Synthesis and biological evaluation of 4-(piperid-3-yl)amino substituted 6-pyridylquinazolines as potent PI3Kδ inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.07.051

PI3Kδ is an intriguing target for developing anti-cancer agent. In this study, a new series of 4-(piperid-3-yl)amino substituted 6-pyridylquinazoline derivatives were synthesized. After biological evaluation, compounds A5 and A8 were identified as potent PI3Kδ inhibitors, with IC₅₀ values of 1.3 and 0.7 nM, respectively, which are equivalent to or better than idelalisib (IC₅₀ = 1.2 nM). Further PI3K isoforms selectivity evaluation showed that compound A5 afforded excellent PI3Kδ selectivity over PI3Kα, PI3Kβ and PI3Kγ. A8 exhibited superior PI3Kδ/γ selectivity over PI3Kα and PI3Kβ. Moreover, compounds A5 and A8 selectively exhibited anti-proliferation against SU-DHL-6 in vitro with IC₅₀ values of 0.16 and 0.12 μM. Western blot analysis indicated that A8 could attenuate the AKT^S473 phosphorylation. Molecular docking study suggested that A8 formed three key H-bonds action with PI3Kδ, which may account for its potent inhibition of PI3Kδ. These findings indicate that 4-(piperid-3-yl)amino substituted 6-pyridylquinazoline derivatives were potent PI3Kδ inhibitors with distinctive PI3K-isoforms and anti-proliferation profiles.

Full text of DOI:10.1016/j.bmc.2019.07.051

Journal Pre-Proof
Synthesis and Biological Evaluation of 4-(Piperid-3-yl)amino Substituted 6-
Pyridylquinazolines as Potent PI3Kδ Inhibitors
Yifan Feng, Weiming Duan, Shu Fan, Hao Zhang, San-Qi Zhang, Minhang Xin
PII:
S0968-0896(19)31006-5
https://doi.org/10.1016/j.bmc.2019.07.051
BMC 15035
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
19 June 2019
29 July 2019
30 July 2019
Please cite this article as: Feng, Y., Duan, W., Fan, S., Zhang, H., Zhang, S-Q., Xin, M., Synthesis and Biological
Evaluation of 4-(Piperid-3-yl)amino Substituted 6-Pyridylquinazolines as Potent PI3Kδ Inhibitors, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.07.051
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JOURNAL PRE-PROOF
Synthesis and Biological Evaluation of 4-(Piperid-3-yl)amino Substituted
6-Pyridylquinazolines as Potent PI3Kδ Inhibitors
Yifan Feng, Weiming Duan, Shu Fan, Hao Zhang, San-Qi Zhang, Minhang Xin *
Department of Medicinal Chemistry, School of Pharmacy, Xi’an Jiaotong University, 76
West Yanta Road, Xi’an, Shaanxi 710061, P.R. China
Footnotes
* Corresponding author: Minhang Xin, E-mail: xmhcpu@163.com
 These authors contributed equally to this work.
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Abstract: PI3Kδ is an intriguing target for developing anti-cancer agent. In this
study, a new series of 4-(piperid-3-yl)amino substituted 6-pyridylquinazoline derivatives
were synthesized. After biological evaluation, compounds A5 and A8 were identified as
potent PI3Kδ inhibitors, with IC50 values of 1.3 and 0.7 nM, respectively, which are
equivalent to or better than idelalisib (IC50 = 1.2 nM). Further PI3K isoforms selectivity
evaluation showed that compound A5 afforded excellent PI3Kδ selectivity over PI3Kα,
PI3Kβ and PI3Kγ. A8 exhibited superior PI3Kδ/γ selectivity over PI3Kα and PI3Kβ.
Moreover, compounds A5 and A8 selectively exhibited anti-proliferation against
SU-DHL-6 in vitro with IC50 values of 0.16 and 0.12 μM. Western blot analysis indicated
that A8 could attenuate the AKTS473 phosphorylation. Molecular docking study suggested
that A8 formed three key H-bonds action with PI3Kδ, which may account for its potent
inhibition of PI3Kδ. These findings indicate that 4-(piperid-3-yl)amino substituted
6-pyridylquinazoline derivatives were potent PI3Kδ inhibitors with distinctive
PI3K-isoforms and anti-proliferation profiles.
Keywords: PI3Kδ inhibitors; PI3K inhibitors; 6-pyridylquinazolines; anti-proliferation.
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1. Introduction
Phosphoinositide 3-kinases (PI3Ks) are members of a family of key intracellular
enzymes. PI3Ks mediate the phosporylation procedure, and trigger the downstream signal
transducer AKT (protein kinase B) and mTOR (Mannalian target of rapamycin) to
regulate cellular activities such as cell proliferation, differentiation, migration and
survival. PI3K family consists of three classes. The class I PI3Ks are well characterized,
which are further individed into PI3Kα, β, δ and γ.1 PI3Kα and β are widely expressed,
whereas PI3Kδ and γ are predominantly restricted in hematopoietic cells.2 Consequently,
inhibition of PI3Kδ (or mixed PI3Kδ/γ) was thought to enable to combat cancers,
immunological and inflammatory conditions and development of small molecule PI3Kδ
inhibitors receives significant interest from industrial and academic community.3
Many classes of PI3Kδ inhibitors have been investigated, and some have been
approved or entered into clinical trials for treatment of leukemia, lymphoma, asthma,
chronic obstructive pulmonary disease (COPD), sjogren’s syndrome, activated PI3Kδ
syndrome (APDS), and rheumatoid arthritis.4 The first-in-class orally potent selective
PI3Kδ inhibitor idelalisib (1) was approved by FDA (US Food and Drug Administration)
in 2014 for treating chronic lymphocytic leukemia (CLL), follicular lymphoma (FL) and
small lymphocytic lymphoma (SLL).5 Although idelalisib gave significant efficacy in
clinic, it showed severe toxicity leading to its approval with black-box warning. 6 Later in
2018, the selective PI3Kδ/γ inhibitors duvelisib (2) was approved by FDA also for
treating CLL, FL and SLL, which was structurally similar to idelalisib.7 Duvelisib was
reported to show lower side effects than idelalisib. Umbralisib (3) was another potent
selective PI3Kδ inhibitor, studied in phase III clinical trials for its efficacy against B-cell
lymphoma. 8 Leniolisib (4), developed by Novartis, was an orally potent selective PI3Kδ
inhibitor which recently entered phase III clinical evaluation for rare autoimmune
diseases such as APDS and Sjogren’s syndrome.9, 10 Nemiralisib (5), developed by GSK
11
(GlaxoSmithKline), was in phase II trials for treatment of asthma and COPD (Figure
1). Overall, these approved and late-stage clinical selective PI3Kδ inhibitors are
categorized into two classes and exemplified as follows: (1) propeller-shaped inhibitors
such as idelalisib, duvelisib, and umbralisib, (2) flat-shaped inhibitors such as leniolisib
and nemiralisib.12 Considering the reported propeller-shaped PI3Kδ inhibitors
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demonstrating severe side effects, there is greater need to develop new classes of PI3Kδ
inhibitors differentiated from propeller-shaped ones, so as to investigate the medication
regimen and improve the safety.13 Herein, we reported our drug effort on development of
PI3Kδ inhibitors which differed from propeller-shaped ones.
Figure 1. Representative structures of approved and late-stage clinical PI3Kδ inhibitors.
