Structurally novel PI3Kδ/γ dual inhibitors characterized by a seven-membered spirocyclic spacer: The SARs investigation and PK evaluation

Article abstract of DOI:10.1016/j.ejmech.2020.112143

Herein, we communicate our recent medicinal chemistry efforts which have culminated in a series of PI3Kδ/γ dual inhibitors structurally featuring a seven-membered spirocyclic spacer. Compound 26, the most potent one among them, exhibited superior PI3Kδ inhibitory activity (IC₅₀ = 1.0 nM) to that of the approved PI3Kδ inhibitor Idelalisib. Besides, it exerted remarkable anti-proliferative efficacy against human malignant B-cell line SU-DHL-6 with GI₅₀ value of 33 nM. The biochemical assay against the other three class I PI3K isoforms identified compound 26 as a potent PI3Kδ/γ dual inhibitor with considerable selectivity over PI3Kα and PI3Kβ. In SU-DHL-6 cells, a dramatic down-regulation of PI3K signaling was observed following compound 26-treatment at the concentration as low as 10 nM. Inspiringly, the pharmacokinetic (PK) study in Sprague-Dawley (SD) rats revealed it was orally available with a favorable bioavailability (F = 87.5%). Overall, compound 26, a promising PI3Kδ/γ dual inhibitor, has the potential to emerge as a clinical candidate for the treatment of leukocyte-mediated malignancies after extensive functional investigation.

Full text of DOI:10.1016/j.ejmech.2020.112143

Journal Pre-proof
Structurally novel PI3Kδ/γ dual inhibitors characterized by a seven-membered
spirocyclic spacer: The SARs investigation and PK evaluation
Qiangqiang Tao, Yuqing Chen, Xiao Liang, Yongzhou Hu, Jiaming Li, Fang Fang,
Huchuan Wang, Chang Meng, Jingtai Liang, Xiaodong Ma, Shuangying Gui
PII:
S0223-5234(20)30110-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112143
EJMECH 112143
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 December 2019
Revised Date: 29 December 2019
Accepted Date: 11 February 2020
Please cite this article as: Q. Tao, Y. Chen, X. Liang, Y. Hu, J. Li, F. Fang, H. Wang, C. Meng, J. Liang,
X. Ma, S. Gui, Structurally novel PI3Kδ/γ dual inhibitors characterized by a seven-membered spirocyclic
spacer: The SARs investigation and PK evaluation, European Journal of Medicinal Chemistry (2020),
doi: https://doi.org/10.1016/j.ejmech.2020.112143.
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Graphical abstract
Structurally novel PI3Kδ/γ dual inhibitors characterized by a seven-membered
spirocyclic spacer: the SARs investigation and PK evaluation
Qiangqiang Tao a, Yuqing Chen a, Xiao Liang a, Yongzhou Hu d, Jiaming Li a, b, Fang Fang a, b, Huchuan
Wang a, b, Chang Meng a, Jingtai Liang a, Xiaodong Ma a, b, *, Shuangying Gui a, c,
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Structurally novel PI3Kδ/γ dual inhibitors characterized by a seven-membered
spirocyclic spacer: the SARs investigation and PK evaluation
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Qiangqiang Tao a, Yuqing Chen a, Xiao Liang a, Yongzhou Hu d, Jiaming Li a, b, Fang Fang a, b, Huchuan
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Wang a, b, Chang Meng a, Jingtai Liang a, Xiaodong Ma a, b, *, Shuangying Gui a, c,
*
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a. College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
b. Institute of Medicinal Chemistry, Anhui Academy of Chinese Medicine, Hefei 230012, China
c. Institute of Pharmaceutics, Anhui Academy of Chinese Medicine, Hefei 230012, China
d. ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of
Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, China
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Abstract: Herein, we communicate our recent medicinal chemistry efforts which have culminated in a
series of PI3Kδ/γ dual inhibitors structurally featuring a seven-membered spirocyclic spacer.
Compound 26, the most potent one among them, exhibited superior PI3Kδ inhibitory activity (IC50
=
1.0 nM) to that of the approved PI3Kδ inhibitor Idelalisib. Besides, it exerted remarkable
anti-proliferative efficacy against human malignant B-cell line SU-DHL-6 with GI50 value of 33 nM.
The biochemical assay against the other three class I PI3K isoforms identified compound 26 as a potent
PI3Kδ/γ dual inhibitor with considerable selectivity over PI3Kα and PI3Kβ. In SU-DHL-6 cells, a
dramatic down-regulation of PI3K signaling was observed following compound 26-treatment at the
concentration as low as 10 nM. Inspiringly, the pharmacokinetic (PK) study in Sprague-Dawley (SD)
rats revealed it was orally available with a favorable bioavailability (F = 87.5%). Overall, compound 26,
a promising PI3Kδ/γ dual inhibitor, has the potential to emerge as a clinical candidate for the treatment
of leukocyte-mediated malignancies after extensive functional investigation.
Key words: Spirocyclic spacer; PI3Kδ/γ dual inhibitor; Anti-proliferative efficacy; PI3K signaling; PK
study
* Corresponding author: Xiaodong Ma
College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China;
Institute of Medicinal Chemistry, Anhui Academy of Chinese Medicine, Hefei 230012, China
E-mail: o-omaxiaodong@163.com
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* Corresponding author: Shuangying Gui
College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China;
Institute of Pharmaceutics, Anhui Academy of Chinese Medicine, Hefei 230012, China
E-mail: guishy0520@126.com
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1. Introduction
The preferential distribution of phosphoinositide 3-kinase δ (PI3Kδ) in leukocytes offers a unique
opportunity for pharmacologically interfering with this isoform in the treatment of leukocyte-mediated
conditions driven by its pathologic activation [1, 2]. Besides the vital role in triggering the proliferation,
survival and trafficking of malignant B-cell [3], PI3Kδ, located downstream of receptor tyrosine
kinases (RTKs) [4], has been proven hyperactive in autoimmune, inflammatory and allergic disorders,
including rheumatoid arthritis (RA), allergic asthma, psoriasis, and systemic lupus erythematosus (SLE)
[5-8]. The approval of Idelalisib (Zydelig, 1, Fig. 1) in 2014 is a testimony to the merit of PI3Kδ
selective inhibitors as therapeutics for relapsed/refractory B-cell malignancies [9, 10]. Additionally, in
the oncology setting, Umbralisib (TGR1202, 2), Dezapelisib (INCB040093, 3), Parsaclisib
(INCB050465, 4) and ME401 5 have been progressed into clinical trials (Fig. 1) [11-13]. Meanwhile,
Seletalisib (UCB5857, 6) [14], Leniolisib (CDZ173, 7) [15], Nemiralisib (GSK2269557, 8) [16, 17]
and GSK2292767 9 [16] are presently under clinical investigation as remedies for non-oncology
diseases, exemplified by respiratory, inflammatory and autoimmune conditions, as well as
immunodeficiency (Fig. 1).
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26
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PI3Kγ, another class I PI3K subtype with restricted expression in leukocytes, is generally coupled to
G protein-coupled receptors (GPCRs) rather than functioning downstream of RTKs [18]. However,
apart from their complementary roles in inflammation and immune response [13], PI3Kγ and PI3Kδ
concurrently mediate the proliferative and survival signaling in certain malignant hematopoietic cells
[13, 19]. In particular, mounting evidences support their participation in modulating tumor
microenvironment [20-23]. For instance, PI3Kγ can serve as a molecular switch that controls immune
suppression via hampering phagocytosis by tumor-associated macrophages (TAMs), which negatively
regulate effector T and natural killer (NK) cells in this state by expressing membrane-bound immune
checkpoint molecules and secreting soluble immunosuppressive factors. Hence, concomitant inhibition
of PI3Kδ and PI3Kγ has the potential to broaden the spectrum of anti-proliferative activity and achieve
therapeutic synergism in hematologic malignancies, as well as inflammatory and autoimmune diseases
[19, 24]. Duvelisib (Copiktra, 10, Fig. 1) [25], marketed in 2018 for the treatment of B-cell mediated
hematopoietic malignancies, is the first-in-class PI3Kδ/γ dual inhibitor. Tenalisib (RP6530, 11) [26],
also featuring PI3Kδ/γ bi-functional inhibitory activity, is currently clinically investigated in a
combination therapy for classical Hodgkin’s lymphomas (Fig. 1). Theoretically, owing to the
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ubiquitous distribution of PI3Kα and PI3Kβ, both PI3Kδ-selective and PI3Kδ/γ dual inhibitors are
beneficial to minimizing the off-target effects of pan-class I PI3Ks inhibitors in treating
leukocyte-mediated conditions [27].
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Fig. 1. Advanced PI3Kδ-selective or PI3Kδ/γ dual inhibitors: approved or under clinical trials
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A considerable number of advanced PI3Kδ-selective and PI3Kδ/γ dual inhibitors are
propeller-shaped molecules (Fig. 1), which share the same pharmacophore and simultaneously occupy
the hinge region and the allosteric pocket of PI3Kδ catalytic cleft [13, 28, 29]. In respect of chemical
structure, hinge binders (HBs) of them are routinely N-containing heteroaryl moiety tethered to a
bicyclic heteroaryl template via a short spacer. While the HB is engaged in H-bond contacts with the
enzyme, the bicyclic template inserts deeply into the allosteric hydrophobic pocket located between
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11
12
13
residues Trp760 and Met752 [30]. In contrast to the structurally diverse bicyclic templates, alteration in
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the short spacer is relatively subtle among these propeller-shaped molecules. Spirocyclic building
blocks are frequently employed in drug discovery for producing conformationally restricted molecules
with structural novelty, and optimizing biological activity, pharmacokinetic (PK) property, as well as
chemical stability [31-34]. During our recent medicinal chemistry campaign to broaden the structural
diversity of the short spacer, a seven-membered spirocycle was introduced to replace the
S-propylamine of 1. We had speculated this structural elaboration, accompanied by modification at
other sites, might affect the class I PI3K specificity profile of compounds. Built upon the resultant
scaffold, structure-activity relationships (SARs) investigation on the replacement at the benzene ring of
the bicyclic template (R1), N-3 substituent (R2) and HB was conducted to attain an optimum compound
for further exploration (Fig. 2). As a consequence, compound 26 was discovered as a potent PI3Kδ/γ
dual inhibitor with favorable in vitro performance and attractive PK profiles.
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Fig. 2. The design rationale of target compounds and the focus of SARs study
2. Results and discussion
2.1. Chemistry
Compounds bearing phenyl as the N-3 substituent were synthesized according to Scheme 1.
Condensation
of
ortho-amino
benzoic
acids
12a-d
with
(S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid in the presence of P(OPh)3,
followed by the in situ treatment with aniline afforded the quinazolone derivatives 13a-d. Afterwards,
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5
6
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removal of the Boc-protecting group and subsequent nucleophilic aromatic substitution (SNAr)
reaction with 6-chloro-9H-purine, 4-amino-6-chloropyrimidine-5-carbonitrile,
2-amino-4-chloro-6-methylpyrimidine-5-carbonitrile, 6-chloro-2-fluoro-9H-purine or 2,
6-dichloro-9H-purine furnished 14-17, 21, 22, 24, 25 and 27-29 as target compounds. In addition,
Boc-deprotected products of 13a and 13b were subjected to SNAr reaction with 2,
4-diamino-6-chloropyrimidine-5-carbonitrile in the presence of KF to generate compound 23 and 26,
respectively.
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Scheme 1. Reagents and conditions:
(a) (1) (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid, P(OPh)3, pyridine, 70 °C,
N2; (2) aniline; (b) (1) trifluoroacetic acid (TFA)/dichloromethane (DCM), 0 °C to rt; (2)
6-chloro-9H-purine,
4-amino-6-chloropyrimidine-5-carbonitrile,
6-chloro-2-fluoro-9H-purine or 2,
2-amino-4-chloro-6-methylpyrimidine-5-carbonitrile,
6-dichloro-9H-purine, N, N-diisopropylethylamine (DIPEA), t-BuOH, 80 °C, N2; (c) (1) TFA/DCM,
0 °C to rt; (2) 2, 4-diamino-6-chloropyrimidine-5-carbonitrile, KF, DIPEA, dimethyl sulfoxide
(DMSO), 90 °C, N2.
Scheme 2 displayed the synthetic route for compounds 18-20. Amides 31a-c were provided via
conversion of 2-fluoro-6-nitrobenzoic acid 30 into the acyl chloride and the following condensation
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5
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with corresponding primary amine. Subsequently, 31a-c were transformed into imidoyl chlorides,
which underwent Mumm rearrangement after coping with
(S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid to generate imides 32a–c. After
reduction of the nitro functionality, the in situ intra-molecular cyclization furnished quinazolone
derivatives 33a-c. Unmasking the nitrogen of the spirocycle and the ultimate SNAr reaction with
6-chloro-9H-purine afforded 18-20 as target compounds.
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Scheme 2. Reagents and conditions: (a) (1) N, N-dimethylformamide (DMF), SOCl2, 80 °C; (2)
corresponding amine, anhydrous triethylamine (TEA), anhydrous DCM; (b) (1) DMF, SOCl2, 80 °C,
N2; (2) (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid, anhydrous TEA,
anhydrous DCM, 0 °C to rt, N2; (c) Zn, AcOH, 40 °C, N2; (d) (1) TFA/DCM, 0 °C to rt; (2)
6-chloro-9H-purine, DIPEA, t-BuOH, 80 °C, N2.
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2.2. PI3Kδ biochemical assay
All the target compounds were firstly assayed against PI3Kδ for their inhibitory activity. According
to the data presented in Table 1, a majority of them exerted remarkable PI3Kδ inhibitory activity with
IC50 values below 50 nM, and six compounds exhibited IC50 values at single-digit nanomolar level.
Among the target compounds, 26 displayed the most vigorous PI3Kδ inhibitory activity with IC50 value
of 1.0 nM, which was more potent than that of Idelalisib.
Some valuable SARs were obtained from the enzymatic activity presented in Table 1. C-5 chloro
replacement at the quinazolone template was more beneficial to PI3Kδ inhibitory potency than fluoro
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replacement, as illustrated by the activity of 15 versus 14, and 26 versus 23. Alteration of C-5 fluoro
substituent into C-6 fluoro substituent did not dramatically affect the activity (17 versus 14). With
respect to the N-3 substituent, replacing the phenyl moiety with benzyl, cyclopentyl and cyclohexyl
significantly weakened the enzymatic activity, as testified by the activity of 14 versus 18-20. Among
the various investigated HBs, it appeared evident that 2, 4-diaminopyrimidine-5-carbonitrile was
optimum in accordance with the activity of 23 versus 14, 21 and 22, as well as 26 versus 15, 24 and 25.
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5
6
7
Besides,
compounds
with
4-aminopyrimidine-5-carbonitrile
or
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2-amino-6-methylpyrimidine-5-carbonitrile as HB were superior to corresponding compounds with
purine as HB in PI3Kδ inhibitory potency (21 and 22 versus 14; 24 and 25 versus 15). As for the purine
moiety, the introduction of C-2 fluoro substituent slightly impaired the activity (28 versus 15), while
the introduction of C-2 chloro substituent considerably lowered the enzymatic activity (29 versus 15).
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Table 1. The PI3Kδ inhibitory activity of target compounds
14
Cpd.
14
R1
R2
HB
PI3Kδ IC50 (nM)
5-F
43
5-Cl
5-Me
6-F
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16
15
16
17
18
19
48
5-F
813
396
5-F
8

