementary data associated with this article can be found, in the online version.
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2
13

Design and synthesis of novel 6-aryl substituted 4-anilinequinazoline derivatives as
potential PI3Kδ inhibitors
Minhang Xin, Yuan-Yuan Hei, Hao Zhang, Ying Shen, San-Qi Zhang *
Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an
Jiaotong University, Xi’an 710061, P.R. China
Graphical Abstract:
CN
O
O
N
HN
N
N
O
N
1
3h
PI3Kdelta: IC50 = 9.3 nM
*
Corresponding author: E-mail: sqzhang@xjtu.edu.cn.
1
4

15