Recently, we disclosed a series of potential PI3Kδ inhibitors containing a quinazoline
scaffold which were significantly different from the propeller-shaped inhibitors.14
Exploring
the
4-substitutents
of
6-pyridylquinazoline
led
to
the
4-aniline-6-pyridylquinazolines exemplified by 6 (PI3Kδ: IC50 = 9.3 nM) and the
4-pyrrolidineamino-6-pyridylquinazolines exemplified by 7 (PI3Kδ: IC50 = 2.7 nM).15, 16
Simply switching the 4-pyrrolidineamino to 4-pyrrolidineoxy (or 4-(piperid-3-yl)amino)
group made the PI3Kδ inhibitory activity retained or enhanced, such as 8 (PI3Kδ: IC50 =
4.9 nM) and 9 (PI3Kδ: IC50 = 3.0 nM).17 In particular, we speculated that larger
4-(piperid-3-yl)amino moiety had
a
bigger twist angle compared to the
4-pyrrolidineamino motif linked to 6-pyridylquinazoline flat, which may contribute to
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balance the PI3Kδ-selectivity (Figure 2). Therefore, we carry out a further structural
investigation by introducing H-bond acceptor groups such as cyano, nitro and
trifluoromethyl at 5-position of 6-pyridyl moiety and several side chains such as
hydrophilic and hydrophobic groups as the tails linked to the 4-(piperid-3-yl)amino
moiety, so as to establish a comprehensive structure-activity relationship (SAR) and the
bioactive profiles. We now report the synthesis and biological evaluation of this series of
new 4-(piperid-3-yl)amino substituted 6-pyridylquinazoline analogues as potent PI3Kδ
inhibitors, notably, of which several compounds showed significant selectivity in both
PI3Kδ inhibition and anti-proliferative activity of cells lines (Figure 3).
(a)
(b)
compound
7
compound 9
Figure 2. (a) Chemical structures of 6, 7, 8 and 9; (b) 3D structures of 7 and 9.
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Figure 3. Current design of 4-(piperid-3-yl)amino substituted 6-pyridylquinazolines as
PI3Kδ inhibitors.
2. Results and discussion
2.1 Chemistry
The synthetic route of theses 4-(piperid-3-yl)amino substituted quinazoline derivatives
A1-14 was outlined in Scheme 1. 6-Bromo-4-chloroquinazoline (10) reacted with
(S)-1-Boc-3-aminopiperidine in the presence of N, N-diisopropylethylamine (DIPEA) in
refluxing
i-PrOH
gave
intermediate
11.
Subsequently,
R1-substituted
2-methoxy-3-bromopyridine reacted with bis(pinacolato)diboron under the Miyaura
borylation condition without further separation to produce R1-substituted
6-methoxypyridinylboronate, which were immediately treated with 11 using
Pd-catalysized Suzuki-coupling reaction to provide 12a-c in one pot. Removal of
Boc-group of 12a-c by TFA at room temperature afforded 13a-c. 13a was acylated with
varied acids to generate compounds A1-A9. Compounds A10 and A12 were prepared
following acylation procedure by TFAA, and compounds A11, A13 and A14 were
prepared following nucleophilic substitution procedure by ethyl bromoacetate and
bromoethanol, respectively (Scheme 1).
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Scheme 1. Reagents and conditions: (a) (S)-1-Boc-3-aminopiperidine, DIPEA, i-PrOH,
reflux, 85%; (b) (i) R1-substituted 3-bromo-6-methoxypyridine KOAc, PdCl2(dppf),
dioxane, reflux, 2.5 h; (ii) R1-substituted 6-methoxypyridinylboronate, Na2CO3,
PdCl2(dppf), dioxane/H2O, reflux, 2.5 h; 57-79 % for two steps; (c) CH2Cl2:TFA = 5:1, rt,
2 h, 23-91 %; (d) diverse acids, DMF, HATU, DIPEA, rt, 12 h, 34-73 %; (e) TFAA,
DMF, DIPEA, rt, 55 % for A10 and 57% for A12; (f) BrCH2CO2Et, CH2Cl2, DIPEA,
reflux, 43 % for A11 and 57 % for A13; (g) BrCH2CH2OH, CH3CN, DIPEA, reflux,
23 %.
2.2 PI3Kδ inhibitory activities and structure-activity relationship (SAR) study
All the newly synthesized 4-(piperid-3-yl)amino substituted 6-pyridylquinazoline
analogues were firstly assessed for their inhibitory activities against PI3Kδ. Idelalisib was
employed as the positive control. The IC50 values were illustrated in Table 1. As expected,
all the 4-(piperid-3-yl)amino substituted 6-pyridylquinazolines showed significantly
potent PI3Kδ inhibitory activities with IC50 values < 100 nM, suggesting the
4-(piperid-3-yl)amino-6-(6-methoxylpyridin-3-yl)quinazoline moiety is critical for
maintaining the PI3Kδ inhibition. Based on the previously observed SAR from
4-pyrrolidineamino-6-6-pyridylquinazoline series, the cyano group was firstly fixed at the
5-position of 6-methoxylpyridin-3-yl moiety, several R side chains including hydrophilic
and hydrophobic group were investigated. Compound A1 bearing a hydrophilic
tetrahydro-2H-pyran-4-carbonyl tail showed potent PI3Kδ inhibitory activity with an IC50
value of 7.7 nM, while replacement of the tetrahydro-2H-pyranyl group with
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4-methylpiperidyl group gave equivalent potency (A2, IC50 = 9.4 nM). Removal of the
methyl group led to compound 12c showing 4-fold improved potency with IC50 value of
2.5 nM, whereas replacement with the 2-aminopropanoyl group retained approximately
potent PI3Kδ activity (A4, IC50 = 3.7 nM). Tuning R to N,N-dimethylaminoacetyl group
resulted in improving PI3Kδ inhibitory activity (A5: IC50 = 1.3 nM) which was equal to
idelalisib (IC50 = 1.2 nM). Subsequently, compound A6 bearing a hydrophobic
chloroacetyl side chain afforded an IC50 value of 8.9 nM, while compound A7 bearing
bromopropionyl tail gave lower PI3Kδ inhibition with an IC50 value of 14.3 nM.
Moreover, the heteroaromatic tails were attempted. Compound A8 with a nicotinic
terminal showed the most potent PI3Kδ inhibitory activity with an IC50 value of 0.7 nM,
two-fold higher than idelalisib. Introduction of the 5-nitro-2-furoyl group gave compound
A9 showing PI3Kδ IC50 value of 3.9 nM. Furthermore, two hydrophobic terminal group
were synthesized leading to compounds A10 and A11 tailed trifluoroacetyl and
ethoxylcarbonylmethyl group, respectively, which displayed comparable potency, with
IC50 value of both 3.2 nM, whereas further changing the cyano group into nitro (A12,
IC50 = 27 nM) and trifluoromethyl group (A13, IC50 = 6.8 nM) on 6-pyridinyl moiety led
to the potency 9- and 2-fold reduced, respectively. An attempt to introduce the
hydroxyethyl tail led to compound A14 showed PI3Kδ inhibition retainable, with an IC50
value of 4.7 nM. Overall, in this first PI3Kδ inhibition evaluation, two compounds A5
and A8 showed IC50 values of < 2 nM, which was equivalent to or better than idelalisib,
and six compounds including A3, A4, A9, A10, A11 and A14 demonstrated IC50 values
between 2 and 5 nM, which was comparable to idelalisib.
All the target compounds were subsequently examined for their cellular
anti-proliferative activity against PI3Kδ-susceptible human malignant B-cell line
SU-DHL-6. The results were summarized in Table 1. In general, except for compound
A12 with weak anti-proliferative efficacy against SU-DHL-6, the other compounds
showed favorable anti-proliferation with micromolar IC50 values or below. Both of
compounds A5 and A8, the most potent PI3Kδ inhibitors, exhibited significantly
anti-proliferative activity against SU-DHL-6 cell line with IC50 values of 0.16 and 0.12
μM, respectively.