5-F
5-F
444
15
20
21
22
23
24
25
26
27
28
5-F
9.4
5.4
3.7
9.6
1.0
9.6
38
CN
N
5-F
H2N
N
NH2
5-Cl
5-Cl
5-Cl
6-F
CN
N
H2N
N
NH2
N
N
5-Cl
N
F
N
H
5-Cl
---
406
3.4
29
---
---
Idelalisib
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4
5
6
7
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2.3. Anti-proliferative assay
As stated in the introduction section, PI3Kδ signaling is critical for the proliferation of malignant
B-cells. Hence, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was
performed to evaluate the anti-proliferative activity of twelve compounds (PI3Kδ IC50s < 50 nM)
against human malignant B-cell line SU-DHL-6 and validate their potential for treating B-cell
malignancies. On the whole, the anti-proliferative activity was consistent with the PI3Kδ inhibitory
activity (Table 2). Compound 26, with the most potent PI3Kδ inhibitory activity, also exerted the most
remarkable anti-proliferative activity throughout this series against SU-DHL-6 cell line with GI50 value
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of 33 nM. Besides, compound 23, another one bearing 2, 4-diaminopyrimidine-5-carbonitrile as HB,
displayed remarkable anti-proliferative activity against SU-DHL-6 cell line with GI50 value of 53 nM.
Although
replacement
of
purine
with
4-aminopyrimidine-5-carbonitrile
or
2-amino-6-methylpyrimidine-5-carbonitrile as HB led to enhancement in PI3Kδ inhibitory activity, this
did not transform into the improvement in cellular potency (21 and 22 versus 14). Hence, compounds
with 4-aminopyrimidine-5-carbonitrile or 2-amino-6-methylpyrimidine-5-carbonitrile as HB were not
selected for further evaluation.
Table 2. The anti-proliferative activity of selected compounds against SU-DHL-6 cell line
Cpd.
14
SU-DHL-6 GI50 (nM)
Cpd.
SU-DHL-6 GI50 (nM)
813
601
107
937
867
1042
53
3417
716
33
24
25
15
16
26
945
2808
34
17
27
21
28
22
Idelalisib
23
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2.4. PI3Kα, β and γ biochemical assays
Table 3. The inhibitory activity of selected compounds against class I PI3K isoforms
Cpd.
15
PI3Kα IC50 (nM) PI3Kβ IC50 (nM) PI3Kγ IC50 (nM) PI3Kδ IC50 (nM)
>5000
(>385-fold) a
221
2641
(203-fold) a
174
113
(9-fold) a
4.3
13
1.0
26
(221-fold) a
(174-fold) a
(4-fold) a
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a. Selectivity fold
Compounds 15 and 26 were subsequently selected as representatives to identify their inhibitory
activities against the other three class I PI3K subtypes. The results demonstrated that both compounds
exhibited favorable selectivity over PI3Kα and PI3Kβ (Table 3). However, less obvious discrepancy
existed between the PI3Kδ and PI3Kγ inhibitory activities of each compound. Therefore, both 15 and
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26 were characterized as PI3Kδ/γ dual inhibitors. In particular, compound 26 also exhibited single-digit
nanomolar inhibitory activity against PI3Kγ.
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2.5. Western blot assay
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To confirm its anti-proliferative activity against SU-DHL-6 cell line was resulted from the
modulation of PI3K signaling, compound 26, the most potent compound throughout this series, was
then investigated in the Western blot analysis. As illustrated by Fig. 3, compound 26 down-regulated
both phos-Akt (Ser473) and phos-S6K1 (Thr389), two well-established biomarkers of PI3K signaling,
in a dose-dependent manner at 2 h post-treatment. In particular, at the dosage as low as 10 nM,
compound 26-treatment led to dramatic decrease in the phosphoration of both Akt and its downstream
signal S6K1. At the dosage of 30 nM, approximately the GI50 value of 26, a more significant decrease
in the phosphoration of Akt and S6K1 was observed. As revealed by the immunoblot assay, the
anti-proliferative activity of 26 against SU-DHL-6 cell line was attributed to its remarkable capability
to attenuate the PI3K signaling.
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Fig. 3. The capability of compound 26 to attenuate PI3K signaling in SU-DHL-6 cells at 2 h
post-treatment: The Akt and S6K1 phosphorylation levels were determined via Western blot assay (A);
Phosphorylation levels of Akt (B) and S6K1 (C) were displayed as folds of control with the bar chart
indicating the quantification of the bands in immunoblotting (results shown as mean ± SD, n=3). *
p<0.05, ** p<0.01 VS control (cells incubated with the medium alone).
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2.6. In vivo PK evaluation
The excellent in vitro performance of 26 provided an incentive for the further PK study in Sprague–
Dawley (SD) rats. Following oral administration at the dosage of 5 mg/kg, compound 26 displayed a
high plasma exposure (AUC0-t = 4878 ± 694 h µg/L), an attractive oral bioavailability (F% = 87.5 ±
12.5), and an acceptable clearance (CL = 0.9 ± 0.1 L/h/kg) (Table 4). Moreover, it exerted a long
elimination half-life (T1/2 = 14.3 ± 4.4 h). Given the favorable PK properties, the remarkable in vitro
potency of 26 was expected to be transformed to favorable therapeutic efficacy in vivo.
Table 4. The PK parameters of compound 26
Cpd.
26
Route
T1/2 (h)a AUC0-t (h µg/L)a Vss (L/kg)a CL (L/h/kg)a
F(%)
IV (5 mg/kg) 3.0 ± 0.3
PO (5 mg/kg) 14.3 ± 4.4
5576 ± 606
4878 ± 694
3.9 ± 0.6
---
0.9 ± 0.1
---
87.5 ± 12.5
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a. Data, displayed as mean ± SD, were calculated according to the results of three biological
replicates. CL, Vss, T1/2, AUC, and F stood for clearance, volume of distribution, half-life, area under
the plasma concentration-time curve, and oral bioavailability, respectively.
2.7. Molecular modeling
With the attempt to unravel its possible binding mode with PI3Kδ catalytic cleft, we conducted the
molecular docking analysis of 26 on the basis of the disclosed PI3Kδ/Idelalisib co-crystal structure
(PDB code 4XE0) [30]. Both Idelalisib (yellow) and compound 26 (magenta) assumed a
propeller-shaped conformation with the bicyclic template sandwiched into the hydrophobic selectivity
pocket that was induced between Trp760 and Met752 (Fig. 4A and 4C). The 2,
4-diaminopyrimidine-5-carbonitrile moiety, as the HB of 26, conferred three H-bonds with hinge
residues Val828 and Glu826 (Fig 4B). Besides, cyclopropyl of the spirocyclic spacer was within the
hydrophobic interaction distance of Met752 and Pro758. These potential contacts provided an
explanation for the remarkable PI3Kδ inhibitory activity of 26.
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Fig. 4. The molecular docking of compound 26 into the ATP-binding site of PI3Kδ
3. Conclusions
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Built upon our insight into the reported propeller-shaped PI3Kδ inhibitors and PI3Kδ/γ dual
inhibitors, a novel structural series of quinazolone derivatives were designed and synthesized via
incorporation of a seven-membered spirocyclic building block as the spacer between the bicyclic
heteroaryl template and HB. The SARs study identified compound 26 as the most potent one
throughout this series, which exerted IC50 value of 1.0 nM against PI3Kδ, and GI50 value of 33 nM
against SU-DHL-6 cell line. Besides, it exhibited single-digit nanomolar inhibitory activity against
PI3Kγ, along with favorable specificity over PI3Kα and PI3Kβ. Further Western blot analysis
illustrated the anti-proliferative potency of 26 was resulted from its remarkable capability to
down-regulate PI3K pathway. Importantly, benefiting from its favorable bioavailability (F = 87.5%),
the in vitro potency of 26 was anticipated to be transformed to desirable in vivo therapeutic efficacy. As
a promising PI3Kδ/γ dual inhibitor, compound 26 deserves extensive functional investigation to assess
its potential as candidate for battling B-cell malignancies.
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4. Experimental section
4.1. Chemistry
Chemical reagents used in this work were all purchased from common commercial suppliers. When
necessary, purification was performed prior to use. NMR spectra were recorded on a Bruker Avance
400 II (400 MHz) spectrometer after dissolving the samples in DMSO-d6 or CDCl3. ESI-MS spectra
were collected by Bruker Esquire-LC-00075 spectrometer, while ESI-HRMS were obtained by Agilent
6230 TOF LC/MS spectrometer. Silica gel (200–300 mesh) was used for performing flash column
chromatography. HPLC was performed using a Shimadzu Essentia LC-16 system with UV detection at
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254 nm. Compounds were eluted with a binary solvent system containing A and B [A: CH3CN; B: H2O
with 0.05% phosphoric acid (W/V)] at the flow rate of 0.8 mL/min. Analytical purity of the target
compounds (all over 95%) was presented in the supplementary material.
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4.1.1. General procedure for preparing quinazolone derivatives 13a-d
5
The mixture of corresponding ortho-amino benzoic acid 12a (12b, 12c or 12d, 1.0 eq),
(S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid (1.1 eq), P(OPh)3 (2.5 eq) and
pyridine (2 mL/1 mmol substrate) was stirred at 70 °C under N2 atmosphere. After the total conversion
of the ortho-amino benzoic acid, aniline (1.2 eq) was added to the mixture, and the resultant mixture
was stirred at the same temperature for 3 h. Following the removal of pyridine in vacuo, the residue
was dissolved in EA, and the solvent was washed successively with HCl (1 N), saturated NaHCO3
solution, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo.
Finally, the residue was subjected to flash column chromatography [petroleum ether (PE)/ethyl acetate
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8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1
(EA) = 40:1-20:1, V/V] to afford the quinazolone derivative (13a-d) as pale solid. The H NMR
spectra of 13a-d indicated the existence of rotamers.
Tert-butyl
(S)-6-(5-fluoro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate (13a)
Pale solid; yield 53%; 1H NMR (400 MHz, CDCl3): δ 7.77–7.65 (m, 1H), 7.63–7.48 (m, 4H), 7.36 (d,
7.6 Hz, 1H), 7.22–7.09 (m, 2H), 4.75–4.65 (m, 0.4H), 4.63–4.56 (m, 0.6H), 3.78–3.58 (m, 1H), 3.43–
3.28 (m 1H), 2.01–1.83 (m, 2H), 1.46 (s, 3H), 1.32 (s, 6H), 0.73–0.62 (m, 1H), 0.61–0.52 (m, 1H),
0.51–0.45 (m, 1H), 0.43–0.35 (m, 1H); ESI-MS: m/z = 436 [M+H]+.
Tert-butyl
(S)-6-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate
(13b) Pale solid; yield 49%; 1H NMR (400 MHz, DMSO-d6): δ 7.83–7.73 (m, 1H), 7.70–7.53 (m, 5H),
7.52–7.43 (m, 1H), 7.38 (d, 7.6 Hz, 0.4H), 7.29 (d, 7.6 Hz, 0.6H), 4.41–4.29 (m, 1H), 3.55–3.45 (m,
1H), 3.30–3.23 (m, 1H), 2.08–1.92 (m, 1H), 1.90–1.78 (m, 1H), 1.36 (s, 3H), 1.22 (s, 6H), 0.64–0.47
(m, 3H), 0.40–0.28 (m, 1H); ESI-MS: m/z = 452 [M+H]+.
Tert-butyl
(S)-6-(5-methyl-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate
(13c) Pale solid; yield 37%; 1H NMR (400 MHz, DMSO-d6): δ 7.73–7.51 (m, 4H), 7.50–7.40 (m, 2H),
7.39–7.34 (m, 0.4H), 7.33–7.25 (m, 1.6H), 4.41–4.30 (m, 1H), 3.54–3.47 (m, 1H), 3.29–3.21 (m, 1H),
14