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Table 1. PI3Kδ inhibitory activity and SAR study of 4-(piperid-3-yl)amino substituted
quinazolines a
R1
R
PI3Kδ
IC50 (nM)
SU-DHL
IC50 (μM)
Compds
7.7 ± 2.1
9.4 ± 0.1
0.15 ± 0.01
0.42 ± 0.06
CN
A1
CN
A2
2.5 ± 0.3
3.7 ± 0.2
2.44 ± 0.43
0.44 ± 0.02
CN
CN
A3
A4
CN
CN
CN
1.3 ± 0.4
8.9 ± 1.0
14.3 ± 1.0
0.16 ± 0.05
0.38 ± 0.14
0.015 ± 0.00
A5
A6
A7
CN
CN
0.7 ± 0.1
3.9 ± 0.7
0.12 ± 0.05
0.14 ± 0.02
A8
A9
3.2 ± 0.2
3.2 ± 0.9
27.0 ± 3.1
0.21 ± 0.02
0.50 ± 0.12
127 ± 32
CN
CN
A10
A11
A12
NO2
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6.8 ± 1.8
0.23 ± 0.05
CF3
A13
CN
-
4.7 ± 2.1
1.2 ± 0.2
2.05 ± 0.32
A14
-
0.033 ± 0.10
Idelalisib
a The IC50 values are shown as the mean ± SD (nM) from two separate experiments.
2.3 Isoform selectivity of the new PI3Kδ inhibitors
To further evaluated the selectivity of the title compounds, four compounds A4, A5,
A8, and A9 were evaluated for their inhibitory activities against a panel of PI3K kinases.
The data were listed in Table 2. Obviously, compounds A4 and A5 were selective PI3Kδ
inhibitors which showed weak inhibition against PI3Kβ and PI3Kγ, and moderate
inhibition against PI3Kα. In particular, A5 displayed 17-fold, 115-fold and 192-fold
PI3Kδ selectivity over PI3Kα, PI3Kβ and PI3Kγ, respectively. A8 was a PI3Kγ/δ
inhibitor with IC50 values of 2.0 and 0.7 nM against PI3Kγ and PI3Kδ, respectively.
Notably, A8 exhibited 170-fold and 13-fold selectivity of PI3Kδ over PI3Kα and PI3Kβ,
respectively. A9 was a PI3Kα/δ inhibitor with IC50 values of 1.8 and 3.9 nM against
PI3Kα and PI3Kδ, respectively, and showed moderate inhibition against PI3Kβ and
PI3Kγ. Compounds selectively targeting PI3Kδ were thought to be capable of attenuating
side effects. However, the orally potent PI3Kδ inhibitor idelalisib afforded severe toxicity
in the clinical validation but the approved pan-PI3K inhibitor copanlisib demonstrated
lower toxicity and fewer side effects compared to idelalisib. Therefore, the PI3K isoforms
selectivity is still an open question. In this series, particularly, compound A5 was a
selective PI3Kδ inhibitor, and A8 was a PI3Kγ/δ inhibitor (Table 2).
Table 2. PI3K isoform selectivity of compounds against Class I PI3Ks a
IC50 (nM)
compds
PI3Kα
PI3Kβ
PI3Kγ
PI3Kδ
18.2 ± 1.4
21.8 ± 2.1
119.5 ± 6.2
1.8 ± 0.3
147.4 ± 9.5
150.1 ± 5.4
9.1 ± 1.2
210.7 ± 7.4
250.4 ± 8.7
2.0 ± 0.2
3.7 ± 0.2
1.3 ± 0.4
0.7 ± 0.1
3.9 ± 0.7
1.2 ± 0.2
A4
A5
A8
31.4 ± 2.1
19.3 ± 2.1
A9
177.2 ± 4.4
139.1 ± 5.2
145.3 ± 7.9
Idelalisib
a The IC50 values are shown as the mean ± SD (nM) from two separate experiments.
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2.4 Cellular anti-proliferation evaluation
Either the pan-PI3K or selective PI3Kδ (or PI3Kδ/γ) inhibitor mainly aimed to treat
cancers. Consequently, furthermore, four compounds A4, A5, A8, and A9 were tested for
their anti-proliferative activities against five cancer lines including B cell lines
SU-DHL-6, Ramos, Raji, and solid cancer lines HCT-116 and MCF-7 in vitro. Idelalisib
and BEZ235 (a clinical PI3K/mTOR dual inhibitor) were used as positive control drugs.
The data were shown in Table 3. It was found four compounds as well as idelalisib
showed significantly selective anti-proliferative activity. They were effective only against
SU-DHL-6 but no efficacy against Ramos, Raji, HCT-116 and MCF-7. Contrarily,
BEZ235 exhibited potent anti-proliferation against all five cell lines. In a word, four
compounds A4, A5, A8, and A9 showed similar anti-proliferative profiles to idelalisib,
which remarkably differed from PI3K/mTOR dual inhibitor BEZ235.
Table 3. Anti-proliferative activities of selected compounds in vitro a
IC50 (μM)
compounds
SU-DHL-6
0.44 ± 0.02
Ramos
>10
Raji
>10
>10
>10
>10
>10
HCT-116
>10
MCF-7
>10
A4
A5
0.16 ± 0.05
0.12 ± 0.05
>10
>10
>10
>10
>10
>10
A8
0.14 ± 0.02
>10
2.12 ± 0.97
>10
>10
A9
0.033 ± 0.10
-
>10
>10
Idelalisib
0.40 ± 0.00 0.52 ± 0.20 0.30 ± 0.12 0.2 ± 0.02
BEZ235
a The IC50 values are average ± SD (nM) of at least three independent experiments in triplicate.
2.5 PI3Kδ pathway cascade evaluation by AKT phosphorylation
Compound A8 was a selective PI3Kδ inhibitor, which was further investigated for
its effects on PI3K-mediated signaling in SU-DHL-6 cell line by western blot analysis.
As shown in Figure 4, compound A8 displayed good reduction of phosphorylation of
AKT (Ser473) with a concentration-dependent manner. This result indicated that
compound A8 enable to inhibit the PI3K/AKT/mTOR signaling pathway (Figure 4).
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Figure 4. Effect of compound A8 on AKT and p-AKT473. SU-DHL-6 cells were treated
with various concentration of A8 for 8h and western blot for indicated proteins was
performed. β-actin was used as a loading control.
2.6 Molecular modeling study
The molecular docking analysis of compound A8 within human PI3Kδ enzyme were
carried out to understand the potent PI3Kδ inhibition. As shown in Figure 5, the docked
pose of A8 showed the favorable interactions within the PI3Kδ binding pocket (PDB
code: 6G6W). The quinazoline scaffold was engaged in H-bond contacts with Val828 in
the hinge region, while the cyano group linked to 6-pyridyl moiety formed another
H-bond with Lys779 in the affinity pocket. Moreover, the 4-(piperid-3-yl)amino side
chain extended to solvent region and the carbonyl group generated an H-bond with
Asn836. This region was close to the PI3Kδ isoform specificity pocket and had a
hydrogen bond with Asn836 which were key factors to generate specific PI3Kδ
selectivity 15. After all, these results may account for the potent PI3Kδ inhibition for A8
(Figure 5).
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Figure 5. Molecular docking studies of compound A8 into the site of PI3Kδ (PDB code:
6G6W). Compound is shown as sticks. H-bonds within 2.5 Å are shown as yellow dashed
lines.
3. Conclusion
In summary, we described the design, synthesis and biological evaluation of a novel
series of 4-(piperid-3-yl)amino substituted 6-pyridylquinazoline derivatives as PI3Kδ
inhibitors. The structure-activity relationship was discussed and many derivatives showed
nanomolar PI3Kδ inhibitory activities. Compounds A5 and A8 displayed significantly
potent PI3Kδ inhibitory activities with IC50 values of 1.3 and 0.7 nM, respectively, which
was equivalent to or better than idelalisib (IC50 = 1.2 nM). Compounds A5 and A8
showed highly efficacious anti-proliferation against PI3Kδ-sensitive human malignant
B-cell line SU-DHL-6 in vitro with IC50 values of 0.16 and 0.12 μM. Compounds A5
showed superior isoform selectivity over PI3Kα, PI3Kβ and PI3Kγ, and A8 showed
favorable PI3Kγ/δ selectivity over PI3Kα and PI3Kβ. Interestingly, both A5 and A8
exhibited similarly potent anti-proliferation profiles to idelalisib. Western blot analysis of
A8-treated SU-DHL-6 cell line indicated that A8 reduced the AKTS473 phosphorylation.
Molecular docking simulation suggested that the high potency of A8 was attributed to the
formation of three key H-bonds. These findings indicate the 4-(piperid-3-yl)amino
substituted 6-pyridylquinazoline derivatives were potent PI3Kδ inhibitors with distinctive
PI3K-isoforms and anti-proliferation profiles.
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4. Experimental section
4.1 General methods
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a 400
Bruker NMR spectrometer with TMS as an internal reference. Mass spectra (MS) were
routinely recorded on an API 3200 LC/MS spectrometer. High-resolution MS (HRMS)
were measured by electrospray ionization (positive mode) on a quadrupole time-of-flight
(QTOF) mass spectrometer (Maxis Q-TOF, Bruker Inc.). The reagents and solvents were
purchased commercially and used without further purification unless specially described
otherwise. The starting material 6-bromo-4-chloroquinazoline (10) was prepared
according to our previously reported methods 15.
4.1.1 (S)-tert-Butyl 3-((6-bromoquinazolin-4-yl) amino) piperidine-1-carboxylate (11)
To a solution of (S)-1-Boc-3-aminopiperidine (6.20 g, 30.99 mmol) in iso-propanol
(20 mL), was slowly added 6-bromo-4-chloroquinazoline (5.00 g, 20.66 mmol) and
DIPEA (5.33 g, 41.32mmol). The mixture was stirred at 85℃ for 2.5 h, and cooled to
room temperature. After the saturated NaCl solution was added, the reaction mixture was
extracted by EtOAc. Dried over anhydrous Na2SO4, the liquid was concentrated and
purified by silica gel chromatography (CH2Cl2/CH3OH, 100:1) to produce compound 11
(7.15 g, 85 %) as a yellow solid. 1H-NMR (400 MHz, CDCl3) δ 8.66 (s, 1H, ArH), 7.98 (s,
1H, ArH), 7.89-7.79 (m, 2H, ArH), 6.98 (s, 1H, NH), 4.41-4.39 (m, 1H, CH), 3.82-3.32
(m, 4H, 2×CH2), 2.45-2.28 (m, 2H, CH2), 1.98-1.75 (m, 2H, CH2), 1.48 (s, 9H, 3×CH3)
ppm. MS (ESI) m/z: [M+H]+ =406.9, 408.9.
4.1.2
(S)-tert-Butyl
3-((6-(6-methoxypyridin-3-yl)quinazolin-4-yl)
amino)
piperidine-1-carboxylate (12a)
Compound 12a was prepared according our reported method. To a solution mixture
of 11 (1.20 g, 2.95mmol) in DME/H2O (8 mL/2 mL), were added the
2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinonitrile (1.01 g, 3.9
mmol), PdCl2(dppf) (270 mg, 0.12 mmol) and Na2CO3 (636 mg, 6.0 mmol). The
mixture was refluxed under argonatmosphere for 4 h, cooled to room temperature and
concentrated. The residue was purified by silica gel chromatography (CH2Cl2/CH3OH,
20:1) to give compound 12a (1.10 g, 80 %) as a brown solid. MS (ESI) m/z: [M+H]+
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=461.2.
4.1.3
(S)-tert-Butyl
3-((6-(6-methoxy-5-nitropyridin-3-yl)quinazolin-4-yl)amino)
piperidine-1-carboxylate (12b)
To a solution mixture of 11 (500 mg, 1.23mmol) in DME/H2O (8 mL/2 mL), were
added
2-methoxy-3-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
(518.2 g, 1.85 mmol), PdCl2(dppf) (75 g, 0.12 mmol) and Na2CO3 (182 mg, 1.72 mmol).
The mixture was refluxed under N2 atmosphere for 4 h and then the mixture was
concentrated and purified by silica gel chromatography (CH2Cl2/CH3OH, 20:1) to give
compound 12b (531 mg, 90 %) as a brown solid. MS (ESI) m/z: [M+H]+ =481.2.
4.1.4
(S)-tert-Butyl
3-((6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)quinazolin-4-yl)amino)piperidine-1-
carboxylate (12c)
To a solution mixture of 11 (350 mg, 0.86 mmol) in DME/H2O (8 mL/2 mL), were
added the 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)
pyridine (561 mg, 1.85 mmol), PdCl2(dppf) (73.2 mg, 0.1 mmol) and Na2CO3 (182 mg,
1.72 mmol). The mixture was refluxed under N2 atmosphere for 4 h and then the mixture
was concentrated and purified by silica gel chromatography (CH2Cl2/CH3OH, 20:1) to
give compound 12c (510 mg, 81 %) as a brown solid. MS (ESI) m/z: [M+H]+ = 504.2.
4.1.5
(S)-2-Methoxy-5-(4-((1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-3-yl)amino)quinaz
olin-6-yl)nicotinonitrile (A1)
To a mixture of 12a (200 mg, 0.43 mmol) in CH2Cl2 (5 mL), was added TFA (1 mL).
The mixture was stirred at room temperature for 2h. The mixture was concentrated, and
then the residue was dissolved in CH2Cl2 (5 mL), were added
tetrahydro-2H-pyran-4-carboxylic acid (85 mg, 0.63 mmol) and HATU (326 mg, 0.86
mmol) and DIPEA (77.4 mg, 0.6mmol). The reaction mixture was stirred at room
temperature for 12h. The mixture concentrated and purified by silica gel
1
chromatography to give compound A1 (107 mg, 41 %) as a white solid. H NMR (400
MHz, DMSO-d6) δ 8.96 (s, 1H, Ar-H), 8.73 (s, 1H, NH), 8.63 (d, J = 19.0 Hz, 1H,
Ar-H), 8.50 (d, J = 7.4 Hz, 1H, Ar-H), 8.19 (s, 1H, Ar-H), 8.09 – 8.00 (m, 1H, Ar-H),
7.79 (s, 1H Ar-H), 4.30-4.25 (m, 1H, CH), 4.08 (s, 3H, OCH3), 4.03 – 3.81 (m, 2H,
15

JOURNAL PRE-PROOF
CH2), 3.47 – 3.36 (m, 2H, CH2), 3.22-3.15 (m, 1H, CH2), 3.05-2.72 (m, 2H, CH2), 2.60–
2.50 (m, 1H, CH2), 2.15-2.10 (m, 1H, CH2), 1.90-1.78 (m, 2H, CH2), 1.71 – 1.46 (m, 4H
CH2×2), 1.29-1.20 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 176.1, 172.7, 163.3,
159.5, 155.5, 150.2, 142.2, 132.6, 131.3, 128.9, 128.7, 120.9, 115.7, 115.4, 96.2, 66.8,
66.7, 55.4, 55.2, 54.1, 49.3, 37.3, 29.0, 18.5, 17.2, 13.0. ESI-HRMS C26H28N6O3, calcd
[M+H]+: 473.2296, found: 473.2306.