1
2
2.78–2.68 (3H, two singlets), 2.04–1.91 (m, 1H), 1.89–1.77 (m, 1H), 1.36 (s, 3H), 1.21 (s, 6H), 0.64–
0.46 (m, 3H), 0.39–0.27 (m, 1H); ESI-MS: m/z = 432 [M+H]+.
3
Tert-butyl
4
(S)-6-(6-fluoro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate (13d)
Pale solid; yield 54%; 1H NMR (400 MHz, DMSO-d6): δ 7.86–7.71 (m, 3H), 7.70–7.53 (m, 3H), 7.52–
7.44 (m, 1H), 7.39 (d, 8.4 Hz, 0.4H), 7.30 (d, 7.6 Hz, 0.6H), 4.45–4.33 (m, 1H), 3.55–3.46 (m, 1H),
3.31–3.22 (m, 1H), 2.09–1.92 (m, 1H), 1.90–1.78 (m, 1H), 1.36 (s, 3H), 1.20 (s, 6H), 0.66–0.47 (m,
3H), 0.39–0.27 (m, 1H); ESI-MS: m/z = 436 [M+H]+.
5
6
7
8
9
4.1.2. General procedure for preparing compounds 14-17, 21, 22, 24, 25 and 27-29
To the solution of corresponding quinazolone intermediate 13a (13b, 13c or 13d) in DCM (4 mL/1
mmol substrate) was added TFA (1 mL/1 mmol substrate) dropwise at 0 °C, and the resultant mixture
was stirred at room temperature. After 13a (13b, 13c or 13d) was totally consumed, saturated NaHCO3
solution was added dropwise at 0 °C to adjust the PH value to 7. Following extraction with DCM, the
organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to
afford the Boc-deprotected secondary amine as slightly yellow solid.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
The mixture of corresponding Boc-deprotected secondary amine (1.0 eq), 6-chloro-9H-purine (1.5
eq), DIPEA (4.0 eq) and t-BuOH (15 mL/1 mmol secondary amine) was stirred at 80 °C under N2
atmosphere. After the secondary amine was totally consumed, t-BuOH was removed in vacuo, and the
residue was dissolved in DCM. The solution was washed with saturated NaHCO3 solution, dried over
anhydrous Na2SO4, and concentrated in vacuo. Finally, the crude product underwent flash column
chromatography (DCM/EA = 5:1-1:1, V/V) to afford the purine derivative (14 or 15-17) as pale solid.
Compounds with 4-aminopyrimidine-5-carbonitrile, 2-amino-6-methylpyrimidine-5-carbonitrile,
2-fluoro-9H-purine or 2-chloro-9H-purine as HBs were preapred via similar procedure to that for 14-17.
However, the eluents utilized in the final flash column chromatography of 21, 22, 24, 25 and 27 were
different (DCM/EA = 15:1-5:1, V/V, for 21, 24 and 27; DCM/EA = 20:1-8:1, V/V, for 22 and 25).The
NMR spectra of 14-17, 28 and 29 indicated the existence of rotamers.
(S)-2-(5-(9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-5-fluoro-3-phenylquinazolin-4(3H)-one (14)
1
Pale solid; yield 61% (for two steps); H NMR (400 MHz, DMSO-d6): δ 13.00 (s, 0.5H), 12.94 (s,
0.5H), 8.26–8.09 (m, 2H), 7.85–7.46 (m, 6H), 7.35–7.18 (m, 2H), 5.57–5.50 (m, 0.5H), 4.83–4.76 (m,
0.5H), 4.31–4.19 (m, 1H), 3.90 (d, 10.8 Hz, 0.5H), 3.73 (d, 10.8 Hz, 0.5H), 2.21–1.94 (m, 2H), 0.88–
15