4.1.6
(S)-2-Methoxy-5-(4-((1-(1-methylpiperidine-4-carbonyl)piperidin-3-yl)amino)quinazol
in-6-yl)nicotinonitrile (A2)
Using the similar procedure of A1, A2 was prepared s a white solid. Yield: 56 %; 1H
NMR (400 MHz, DMSO-d6) δ 8.94 (d, J = 2.2 Hz, 1H, Ar-H), 8.70 (d, J = 2.3 Hz, 1H,
NH), 8.61 (d, J = 14.0 Hz, 1H, Ar-H), 8.47 (d, J = 4.4 Hz, 1H, Ar-H), 8.15 (d, J = 8.6 Hz,
1H, Ar-H), 8.04 (dd, J = 18.6, 6.8 Hz, 1H, Ar-H), 7.76 (d, J = 8.6 Hz, 1H, Ar-H), 4.30 –
4.23 (m, 1H, CH), 4.06 (s, 3H, OCH3), 3.18 – 2.92 (m, 4H CH2×2), 2.80 – 2.53 (m, 4H
CH2×2), 2.16 (s, 3H CH3), 2.13 – 1.85 (m, 3H CH2+CH), 1.84 – 1.70 (m, 2H CH2), 1.67
– 1.58 (m, 2H CH2), 1.52 – 1.31 (m, 2H CH2). 13C NMR (100 MHz, DMSO-d6) δ 171.8,
163.4, 159.5, 155.8, 155.7, 150.3, 149.4, 142.3, 132.7, 131.4, 128.9, 121.0, 115.7, 115.5,
96.3, 55.2, 53.6, 53.4, 49.3, 47.4, 46.1, 43.5, 42.1, 35.4, 30.2, 26.4, 24.4. ESI-HRMS
C27H31N7O2, calcd [M+H]+: 486.2612, found: 486.2617.
4.1.7
(S)-2-Methoxy-5-(4-((1-(piperidine-4-carbonyl)piperidin-3-yl)amino)
quinazolin-6-yl)nicotinonitrile (A3)
Using the similar procedure of A1, A3 was prepared as a white solid. Yield: 56 %;
1H NMR (400 MHz, DMSO-d6) δ 8.99 (t, J = 1.8 Hz, 1H, Ar-H), 8.85 – 8.80 (m, 1H,
NH), 8.76 (d, J = 1.8 Hz, 1H, Ar-H), 8.65 – 8.60 (m, 1H, Ar-H), 8.39 – 8.34 (m, 2H,
Ar-H), 7.88 (dd, J = 5.7, 2.6 Hz, 1H, Ar-H), 4.40 – 4.35 (m, 1H, CH), 4.26 – 4.20 (m, 1H,
NH), 4.06 (s, 3H, OCH3), 4.05 – 3.95 (m, 2H, CH2), 3.40 – 3.20 (m, 2H, CH2), 3.18 –
2.92 (m, 4H, CH2×2), 2.80 – 2.53 (m, 4H, CH2), 2.20 – 2.10 (m, 1H CH), 1.95 – 1.65 (m,
13
6H CH2×3), 1.60 – 1.40 (m, 1H, CH2). C NMR (101 MHz, DMSO-d6) δ 171.9, 163.3,
159.5, 158.6, 155.7, 150.2, 142.2, 132.6, 131.3, 128.9, 128.7, 121.1, 119.2, 115.7, 115.5,
96.2, 55.2, 49.3, 46.0, 43.1, 42.9, 42.6, 35.7, 34.6, 25.8, 25.5, 16.0. ESI-HRMS
C26H29N7O2, calcd [M+H]+: 472.2456, found: 472.2461.
16

JOURNAL PRE-PROOF
4.1.8
(S)-5-(4-((1-Alanylpiperidin-3-yl)amino)quinazolin-6-yl)-2-
methoxynicotinonitrile (A4)
Using the similar procedure of A1, A4 was prepared as a white solid. Yield: 64 %;
1H NMR (400 MHz, DMSO-d6) δ 8.96 (s, 1H, Ar-H), 8.86 (d, J = 6.4 Hz, 1H, Ar-H),
＋
8.73 (d, J = 2.0 Hz, 1H, Ar-H), 8.38 (d, J = 8.7 Hz, 1H, Ar-H), 8.17 (s, 2H, NH2 ), 7.92
(dd, J = 8.6, 4.8 Hz, 1H, Ar-H), 4.67 – 4.38 (m, 3H, CH+NH2), 4.08 (s, 3H OCH3), 4.04
–3.89 (m, 1H, CH), 3.20-2.72 (m, 2H, CH2), 2.20-2.00 (m, 1H, CH2), 2.00-1.80 (m, 2H,
13
CH2), 1.55 – 1.45 (m, 1H CH2), 1.40-1.30 (m, 3H, CH3), 1.30-1.25 (m, 2H, CH2). C
NMR (100 MHz, DMSO-d6) δ 173.8, 163.7, 159.4, 155.5, 155.3, 149.0, 145.6, 138.2,
134.8, 131.4, 128.8, 128.7, 120.4, 115.5, 111.1, 53.8, 49.3, 48.8, 46.5, 42.4, 30.3, 24.3,
21.1. ESI-HRMS C23H25N7O2, calcd [M+H]+: 432.2143, found: 432.2140.
4.1.9
(S)-5-(4-((1-(Dimethylglycyl)piperidin-3-yl)amino)quinazolin-6-yl)-2-methoxynicotinon
itrile (A5)
Using the similar procedure of A1, A5 was prepared as a white solid. Yield: 64 %;
1H NMR (400 MHz, DMSO-d6) δ 8.94 (d, J = 2.2 Hz, 1H Ar-H), 8.70 (s, 1H NH), 8.62
(d, J = 10.4 Hz, 1H Ar-H), 8.56 – 8.47 (m, 1H Ar-H), 8.18 – 8.13 (m, 1H Ar-H), 8.04 (d,
J = 6.6 Hz, 1H Ar-H), 7.76 (dd, J = 8.6, 5.4 Hz, 1H Ar-H), 4.25 – -4.10 (m, 1H, CH),
4.06 (s, 3H, OCH3), 4.00 – 3.50 (m, 2H, CH2), 3.45 – 3.20 (m, 2H, CH2), 3.10 – 2.70 (m,
2H, CH2), 2.45 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.11 – 1.75 (m, 2H, CH2), 1.74 – 1.43 (m,
13
2H, CH2). C NMR (100 MHz, DMSO-d6) δ 163.3, 159.4, 159.4, 155.8, 150.3, 149.3,
142.3, 132.6, 131.4, 129.0, 128.8, 121.0, 115.7, 115.4, 96.2, 55.4, 55.2, 49.1, 48.5, 47.35,
46.0, 44.9, 44.7, 24.7, 24.1. ESI-HRMS C24H27N7O2, calcd [M+H]+: 446.2299, found:
446.2301.
4.1.10
(S)-5-(4-((1-(2-Chloroacetyl)piperidin-3-yl)amino)quinazolin-6-yl)-2-methoxynicotino
nitrile (A6)
Using the similar procedure of A1, A6 was prepared as a white solid. Yield: 70 %;
1H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H Ar-H), 8.71 (d, J = 2.3 Hz, 1H, NH), 8.62
(d, J = 11.2 Hz, 1H, Ar-H), 8.51 (d, J = 11.9 Hz, 1H, Ar-H), 8.16 (d, J = 8.4 Hz, 1H,
Ar-H), 8.07 (dd, J = 11.6, 7.4 Hz, 1H Ar-H), 7.79 – 7.74 (m, 1H Ar-H), 4.55 – 4.50 (m,
17

JOURNAL PRE-PROOF
1H, CH), 4.44 – 4.29 (m, 2H CH2), 4.26-4.18 (m, 2H, CH2), 4.07 (s, 3H OCH3), 3.17 –
3.06 (m, 1H, CH2), 2.82 – 2.65 (m, 1H, CH2), 2.15 – 1.75 (s, 2H, CH2), 1.78 – 1.51 (m,
2H CH2).13C NMR (100 MHz, DMSO-d6) δ 165.2, 163.3, 159.4, 155.8, 150.3, 149.2,
142.3, 132.7, 131.3, 129.0, 129.0, 128.7, 121.0, 115.7, 115.4, 96.2, 55.4, 55.2, 49.1, 47.4,
42.7, 42.5, 30.3, 24.0. ESI-HRMS C22H21ClN6O2, calcd [M+H]+: 437.1487, found:
437.1487.
4.1.11
(S)-5-(4-((1-(2-Bromopropionyl)piperidin-3-yl)amino)quinazolin-6-yl)-2-methoxynico
tinonitrile (A7)
Using the similar procedure of A1, A7 was prepared as a white solid. Yield: 35 %;
1H NMR (400 MHz, DMSO-d6) δ 8.93 (d, J = 2.4 Hz, 1H, Ar-H), 8.69 (d, J = 1.8 Hz, 1H,
Ar-H), 8.59 (s, 1H, NH), 8.51 (d, J = 12.8 Hz, 1H, Ar-H), 8.15 (dd, J = 8.7, 1.3 Hz, 1H
Ar-H), 8.05 – 8.01 (m, 1H, Ar-H), 7.76 (d, J = 8.7 Hz, 1H Ar-H), 6.89 – 6.74 (m, 1H,
CH2), 6.15 – 6.08 (m, 1H, CH2), 5.74 – 5.60 (m, 1H, CH2), 4.67 – 4.20 (m, 3H CH +
CH2), 4.06 (s, 3H OCH3), 3.17 – 2.68 (m, 2H, CH2), 2.14 – 1.85 (m, 2H, CH2), 1.85 –
13
1.53 (m, 2H, CH2), 1.28-1.07 (m, 1H, CH2). C NMR (100 MHz, DMSO-d6) δ 164.9,
163.3, 155.8, 150.2, 149.2, 142.2, 132.6, 131.3, 129.0, 128.7, 127.7, 120.9, 115.7, 115.4,
96.20, 55.2, 49.7, 48.7, 47.5, 30.3, 25.4, 24.1. ESI-HRMS C23H24BrN6O2, calcd [M+H]+:
495.1139, found: 495.1158.
4.1.12
nicotinonitrile (A8)
Using the similar procedure of A1, A8 was prepared as a white solid. Yield: 67 %;
(S)-2-Methoxy-5-(4-((1-nicotinoylpiperidin-3-yl)amino)quinazolin-6-yl)
1H-NMR (400 MHz, DMSO-d6) δ 9.30 – 8.97 (m, 2H, Ar-H), 8.74 – 8.48 (m, 5H,
NH+Ar-H), 8.29 – 8.15 (m, 1H Ar-H), 7.90 – 7.76 (m, 2H, Ar-H), 7.52 – 7.40 (m, 1H
Ar-H), 4.30 – 4.25 (m, 1H, CH), 4.04 (s, 3H OCH3), 3.90 – 3.51 (m, 2H CH2), 3.25 –
2.90 (m, 2H, CH2), 2.23 – 2.00 (m, 1H, CH2), 2.00 – 1.75 (m, 2H, CH2), 1.75 – 1.60 (m,
13
1H, CH2). C NMR (100 MHz, DMSO-d6) δ 167.5, 163.4, 159.7 154.1, 154.0, 150.8,
150.7, 150.7, 150.3, 148.1, 147.9, 142.3, 135.2, 132.5, 132.2, 128.5, 126.2, 123.0, 121.7,
121.5, 115.6, 114.8, 96.2, 55.2, 53.8, 49.0, 42.1, 18.5, 17.2. ESI-HRMS C26H23N7O2,
calcd [M+H]+: 466.1986, found: 466.1984.
4.1.13
(S)-2-Methoxy-5-(4-((1-(5-nitrofuran-2-carbonyl)piperidin-3-yl)amino)
18

JOURNAL PRE-PROOF
quinazolin-6-yl)nicotinonitrile (A9)
Using the similar procedure of A1, A9 was prepared as a white solid. Yield: 36 %;
1H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H, Ar-H), 8.70 (s, 1H, Ar-H), 8.60 (s, 1H,
Ar-H), 8.50 (s, 1H, NH), 8.15 (d, J = 8.4 Hz, 1H, Ar-H), 8.01 (s, 1H, Ar-H), 7.79 (d, J =
3.8 Hz, 1H, Ar-H), 7.75 (d, J = 8.0 Hz, 1H, Ar-H), 7.58 (s, 1H Ar-H), 4.38 – 4.30 (m, 1H,
CH), 4.29 – 4.09 (m, 2H, CH2), 4.06 (s, 3H, OCH3), 3.40 – 3.30 (m, 2H CH2), 2.20 – 2.13
(m, 1H, CH2), 1.98 – 1.75 (m, 2H, CH2), 1.70 – 1.65 (m, 1H, CH2). 13C NMR (100 MHz,
DMSO-d6) δ 163.3, 159.3, 157.4, 155.4, 151.6, 150.3, 149.3, 142.3, 140.8, 132.7, 131.3,
129.0, 128.8, 121.0, 116.9, 115.7, 115.3, 113.3, 96.2, 55.4, 55.2, 50.6, 43.5, 29.8, 23.8.
ESI-HRMS C25H21N7O5, calcd [M+H]+: 500.1677, found: 500.1679.
4.1.14
(S)-2-Methoxy-5-(4-((1-(2,2,2-trifluoroacetyl)piperidin-3-yl)amino)
quinazolin-6-yl)nicotinonitrile (A10)
Using the similar procedure of A1, A10 was prepared as a white solid. Yield: 20 %;
1H NMR (400 MHz, DMSO-d6) δ 8.99 (d, J = 1.5 Hz, 1H, Ar-H), 8.83 (d, J = 2.3 Hz, 1H,
Ar-H), 8.65 (dd, J = 6.4, 1.6 Hz, 1H, Ar-H), 8.52 (d, J = 4.0 Hz, 1H, NH), 8.20 (dd, J =
8.7, 1.8 Hz, 1H, Ar-H), 8.19 – 8.10 (m, 1H, Ar-H), 7.80 (d, J = 8.7 Hz, 1H, Ar-H), 4.38
– 4.30 (m, 1H, CH), 4.11 (s, 3H OCH3), 3.33 – 3.10 (m, 2H CH2), 2.15 – 2.05 (m, 1H,
CH2), 2.00 – 1.94 (m, 1H, CH2), 1.88 – 1.75 (m, 1H, CH2), 1.70 – 1.55 (m, 1H, CH2),
1.30 – 1.20 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 163.4, 159.6, 159.5, 155.4,
150.3, 142.4, 133.0, 133.0, 131.7, 129.0, 128.2, 128.1, 121.1, 115.7, 115.2, 96.3, 55.2,
54.1, 42.3, 18.5, 17.2, 13.0. ESI-HRMS C22H19F3N6O2, calcd [M+H]+: 457.1594, found:
457.1597.