1
2
0.76 (m, 1H), 0.68–0.58 (m, 2H), 0.49–0.40 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ 161.03 (d,
JC-F = 263.0 Hz), 160.96 (d, JC-F = 261.0 Hz), 160.25, 159.29 (d, JC-F = 4.00 Hz), 158.90 (d, JC-F = 5.00
Hz), 158.79, 152.78, 152.62, 152.33, 151.72, 151.47, 150.94, 149.97, 149.55, 139.37, 139.30, 137.07,
136.60, 135.66, 130.69, 130.01, 129.96, 129.90, 129.76, 129.71, 129.57, 129.49, 129.38, 123.84 (d,
3
4
5
JC-F = 3.00 Hz), 123.72 (d, JC-F = 3.00 Hz), 119.45, 119.38, 113.34 (d, JC-F = 21.0 Hz), 113.21 (d, JC-F =
6
19.0 Hz), 110.61 (d, JC-F = 6.00 Hz), 61.54, 60.35, 57.68, 56.36, 38.82, 21.68, 18.94, 13.84, 11.64,
10.85, 8.42; ESI-HRMS: m/z calcd for C25H20FN7O [M+H]+ 454.1792; found 454.1803; HPLC: tR =
9.08 min, Agilent TC-C18(2) 250 × 4.6mm 5µm, 30 °C, eluent A-40%, eluent B-60%.
7
8
9
(S)-2-(5-(9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-5-chloro-3-phenylquinazolin-4(3H)-one (15)
Pale solid; yield 66% (for two steps); 1H NMR (400 MHz, DMSO-d6): δ 12.98 (brs, 1H), 8.28–8.04
(m, 2.5H), 7.81–7.75 (m, 0.5H), 7.73–7.30 (m, 7H), 5.56–5.49 (m, 0.5H), 4.80–4.73 (m, 0.5H), 4.30–
4.19 (m, 1H), 3.90 (d, 10.8 Hz, 1H), 3.73 (d, 10.8 Hz, 1H), 2.21–1.92 (m, 2H), 0.89–0.74 (m, 1H),
0.68–0.55 (m, 2H), 0.49–0.40 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ 160.32, 159.96, 159.92,
158.48, 152.76, 152.52, 152.22, 151.76, 151.63, 150.96, 150.34, 149.91, 139.77, 139.48, 137.22,
136.75, 134.86, 133.15, 133.11, 130.58, 130.04, 129.98, 129.88, 129.75, 129.70, 129.50, 129.44,
129.32, 129.28, 127.33, 127.22, 119.40, 119.21, 117.91, 61.56, 60.38, 57.63, 56.35, 38.77, 21.64, 18.90,
13.87, 11.67, 10.77, 8.33; ESI-HRMS: m/z calcd for C25H20ClN7O [M+H]+ 470.1496; found 470.1495;
HPLC: tR = 6.74 min, Agilent TC-C18(2) 250 × 4.6mm 5µm, 30 °C, eluent A-55%, eluent B-45%.
(S)-2-(5-(9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-5-methyl-3-phenylquinazolin-4(3H)-one (16)
Pale solid; yield 59% (for two steps); 1H NMR (400 MHz, DMSO-d6): δ 8.32–8.00 (m, 2.5H), 7.81–
7.44 (m, 5.5H), 7.37–7.11 (m, 2H), 5.67–5.49 (m, 0.5H), 4.86–4.72 (m, 0.5H), 4.31–4.20 (m, 1H),
3.95–3.87 (m, 0.5H), 3.79–3.72 (m, 0.5H), 2.78–2.63 (m, 3H), 2.17–1.92 (m, 2H), 0.85–0.72 (m, 0.5H),
0.69–0.52 (m, 2.5H), 0.49–0.39 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ 162.87, 162.50, 158.65,
157.14, 152.82, 152.47, 152.16, 151.96, 151.69, 151.17, 149.42, 149.00, 140.70, 140.62, 139.85,
139.58, 137.66, 137.18, 134.04, 133.98, 130.75, 130.10, 129.92, 129.87, 129.68, 129.63, 129.48,
129.43, 129.31, 129.21, 129.13, 126.02, 125.88, 119.49, 119.43, 119.37, 61.45, 60.24, 57.69, 56.37,
38.97, 23.11, 21.64, 18.89, 14.07, 14.01, 11.74, 10.86, 8.42; ESI-HRMS: m/z calcd for C26H23N7O
[M+H]+ 450.2042; found 450.2035; HPLC: tR = 12.05 min, Agilent TC-C18(2) 250 × 4.6mm 5µm,
30 °C, eluent A-40%, eluent B-60%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
(S)-2-(5-(9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-6-fluoro-3-phenylquinazolin-4(3H)-one (17)
16

1
2
Pale solid; yield 62% (for two steps); 1H NMR (400 MHz, DMSO-d6): δ 12.96 (s, 0.5H), 12.90 (s,
0.5H), 8.31–8.04 (m, 2.5H), 7.84–7.72 (m, 1.5H), 7.71–7.46 (m, 6H), 5.63–5.54 (m, 0.5H), 4.88–4.80
(m, 0.5H), 4.33–4.21 (m, 1H), 3.92 (d, 11.2 Hz, 0.5H), 3.76 (d, 11.2 Hz, 0.5H), 2.20–2.08 (m, 1H),
2.04–1.96 (m, 1H), 0.83–0.76 (m, 0.5H), 0.69–0.53 (m, 2.5H), 0.49–0.40 (m, 1H); 13C NMR (100 MHz,
DMSO-d6): δ 161.71 (d, JC-F = 3.00 Hz), 161.34 (d, JC-F = 3.00 Hz), 160.40 (d, JC-F = 244 Hz), 158.58,
157.13, 152.77, 152.62, 152.31, 151.71, 151.44, 150.92, 144.71, 144.29, 139.35, 139.27, 137.25,
136.79, 130.59, 130.54, 130.42, 130.04, 129.98, 129.74, 129.52, 129.45, 129.23, 123.58 (d, JC-F = 10.0
Hz), 123.34 (d, JC-F = 10.0 Hz), 122.24, 122.17, 119.45, 119.36, 111.34 (d, JC-F = 23.0 Hz), 61.51,
60.28, 57.66, 56.35, 38.94, 21.66, 18.90, 13.89, 11.62, 10.83, 8.35; ESI-HRMS: m/z calcd for
C25H20FN7O [M+H]+ 454.1792; found 454.1805; HPLC: tR = 6.41 min, Agilent TC-C18(2) 250 ×
4.6mm 5µm, 30 °C, eluent A-50%, eluent B-50%.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
(S)-4-amino-6-(6-(5-fluoro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptan-5-yl)
pyrimidine-5-carbonitrile (21)
1
Pale solid; yield 71%; H NMR (400 MHz, DMSO-d6): δ 8.02 (s, 1H), 7.82–7.53 (m, 5H), 7.48 (d,
6.8 Hz, 1H), 7.42 (d, 8.0 Hz, 1H), 7.38–7.02 (m, 3H), 4.79–4.65 (m, 1H), 3.96 (d, 10.0 Hz, 1H), 3.85 (d,
9.6 Hz, 1H), 2.09–1.99 (m, 1H), 1.96–1.86 (m, 1H), 0.79–0.69 (m, 1H), 0.67–0.57 (m, 2H), 0.43–0.35
(m, 1H); 13C NMR (100 MHz, DMSO-d6): δ 166.12, 161.02 (d, JC-F = 262 Hz), 159.47, 159.31, 159.11,
158.86 (d, JC-F = 3.00 Hz), 149.86, 136.85, 135.75 (d, JC-F = 10.0 Hz), 131.98, 130.00, 129.89, 129.78,
129.35, 123.79 (d, JC-F = 2.00 Hz), 117.72, 113.32 (d, JC-F = 19.0 Hz), 110.61 (d, JC-F = 5.00 Hz), 68.32,
61.63, 56.79, 38.23, 21.48, 12.11, 10.21; ESI-HRMS: m/z calcd for C25H20FN7O [M+H]+ 454.1792;
found 454.1780; HPLC: tR = 10.97 min, Agilent TC-C18(2) 250 × 4.6mm 5µm, 30 °C, eluent A-50%,
eluent B-50%.
(S)-2-amino-4-(6-(5-fluoro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptan-5-yl)
-6-methylpyrimidine-5-carbonitrile (22)
1
Pale solid; yield 74%; H NMR (400 MHz, DMSO-d6): δ 7.89 (d, 7.2 Hz, 1H), 7.83–7.72 (m, 1H),
7.68–7.46 (m, 4H), 7.40 (d, 8.0 Hz, 1H), 7.26 (t, 9.2 Hz, 1H), 7.02 (brs, 1H), 6.70 (brs, 1H), 4.86–4.68
(m, 1H), 4.05–3.77 (m, 2H), 2.28 (s, 3H), 1.96–1.77 (m, 2H), 0.73–0.48 (m, 3H), 0.44–0.29 (m, 1H);
13C NMR (100 MHz, DMSO-d6): δ 172.81, 162.08, 161.06 (d, JC-F = 261 Hz), 159.81, 159.02, 158.47
(d, JC-F = 3.00 Hz), 149.78, 136.71, 135.65 (d, JC-F = 9.00 Hz), 130.18, 129.94, 129.87, 129.73, 129.67,
123.90 (d, JC-F = 3.00 Hz), 119.87, 113.29 (d, JC-F = 20.0 Hz), 110.76 (d, JC-F = 5.00 Hz), 77.92, 61.11,
17

1
2
57.05, 38.18, 23.63, 21.11, 12.92, 9.21; ESI-HRMS: m/z calcd for C26H22FN7O [M+H]+ 468.1948;
found 468.1951; HPLC: tR = 7.18 min, Agilent TC-C18(2) 250 × 4.6mm 5µm, 30 °C, eluent A-50%,
eluent B-50%.
3
4
(S)-4-amino-6-(6-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptan-5-yl)
pyrimidine-5-carbonitrile (24)
5
1
6
Pale solid; yield 70%; H NMR (400 MHz, DMSO-d6): δ 8.02 (s, 1H), 7.75–7.51 (m, 7H), 7.48 (d,
7
7.2 Hz, 1H), 7.28 (brs, 2H), 4.77–4.64 (m, 1H), 3.95 (d, 9.6 Hz, 1H), 3.86 (d, 9.6 Hz, 1H), 2.08–2.01
(m, 1H), 1.96–1.87 (m, 1H), 0.79–0.68 (m, 1H), 0.65–0.57 (m, 2H), 0.44–0.35 (m, 1H); 13C NMR (100
MHz, DMSO-d6): δ 166.12, 159.88, 159.30, 159.12, 150.29, 137.08, 134.90, 133.20, 131.98, 130.00,
129.89, 129.75, 129.40, 129.29, 129.13, 127.33, 118.01, 117.73, 68.32, 61.64, 56.81, 38.16, 21.47,
12.27, 10.10; ESI-HRMS: m/z calcd for C25H20ClN7O [M+H]+ 470.1496; found 470.1503; HPLC: tR =
11.54 min, Agilent TC-C18(2) 250 × 4.6mm 5µm, 30 °C, eluent A-55%, eluent B-45%.
(S)-2-amino-4-(6-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptan-5-yl)
-6-methylpyrimidine-5-carbonitrile (25)
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Pale solid; yield 68%; 1H NMR (400 MHz, DMSO-d6): δ 7.89 (d, 7.6 Hz, 1H), 7.71 (t, 8.0 Hz, 1H),
7.65–7.60 (m, 1H), 7.59–7.47 (m, 5H), 7.03 (brs, 1H), 6.70 (brs, 1H), 4.82–4.70 (m, 1H), 3.98–3.84 (m,
2H), 2.28 (s, 3H), 1.94–1.80 (m, 2H), 0.71–0.51 (m, 3H), 0.41–0.32 (m, 1H); 13C NMR (100 MHz,
DMSO-d6): δ 172.80, 162.07, 160.01, 159.78, 158.12, 150.20, 136.93, 134.82, 133.21, 130.18, 129.89,
129.78, 129.69, 129.67, 129.38, 127.44, 119.87, 118.14, 77.90, 61.10, 57.05, 38.05, 23.63, 21.11, 13.05,
9.08; ESI-HRMS: m/z calcd for C26H22ClN7O [M+H]+ 484.1653; found 484.1666; HPLC: tR = 9.55
min, Agilent TC-C18(2) 250 × 4.6mm 5µm, 30 °C, eluent A-40%, eluent B-60%.
(S)-4-amino-6-(6-(6-fluoro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptan-5-yl)
pyrimidine-5-carbonitrile (27)
1
Pale solid; yield 65%; H NMR (400 MHz, DMSO-d6): δ 8.00 (s, 1H), 7.78 (d, 8.0 Hz, 1H), 7.74–
7.44 (m, 7H), 7.21 (brs, 2H), 4.87–4.69 (m, 1H), 4.06–3.91 (m, 1H), 3.86 (d, 10.0 Hz, 1H), 2.06–1.98
(m, 1H), 1.97–1.88 (m, 1H), 0.80–0.69 (m, 1H), 0.67–0.55 (m, 2H), 0.45–0.34 (m, 1H); 13C NMR (100
MHz, DMSO-d6): δ 166.12, 161.28, 160.48 (d, JC-F = 243 Hz), 159.31, 159.10, 157.83, 144.63, 137.05,
130.60, 130.52, 130.03, 129.91, 129.82, 129.24, 123.56 (d, JC-F = 24.0 Hz), 122.23 (d, JC-F = 8.00 Hz),
117.76, 111.37 (d, JC-F = 24.0 Hz), 68.34, 61.55, 56.80, 38.34, 21.48, 12.10, 10.20; ESI-HRMS: m/z
calcd for C25H20FN7O [M+H]+ 454.1792; found 454.1785; HPLC: tR = 11.75 min, Agilent TC-C18(2)
18