4.1.15
Ethyl
(S)-2-(3-((6-(5-cyano-6-methoxypyridin-3-yl)quinazolin-4-yl)amino)
piperidin-1-yl)acetate (A11)
Using the similar procedure of A1, A11 was prepared as a white solid. Yield: 57 %;
1H NMR (400 MHz, DMSO-d6) δ 8.92 (d, J = 2.5 Hz, 1H), 8.69 (d, J = 2.5 Hz, 1H, Ar-H),
8.58 (d, J = 1.6 Hz, 1H, Ar-H), 8.47 (s, 1H, NH), 8.13 (dd, J = 8.7, 1.8 Hz, 1H, Ar-H),
7.91 (d, J = 7.8 Hz, 1H, Ar-H), 7.73 (d, J = 8.7 Hz, 1H, Ar-H), 4.43 – 4.33 (m, 1H, CH),
4.12 – 4.06 (m, 2H CH2), 4.06 (s, 3H, OCH3), 3.32 – 3.25 (m, 2H, CH2), 3.08 – 3.00 (m,
1H, CH2), 2.81 – 2.75 (m, 1H, CH2), 2.36 – 2.30 (m, 2H, CH2), 2.01 – 1.91 (m, 1H, CH2),
13
1.79 – 1.71 (m, 1H, CH2), 1.64 – 1.38 (m, 2H CH2), 1.18 (t, J = 7.1 Hz, 3H, CH3). C
19

JOURNAL PRE-PROOF
NMR (100 MHz, DMSO-d6) δ 170.7, 163.3, 159.3, 155.9, 150.2, 149.2, 142.2, 132.5,
131.1, 129.0, 128.7, 120.8, 115.7, 115.4, 96.2, 60.3, 58.9, 57.3, 55.2, 52.4, 47.9, 29.9,
24.5, 14.6. ESI-HRMS C24H26N6O3, calcd [M+H]+: 447.2139, found: 447.2132.
4.1.16 Ethyl (S)-2,2,2-trifluoro-1-(3-((6-(6-methoxy-5-nitropyridin-3-yl)quinazolin-4-yl)
amino)piperidin-1-yl)ethan-1-one (A12)
Using the similar procedure of A1, A12 was prepared as a white solid. Yield: 52 %;
1H NMR (400 MHz, DMSO-d6) δ 8.99 (d, J = 1.5 Hz, 1H, Ar-H), 8.82 (d, J = 2.3 Hz, 1H,
NH), 8.65 (dd, J = 6.4, 1.6 Hz, 1H, Ar-H), 8.52 (d, J = 4.0 Hz, 1H, Ar-H), 8.20 (dd, J =
8.7, 1.8 Hz, 1H, Ar-H), 8.15 – 8.06 (m, 1H, Ar-H), 7.79 (d, J = 8.7 Hz, 1H, Ar-H), 4.43 –
4.33 (m, 1H, CH), 4.10 (s, 3H OCH3), 3.33 – 3.11 (m, 2H CH2), 2.14 – 2.00 (m, 1H,
CH2), 2.00 – 1.93 (m, 1H, CH2), 1.90 – 1.75 (m, 1H, CH2), 1.65 – 1.50 (m, 1H, CH2),
1.27 – 1.20 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 159.5, 159.4, 155.8, 155.4,
150.2, 149.3, 134.3, 133.4, 132.4, 131.5, 129.3, 128.8, 121.2, 121.2, 115.4, 55.3, 47.9,
47.2, 43.8, 24.8, 23.4. ESI-HRMS C21H19F3N6O4, calcd [M+H]+: 477.1493, found:
477.1493.
4.1.17
Ethyl
(S)-2-(3-((6-(5-cyano-6-methoxypyridin-3-yl)quinazolin-4-yl)amino)piperidin-1-yl)acet
ate (A13)
Using the similar procedure of A1, A13 was prepared as a white solid. Yield: 50 %;
1H NMR (400 MHz, DMSO-d6) δ 8.92 (s, 1H Ar-H), 8.62 (s, 1H NH), 8.49 (s, 1H Ar-H),
8.45 (s, 1H Ar-H), 8.17 (d, J = 8.5 Hz, 1H Ar-H), 7.96 (d, J = 7.6 Hz, 1H Ar-H), 7.76 (d,
J = 8.6 Hz, 1H Ar-H), 4.43 – 4.33 (m, 1H, CH), 4.12 – 4.06 (m, 2H CH2), 4.06 (s, 3H,
OCH3), 3.32 – 3.25 (m, 2H, CH2), 3.08 – 3.00 (m, 1H, CH2), 2.81 – 2.75 (m, 1H, CH2),
2.36 – 2.30 (m, 2H, CH2), 2.01 – 1.91 (m, 1H, CH2), 1.79 – 1.71 (m, 1H, CH2), 1.64 –
13
1.38 (m, 2H CH2), 1.19 (t, J = 7.1 Hz, 3H, CH3). C NMR (100 MHz, DMSO-d6) δ
170.7, 160.0, 159.3, 155.8, 149.8, 149.2, 135.7, 133.1, 131.5, 128.8, 128.7, 122.2, 121.0,
115.5, 111.8, 60.3, 58.9, 57.3, 54.8, 52.4, 47.9, 29.9, 24.5, 14.6. ESI-HRMS
C24H26F3N5O3, calcd [M+H]+: 490.2061, found: 490.2058.
4.1.18
(S)-5-(4-((1-(2-Hydroxyethyl)piperidin-3-yl)amino)quinazolin-6-yl)-2-methoxynicotino
nitrile (A14)
20

JOURNAL PRE-PROOF
Using the similar procedure of A1, A14 was prepared as a white solid. Yield: 53 %;
1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H Ar-H), 8.74 – 8.68 (m, 1H Ar-H), 8.64 –
8.58 (m, 1H Ar-H), 8.51 – 8.47 (m, 1H Ar-H), 8.19 – 8.13 (m, 1H Ar-H), 8.07 – 7.95 (m,
1H NH), 7.78 – 7.73 (m, 1H Ar-H), 4.55 – 4.35 (m, 1H CH), 4.07 (s, 3H OCH3), 3.60 –
3.50 (m, 2H CH2), 3.40 – 3.30 (m, 2H CH2), 3.07 (s, 1H OH), 2.75 – 2.65 (m, 2H CH2),
2.40 – 2.25 (m, 2H, CH2), 2.05 – 1.90 (m, 1H, CH2), 1.85 – 1.75 (m, 1H, CH2), 1.70 –
1.45 (m, 1H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 163.5, 160.1, 153.8, 150.4, 142.4,
134.1, 133.1, 128.5, 121.6, 119.0, 116.0, 115.6, 114.6, 113.1, 96.3, 58.9, 55.4, 55.3, 52.3,
46.08, 39.9, 27.8, 21.6. ESI-HRMS C22H24N6O2, calcd [M+H]+: 405.2034, found:
405.2062.
4.2 PI3K enzymatic activity assay
The PI3K isoform activity assay was performed as our previous procedures. 18 All
o
the PI3K isoform reaction was conducted at 30 C for 60 min. The 50 μL reaction
mixture contains 50 mM HEPES (pH 7.5), 50 mM NaCl, 3 mM MgCl2, 0.025 mg/mL
BSA, 0.2 μg/mL PI3K, 10 μM ATP and 100 μM PI substrate. The tested compounds
were diluted in 10% DMSO and 5 μL of the dilution was added to a 50 μL reaction so
that the final concentration of DMSO is 1% in all the reactions. After the plate was
cooled at room temperature for 5ꢀmin, 5ꢀμL of ADP-Glo reagent was added into each well
to stop the reaction and consume the remaining ADP within 40 min. At the end, 10ꢀμL of
kinase detection reagent was added into the well and incubated for 30ꢀmin. The assay was
performed using Kinase-Glo Plus luminescence kinase assay. The luminescence signal
was detected by VICTOR-X multi-label plate reader. The IC50 values were calculated
using nonlinear regression with normalized dose−response fit using GraphPad Prism 5.0
sofeware.