1
2
250 × 4.6mm 5µm, 30 °C, eluent A-55%, eluent B-45%.
(S)-5-chloro-2-(5-(2-fluoro-9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-3-phenylquinazolin-4(3H)-o
ne (28)
3
4
Pale solid; yield 67%; 1H NMR (400 MHz, DMSO-d6): δ 14.12 (brs, 0.2H), 13.16 (s, 0.4H), 13.08 (s,
0.4H), 8.70 (s, 0.4H), 8.27–8.01 (m, 1.6H), 7.77–7.33 (m, 7H), 5.57–5.48 (m, 0.5H), 4.77–4.69 (m,
0.5H), 4.31–4.19 (m, 1H), 3.84 (d, 11.2 Hz, 0.5H), 3.68 (d, 11.2 Hz, 0.5H), 2.21–2.01 (m, 2H), 0.88–
0.74 (m, 1H), 0.71–0.55 (m, 2H), 0.49–0.40 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ 160.25,
159.85, 159.05, 158.71 (d, JC-F = 200 Hz), 158.24 (d, JC-F = 202 Hz), 157.87, 154.13, 153.92, 152.32,
152.12, 150.24, 149.86, 139.95, 139.88, 139.86, 137.12, 136.71, 134.92, 134.83, 133.24, 133.15,
130.67, 130.15, 130.08, 129.88, 129.79, 129.76, 129.74, 129.53, 129.38, 129.13, 129.02, 127.41,
127.29, 117.99 (d, JC-F = 2.00 Hz), 117.93 (d, JC-F = 4.00 Hz), 117.86 (d, JC-F = 4.00 Hz), 61.68, 60.65,
60.22, 57.73, 56.54, 38.50, 21.52, 18.82, 13.85, 12.08, 10.24, 8.07; ESI-HRMS: m/z calcd for
C25H19ClFN7O [M+H]+ 488.1402; found 488.1416; HPLC: tR = 7.37 min, Agilent TC-C18(2) 250 ×
4.6mm 5µm, 30 °C, eluent A-70%, eluent B-30%.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
(S)-5-chloro-2-(5-(2-chloro-9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-3-phenylquinazolin-4(3H)-o
ne (29)
Pale solid; yield 62%; 1H NMR (400 MHz, DMSO-d6): δ 14.14 (brs, 0.2H), 13.21 (s, 0.4H), 13.13 (s,
0.4H), 8.74 (s, 0.6H), 8.32–8.04 (m, 1.4H), 7.86–7.79 (m, 0.5H), 7.77–7.42 (m, 6H), 7.39 (dd, 1.5 Hz,
8.4 Hz, 0.5H), 5.53–5.46 (m, 0.4H), 4.78–4.69 (m, 0.6H), 4.29–4.16 (m, 1H), 3.85 (d, 11.2 Hz, 0.5H),
3.69 (d, 11.2 Hz, 0.5H), 2.18–1.94 (m, 2H), 0.91–0.71 (m, 1H), 0.69–0.56 (m, 2H), 0.51–0.39 (m, 1H);
13C NMR (100 MHz, DMSO-d6): δ 160.25, 159.83, 159.04, 157.88, 153.25, 153.05, 152.53, 151.97,
151.23, 150.26, 149.85, 148.19, 140.08, 137.16, 136.71, 134.91, 134.82, 133.23, 133.14, 130.66,
130.16, 130.06, 129.88, 129.79, 129.78, 129.73, 129.52, 129.48, 129.35, 129.32, 127.41, 127.29,
118.48, 117.97, 61.69, 60.59, 57.73, 56.56, 38.45, 21.46, 18.85, 13.84, 12.25, 10.10, 8.05; ESI-HRMS:
m/z calcd for C25H19Cl2N7O [M+H]+ 504.1106; found 504.1098; HPLC: tR = 9.06 min, Agilent
TC-C18(2) 250 × 4.6mm 5µm, 30 °C, eluent A-40%, eluent B-60%.
4.1.3. General procedure for preparing compounds 23 and 26
The mixture of corresponding Boc-deprotected secondary amine obtained in section 4.1.2 (1.1 eq), 2,
4-diamino-6-chloropyrimidine-5-carbonitrile (1.0 eq), DIPEA (5.5 eq), KF (2.0 eq) and DMSO (10
mL/1 mmol substrate) was stirred at 90 °C under N2 atmosphere. After the total consumption of 2,
19

1
2
4-diamino-6-chloropyrimidine-5-carbonitrile, water was added to the mixture, and the crude product
was obtained via filtration. Finally, the crude product underwent flash column chromatography
(DCM/EA = 10:1-4:1, V/V) to afford the title compound as pale solid.
3
4
(S)-2,4-diamino-6-(6-(5-fluoro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptan-5
-yl)pyrimidine-5-carbonitrile (23)
5
1
6
Pale solid; yield 51%; H NMR (400 MHz, DMSO-d6): δ 7.87 (d, 6.8 Hz, 1H), 7.82–7.72 (m, 1H),
7
7.67–7.39 (m, 5H), 7.26 (t, 9.2 Hz, 1H), 6.50 (brs, 2H), 6.21 (brs, 2H), 4.89–4.68 (m, 1H), 3.97–3.75
(m, 2H), 1.95–1.76 (m, 2H), 0.71–0.50 (m, 3H), 0.43–0.29 (m, 1H); 13C NMR (100 MHz, DMSO-d6):
δ 166.99, 162.46, 161.06 (d, JC-F = 262.0 Hz), 160.98, 159.07, 159.00 (d, JC-F = 4.00 Hz), 149.88,
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
136.75, 135.62 (d, JC-F = 11.0 Hz), 130.12, 129.90, 129.79, 129.64 (d, JC-F = 2.00 Hz), 123.88 (d, JC-F =
3.00 Hz), 120.02, 113.19 (d, JC-F = 20.0 Hz), 110.75, 110.70, 60.90, 60.82, 57.07, 38.36, 20.93, 12.87,
9.40; ESI-HRMS: m/z calcd for C25H21FN8O [M+H]+ 469.1901; found 469.1916; HPLC: tR = 7.26 min,
Agilent TC-C18(2) 250 × 4.6mm 5µm, 30 °C, eluent A-40%, eluent B-60%.
(S)-2,4-diamino-6-(6-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptan-5
-yl)pyrimidine-5-carbonitrile (26)
Pale solid; yield 54%; 1H NMR (400 MHz, DMSO-d6): δ 7.87 (d, 7.2 Hz, 1H), 7.71 (t, 8.0 Hz, 1H),
7.66–7.38 (m, 6H), 6.55 (brs, 2H), 6.25 (brs, 2H), 4.84–4.69 (m, 1H), 3.86 (s, 2H), 1.93–1.73 (m, 2H),
0.68–0.51 (m, 3H), 0.41–0.28 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ 166.96, 162.40, 160.96,
160.03, 158.75, 150.29, 136.98, 134.80, 133.19, 130.12, 129.89, 129.64, 129.31, 127.45, 120.02,
118.10, 60.92, 60.83, 57.09, 38.29, 20.99, 13.03, 9.28; ESI-HRMS: m/z calcd for C25H21ClN8O
[M+H]+ 485.1605; found 485.1622; HPLC: tR = 7.83 min, Agilent TC-C18(2) 250 × 4.6mm 5µm,
30 °C, eluent A-50%, eluent B-50%.
4.1.4. General procedure for preparing amide intermediates 31a-c
The mixture of 2-fluoro-6-nitrobenzoic acid (1.0 eq), catalytic amount of DMF, and SOCl2 (0.5
mL/1 mmol substrate) was refluxed for 5 h. Afterwards, it was concentrated, and the residue was
dissolved in anhydrous DCM. The resultant solution was added dropwise to a solution of
corresponding amine (1.0 eq) and TEA (1.2 eq) in anhydrous DCM at 0 °C. After being stirred at room
temperature for 3 h, the reaction mixture was quenched by saturated NaHCO3 solution, and extracted
with DCM. The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated
in vacuo. Finally, the crude product underwent flash column chromatography (PE/EA = 5:1, V/V) to
20

1
2
afford the amide (31a, 31b or 31c) as slight yellow solid.
N-benzyl-2-fluoro-6-nitrobenzamide (31a) Slight yellow solid; yield 61%.
3
N-cyclopentyl-2-fluoro-6-nitrobenzamide (31b) Slight yellow solid; yield 74%.
N- cyclohexyl-2-fluoro-6-nitrobenzamide (31c) Slight yellow solid; yield 71%.
4.1.5. General procedure for preparing imide intermediates 32a-c
4
5
6
The mixture of corresponding amide (31a, 31b or 31c, 1.0 eq), catalytic amount of DMF, and SOCl2
(3 mL/1 mmol amide) was stirred at 80 °C under N2 atmosphere for 2 h. Afterwards, it was
concentrated in vacuo, and the residue was dissolved in anhydrous DCM (6 mL/1 mmol amide). The
7
8
9
resultant
mixture
was
added
dropwise
to
a
solution
of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
(S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid (1.1 eq) and anhydrous TEA (3.0
eq) in anhydrous DCM (6 mL/1 mmol amide) at 0 °C under N2 atmosphere. After being stirred at the
same temperature for 1 h, the reaction mixture was stirred at room temperature for 6 h. Then, it was
quenched by saturated NaHCO3 solution and extracted with DCM. The organic layer was washed with
brine, dried over anhydrous Na2SO4, and concentrated in vacuo. Finally, the residue underwent flash
column chromatography (PE/EA = 4:1, V/V) to afford the imide (32a, 32b or 32c) as slightly brown
foam or pale foam. The 1H NMR spectra of 32a-c indicated the existence of rotamers.
Tert-butyl (S)-6-(benzyl(2-fluoro-6-nitrobenzoyl)carbamoyl)-5-azaspiro[2.4]heptane-5-carboxylate
(32a)
1
Slightly brown foam; yield 45%; H NMR (400 MHz, DMSO-d6): δ 8.27–8.15 (m, 1H), 7.93–7.73
(m, 2H), 7.64–7.08 (m, 5H), 5.45–4.70 (m, 3H), 3.29–3.06 (m, 2H), 2.45–2.22 (m, 1H), 1.64–1.43 (m,
1H), 1.40–1.15 (9H, three singlets), 0.61–0.36 (m, 3H), 0.34–0.21 (m, 1H); ESI-MS: m/z = 498
[M+H]+.
Tert-butyl
(S)-6-(cyclopentyl(2-fluoro-6-nitrobenzoyl)carbamoyl)-5-azaspiro[2.4]heptane-5-carboxylate (32b)
1
Pale foam; yield 53%; H NMR (400 MHz, DMSO-d6): δ 8.22–8.11 (m, 1H), 7.91–7.84 (m, 1H),
7.81–7.70 (m, 1H), 5.24–5.08 (m, 1H), 4.56–4.33 (m, 1H), 3.40–3.36 (m, 1H), 3.12 (d, 10.0 Hz, 1H),
2.63–2.56 (m, 0.7H), 2.49–2.41 (m, 0.3H), 2.16–1.72 (m, 6H), 1.63–1.43 (m, 3H), 1.39–1.28 (9H, three
singlets), 0.61–0.39 (m, 3H), 0.38–0.24 (m, 1H); ESI-MS: m/z = 476 [M+H]+.
Tert-butyl
(S)-6-(cyclohexyl(2-fluoro-6-nitrobenzoyl)carbamoyl)-5-azaspiro[2.4]heptane-5-carboxylate (32c)
21

1
1
2
Pale foam; yield 56%; H NMR (400 MHz, DMSO-d6): δ 8.22–8.10 (m, 1H), 7.89–7.81 (m, 1H),
7.79–7.70 (m, 1H), 5.16–5.00 (m, 1H), 3.91–3.69 (m, 1H), 3.28–3.25 (m, 1H), 3.17–3.04 (m, 1H),
2.71–2.54 (m, 1H), 2.42–2.23 (m, 2H), 1.86–1.66 (m, 4H), 1.65–1.45 (m, 2H), 1.40–1.26 (11H), 1.18–
1.06 (m, 1H), 0.63–0.51 (m, 2H), 0.50–0.23 (m, 2H); ESI-MS: m/z = 490 [M+H]+.
3
4
5
4.1.6. General procedure for preparing quinazolone intermediates 33a-c
6
To a solution of corresponding imide (32a, 32b or 32c, 1.0 eq) in AcOH (10 mL/1 mmol imide) was
carefully added activated zinc powder (10 eq) at room temperature. The resultant mixture was sttired at
40 °C under N2 atmosphere for 8 h. After filtration, the filtrate was concentrated in vacuo, and the
residue was dissolved in DCM. Then, the solution was washed with saturated NaHCO3 solution and
brine. The organic layer was dried over anhydrous Na2SO4, and concentrated in vacuo. Finally, the
residue underwent flash column chromatography (PE/EA = 6:1, V/V) to afford the quinazolone
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1
derivative (33a, 33b or 33c) as white foam. The H NMR spectra of 33a-c indicated the existence of
rotamers.
Tert-butyl
(S)-6-(3-benzyl-5-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate
(33a)
White foam; yield 83%; 1H NMR (400 MHz, DMSO-d6): δ 7.87–7.76 (m, 1H), 7.42 (d, 8.0 Hz, 1H),
7.39–7.23 (m, 6H), 5.47–5.20 (m, 2H), 5.10–4.98 (m, 1H), 3.57–3.47 (m, 1H), 3.41–3.37 (m, 0.3H),
3.32–3.26 (m, 0.7H), 1.44–1.21 (m, 5H), 1.06 (s, 6H), 0.61–0.51 (m, 2H), 0.43–0.35 (m, 2H); ESI-MS:
m/z = 450 [M+H]+.
Tert-butyl
(S)-6-(3-cyclopentyl-5-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate
(33b)
1
White foam; yield 72%; H NMR (400 MHz, CDCl3): δ 7.68–7.53 (m, 1H), 7.47–7.35 (m, 1H),
7.16–7.01 (m, 1H), 5.35–5.25 (m, 0.4H), 5.24–5.14 (m, 0.6H), 4.69–4.46 (m, 1H), 3.69 (d, 10.0 Hz,
0.7H), 3.61–3.48 (m, 1.3H), 2.54–2.37 (m, 2H), 2.26–2.08 (m, 2H), 2.00–1.92 (m, 1H), 1.85–1.75 (m,
1H), 1.74–1.57 (m, 4H), 1.49 (s, 3H), 1.30–1.15 (m, 6H), 0.75–0.53 (m, 3H), 0.52–0.34 (m, 1H);
ESI-MS: m/z = 428 [M+H]+.
Tert-butyl
(S)-6-(3-cyclohexyl-5-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate
22