4.3 Anti-proliferative assays using a MTT method in vitro
Cellular proliferation was determined using a MTT method in vitro.19
4.4 Western blot assay
The levels of Akt and phospho-Akt (p-Akt, S473) in SU-DHL-6 cells were
determined by Western blot. SU-DHL-6 cells were treated with final concentrations of
1000 nM, 100 nM, and 10 nM of A8 and with the concentrations of 1000 and 100 nM of
idelalisib for 8 h. Cell lysates were clarified by centrifugation at 12,000 rpm for 20 min at
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4 ℃, and supernatant was collected. Equal amounts of protein were subjected to
SDS-PAGE and transferred to nitrocellulose membranes (Merck Millipore, Billerica, MA,
USA). The membranes were blocked and incubated overnight at 4 °C with primary
antibodies against p-Akt, Akt, and beta-actin, followed by incubation with appropriate
secondary antibodies. Antibody binding was detected with chemiluminescence reagents
(Sigma-Aldrich, St. Louis, MO, USA).
4.5 Molecular docking simulation
The molecular docking was performed as our previous reported methods.19
Acknowledgements
This work was supported by the Natural Science Foundation of Shaanxi Province,
China (2019JM-023). The authors also thank the Talent funding Program of Health
Science Center, Xi’an Jiaotong University.
Appendix A. Supplementary data
Supplementary data to this article can be found attached.
References
[1] Liu P, Cheng H, Roberts TM, Zhao J. Targeting the phosphoinositide 3-kinase
pathway in cancer. Nat. Rev. Drug. Disc. 2009, 8(8): 627-644.
[2] Garces AE, Stocks MJ, Class 1 PI3K clinical candidates and recent inhibitor design
strategies: a medicinal chemistry perspective. J. Med. Chem. 2019, 62(10):
4815-4850.
[3] Wei M, Wang X, Song Z, Jiao M, Ding J, Meng LH, Zhang A. Targeting PI3Kδ:
emerging therapy for chronic lymphocytic leukemia and beyond. Med. Res. Rev.
2015, 35(4): 720-752.
[4] Perry MWD, Abdulai R, Mogemark M, Petersen J, Thomas MJ, Valastro B, Westin
Eriksson A. Evolution of PI3Kγ and δ inhibitors for inflammatory and autoimmune
diseases. J. Med. Chem. 2019, 62(10): 4783-4814.
[5] Fruman DA, Cantley LC. Idelalisib - a PI3Kδ inhibitor for B-cell cancers. N. Eng. J.
Med. 2014, 370(11): 1061-1062.
[6] Markham A. Idelalisib: first global approval. Drugs 2014, 74(14): 1701-1707.
[7] Blair HA. Duvelisib: first global approval. Drugs 2018, 78(17): 1847-1853.
[8] Barrientos JC. Can umbralisib bring PI3Kδ out of the shadows? Lancet Oncol. 2018,
19(4): 432-434.
[9] Hoegenauer K, Soldermann N, Stauffer F, Furet P, Graveleau N, Smith AB, Hebach
C, Hollingworth GJ, Lewis I, Gutmann S, Rummel G, Knapp M, Wolf RM,
Blanz J, Feifel R, Burkhart C, Zecri F. Discovery and pharmacological
characterization of novel quinazoline-based PI3K delta-selective inhibitors. ACS
22

JOURNAL PRE-PROOF
Med. Chem. Lett. 2016, 7(8): 762–767.
[10]Hoegenauer K, Soldermann N, Zecri F, Strang RS, Graveleau N, Wolf RM, Cooke
NG, Smith AB, Hollingworth GJ, Blanz J, Gutmann S, Rummel G,
Littlewood-Evans A, Burkhart C. Discovery of CDZ173 (Leniolisib), representing a
structurally novel class of PI3K delta-selective inhibitors, ACS Med. Chem.
Lett. 2017, 8(9): 975-980.
[11]Down K, Amour A, Baldwin IR, Cooper AW, Deakin AM, Felton LM, Guntrip SB,
Hardy C, Harrison ZA, Jones KL, Jones P, Keeling SE, Le J, Livia S, Lucas F,
Lunniss CJ, Parr NJ, Robinson E, Rowland P, Smith S, Thomas DA, Vitulli G,
Washio Y, Hamblin JN. Optimization of Novel Indazoles as Highly Potent and
Selective Inhibitors of Phosphoinositide 3-Kinase δ for the Treatment of Respiratory
Disease. J Med Chem. 2015, 58(18): 7381-7399.
[12]Berndt A, Miller S, Williams O, Le DD, Houseman BT, Pacold JI, Gorrec F, Hon
WC, Liu Y, Rommel C, Gaillard P, Rückle T, Schwarz MK, Shokat KM, Shaw
JP, Williams
RL.
The p110 delta structure: mechanisms for selectivity and potency of new PI(3)K inhi
bitors. Nat Chem Biol. 2010, 6(2):117-24.
[13]Hoegenauer K, Soldermann N, Hebach C, Hollingworth GJ, Lewis I, Von Matt A,
Burkhart C. Discovery of novel pyrrolidineoxy-substituted heteroaromatics as potent
and selective PI3K delta inhibitors with improved physicochemical
properties. Bioorg. Med. Chem. Lett, 2016, 26(23): 5657-5662.
[14]Xin M, Hei YY, Zhang H, Xie XX, Mao S, Zuo SJ, Zhang SQ. Discovery of
6-benzamide containing 4-phenylquinazoline derivatives as novel PI3Kδ inhibitors.
Lett. Drug Des. Dis. 2017, 14(2): 167-174.
[15]Xin M, Hei YY, Zhang H, Shen Y, Zhang SQ. Design and synthesis of novel 6-aryl
substituted 4-anilinequinazoline derivatives as potential PI3Kδ inhibitors. Bioorg.
Med. Chem. Lett. 2017, 27(9): 1972-1977.
[16]Xin M, Hei YY, Zhang H, Shen Y, Zhao HY, Mao S, Zhang SQ. Novel 6-aryl
substituted 4-pyrrolidineaminoquinazoline derivatives as potent phosphoinositide
3-kinase delta (PI3Kδ) inhibitors Bioorg. Med. Chem. 2018, 26(8): 2028-2040.
[17]Xin M, Duan W, Feng Y, Hei YY, Zhang H, Shen Y, Zhao HY, Mao S, Zhang SQ.
Introduction of pyrrolidineoxy or piperidineamino group at the 4-position of
quinazoline leading to novel quinazoline-based phosphoinositide 3-kinase delta
(PI3Kδ) inhibitors. J. Enzyme Inhib. Med. Chem, 2018, 33(1): 651-656.
[18]Hei YY, Zhang SQ, Feng Y, Wang J, Duan W. Zhang H, Mao S, Sun H, Xin M.
Alkylsulfonamide-containing quinazoline derivatives as potent and orally
bioavailable
PI3Ks
inhibitors.
Bioorg.
Med.
Chem.
2019,
https://doi.org/10.1016/j.bmc.2019.05.043
[19]Hei YY, Xin M, Zhang H, Xie XX, Mao S, Zhang SQ. Synthesis and antitumor
activity evaluation of 4,6-disubstituted quinazoline derivatives as novel PI3K
inhibitors. Bioorg. Med. Chem. Lett. 2016, 26(18): 4408-4413.
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JOURNAL PRE-PROOF
Synthesis and Biological Evaluation of 4-(Piperid-3-yl)amino Substituted
6-Pyridylquinazolines as Potent PI3Kδ Inhibitors
Yifan Feng, Weiming Duan, Shu Fan, Hao Zhang, San-Qi Zhang, Minhang Xin *
Department of Medicinal Chemistry, School of Pharmacy, Health Science Center,
Xi’an Jiaotong University, 76 West Yanta Road, Xi’an, Shaanxi 710061, P.R. China
Graphical Abstract:
* Corresponding author: E-mail: xmhcpu@163.com.