1
2
(33c)
White foam; yield 84%; 1H NMR (400 MHz, DMSO-d6): δ 7.82–7.70 (m, 1H), 7.32 (d, 8.4 Hz, 1H),
3
7.27–7.15 (m, 1H), 5.25–5.09 (m, 1H), 4.13–3.86 (m, 1H), 3.59–3.37 (m, 2H), 2.75–2.54 (m, 2H),
1.87–1.71 (m, 4H), 1.69–1.58 (m, 2H), 1.57–1.43 (m, 1H), 1.41–1.16 (m, 6H), 1.10 (s, 6H), 0.70-0.39
(m, 4H); ESI-MS: m/z = 442 [M+H]+.
4
5
6
4.1.7. General procedure for preparing target compounds 18-20
7
Compounds 18-20 were prepared via similar procedure to that for compounds 14-17 as pale solid.
The NMR spectra of 18-20 indicated the existence of rotamers.
8
9
(S)-2-(5-(9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-3-benzyl-5-fluoroquinazolin-4(3H)-one (18)
1
10
11
12
13
14
15
16
17
18
Pale solid; yield 57% (for two steps); H NMR (400 MHz, DMSO-d6): δ 8.29–7.83 (m, 2H), 7.76–
7.61 (m, 1H), 7.60–7.07 (m, 7H), 6.32–6.20 (m, 0.4H), 5.82–5.67 (m, 0.6H), 5.64–5.41 (m, 2H), 4.36
(d, 10.0 Hz, 0.5H), 4.23 (d, 11.2 Hz, 0.5H), 3.99–3.88 (m, 0.5H), 3.79–3.70 (m, 0.5H), 2.29–2.02 (m,
1.3H), 1.95–1.85 (m, 0.7H), 0.79–0.21 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ 160.82 (d, JC-F
=
261.0 Hz), 160.32, 158.98, 152.18, 151.85, 151.77, 149.57, 140.17, 136.96, 135.54 (d, JC-F = 10.0 Hz),
128.96, 127.70, 127.43, 123.67, 119.47, 113.19 (d, JC-F = 20.0 Hz), 109.99 (d, JC-F = 6.00 Hz), 60.28,
57.64, 46.33, 39.04, 24.78, 21.49, 18.82, 12.02, 10.39; ESI-HRMS: m/z calcd for C26H22FN7O [M+H]+
468.1948; found 468.1942; HPLC: tR = 9.54 min, Agilent TC-C18(2) 250 × 4.6mm 5µm, 30 °C, eluent
A-40%, eluent B-60%.
19
20
(S)-2-(5-(9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-3-cyclopentyl-5-fluoroquinazolin-4(3H)-one
(19)
21
22
23
24
25
26
27
28
29
30
Pale solid; yield 60% (for two steps); 1H NMR (400 MHz, CDCl3): δ 13.17 (brs, 1H), 8.44 (s, 0.6H),
8.18 (s, 0.4H), 8.00 (s, 0.4H), 7.69 (s, 0.6H), 7.54–7.36 (m, 1H), 7.20 (d, 8.0 Hz, 0.4H), 7.14 (d, 8.0 Hz,
0.6H), 7.06–6.91 (m, 1H), 6.72–6.57 (m, 0.6H), 5.97–5.86 (m, 0.4H), 4.96–4.77 (m, 1H), 4.54 (d, 10.8
Hz, 0.4H), 4.44 (d, 10.8 Hz, 0.4H), 4.13 (d, 10.8 Hz, 0.6H), 3.97 (d, 10.8 Hz, 0.6H), 2.63–2.30 (m, 3H),
2.29–2.11 (m, 2H), 1.93–1.65 (m, 5H), 0.87–0.64 (m, 3H), 0.61–0.40 (m, 1H); 13C NMR (100 MHz,
DMSO-d6): δ 160.73 (d, JC-F = 262.0 Hz), 159.97, 158.93 (d, JC-F = 3.00 Hz), 152.83, 152.56, 151.09,
149.02, 139.18, 134.92 (d, JC-F = 10.0 Hz), 123.37, 119.72, 112.88 (d, JC-F = 21.0 Hz), 110.89 (d, JC-F
=
5.00 Hz), 61.74, 58.57, 56.12, 39.03, 28.92, 28.30, 26.19, 26.08, 19.04, 14.82, 8.24; ESI-HRMS: m/z
calcd for C24H24FN7O [M+H]+ 446.2105; found 446.2108; HPLC: tR = 6.94 min, Agilent TC-C18(2)
250 × 4.6mm 5µm, 30 °C, eluent A-50%, eluent B-50%.
23

1
2
(S)-2-(5-(9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-3-cyclohexyl-5-fluoroquinazolin-4(3H)-one
(20)
1
3
Pale solid; yield 63% (for two steps); H NMR (400 MHz, DMSO-d6): δ 12.99 (s, 0.3H), 12.86 (s,
4
0.7H), 8.31–7.86 (m, 2H), 7.69–7.51 (m, 1H), 7.22–7.00 (m, 2H), 6.49–6.32 (m, 0.7H), 5.85–5.72 (m,
0.3H), 4.47 (d, 11.2 Hz, 0.3H), 4.40–4.16 (m, 1.3H), 4.02 (d, 10.8 Hz, 0.7H), 3.76 (d, 10.8 Hz, 0.7H),
3.00–2.84 (m, 0.7H), 2.80–2.56 (m, 2.3H), 2.49–2.43 (m, 1H), 2.15–1.33 (m, 8H), 0.78–0.42 (m, 4H);
13C NMR (100 MHz, DMSO-d6): δ 160.73 (d, JC-F = 263.0 Hz), 159.60 (d, JC-F = 4.00 Hz), 159.33,
152.89, 152.67, 150.87, 149.19, 148.93, 139.31, 138.94, 134.98 (d, JC-F = 10.0 Hz), 123.38, 123.28,
119.59, 113.96 (d, JC-F = 21.0 Hz), 111.03 (d, JC-F = 4.00 Hz), 61.70, 60.12, 59.71, 57.82, 56.21, 38.81,
28.27, 27.89, 26.34, 26.19, 26.05, 25.61, 25.47, 18.93, 14.80, 10.27, 8.25; ESI-HRMS: m/z calcd for
C25H26FN7O [M+H]+ 460.2261; found 460.2267; HPLC: tR = 11.43 min, Agilent TC-C18(2) 250 ×
4.6mm 5µm, 30 °C, eluent A-50%, eluent B-50%.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
4.2. Biology
4.2.1. Class I PI3Ks biochemical assay
The inhibitory activity against class I PI3Ks was evaluated via ADP-Glo assay according to a
reported protocol with minor modification [35].
4.2.2. Anti-proliferative assay
The anti-proliferative activity of compounds against SU-DHL-6 cell line was evaluated via MTT
assay. SU-DHL-6 cells (ATCC) seeded in 96-well plates at the density of 1×105/well were cultured
(37 °C, 5% CO2) till 90% of the cells were fused. Following 2 h-incubation in serum-free medium, the
supernatant was discarded. The cells were incubated with RPMI 1640 medium alone or with the tested
compounds at the indicated concentrations for 72 h. At 68 h post-compound treatment, 20 µL MTT
solution (5 mg/mL) was added. The 96-well plates were centrifuged at 1500 rpm for 3 min, and the
supernatant was discarded. After adding 150 µL DMSO to each well, the plates were shaken for 10 min.
The OD570 value was read and the inhibition rates calculated accordingly. GI50 values were calculated
using GraphPad Prism 6.
4.2.3. Western blot assay
Anti-phos-Akt (S473), Anti-Akt, Anti-phos-S6K1 (T389), Anti-S6K1, and Anti-GAPDH purchased
from Abcam Inc., as well as corresponding secondary antibodies were utilized in Western blot analysis.
GAPDH served as a loading control. SU-DHL-6 cells seeded into six-well plate at a density of 1 ×
24

1
2
106/well were cultured overnight (37 °C, 5% CO2). Following exposure to compound 26 or Idelalisib at
the desired concentrations for 2 h, cells were harvested for immunoblotting. The proteins were
separated on SDS-PAGE, and subsequently transferred onto PVDF membrane. After successive
incubation of the membranes with antibodies and secondary antibodies, the membranes were imaged
and the optical density measured.
3
4
5
6
4.2.4. PK study
7
The dosage and the number of animals for each group were presented in Table 4. Male SD rats were
used in this experiment. Both the intravenous and oral doses were formulated in a solution of 5%
DMSO, 10% Solutol, 10% EtOH and 75% Saline. At the designated time points, the animal was
anaesthetized with isoflurane, and blood sample was collected via ophthalmic vein for terminal
bleeding into heparin-coated EP tubes. The plasma samples were stored at –20 °C till the analysis, and
the concentration of the tested compound in plasma was determined with UHPLC-MS/MS.
4.3. Molecular docking
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
C-DOCKER module of Discovery Studio (version 2.5; Accelrys, San Diego, CA, USA, 2008) was
utilized for the molecular modeling. The CHARMm-force field was applied to the protein after the
removal of original ligand and solvent molecules from the PI3Kδ/Idelalisib co-crystal structure. 3D
structure of the ligand was generated, and the energy minimization performed. The ligand was then
docked into the active site of the receptor, which was defined according to the location of Idelalisib in
the enzyme. The ultimate binding conformation was determined on the basis of the calculated
C-DOCKING ENERGE.
Conflict of interest
The authors confirm that this article content has no conflicts of interest.
Acknowledgements
This research was financially spported by Natural Science Foundation of Anhui Province (No.
1808085QH261 and 1808085QH289), the Key Project of Natural Science Research in Universities of
Anhui Province (No. KJ2019A1004), and University-Enterprise Cooperative Projects (No. 2018HZ6
and No. 2019HZ078).
25

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2
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