Discovery of (S)-2-(1-(4-Amino-3-(3-fluoro-4-methoxyphenyl)-1 H-pyrazolo[3,4- d]pyrimidin-1-yl)propyl)-3-cyclopropyl-5-fluoroquinazolin-4(3 H)-one (IHMT-PI3Kδ-372) as a Potent and Selective PI3KδInhibitor for the Treatment of Chronic Obstructive Pulmonary Disease

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Discovery of (S)‑2-(1-(4-Amino-3-(3-ﬂuoro-4-

methoxyphenyl)‑1H‑pyrazolo[3,4‑d]pyrimidin-1-yl)propyl)-3-

cyclopropyl-5-ﬂuoroquinazolin-4(3H)‑one (IHMT-PI3Kδ-372) as a

Potent and Selective PI3Kδ Inhibitor for the Treatment of Chronic

Obstructive Pulmonary Disease

Feng Li,^#Xiaofei Liang,^#Zongru Jiang,^#Aoli Wang,^#Junjie Wang, Cheng Chen, Wenliang Wang,

Fengming Zou, Ziping Qi, Qingwang Liu, Zhenquan Hu, Jiangyan Cao, Hong Wu, Beilei Wang,

Li Wang, Jing Liu,* and Qingsong Liu *

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ABSTRACT: Accumulated pieces of evidence have shown that

PI3Kδ plays a critical role in chronic obstructive pulmonary disease

(COPD). Using a fragment-hybrid approach, we discovered a

potent and selective PI3Kδ inhibitor (S)-18. In the biochemical

assay, (S)-18 inhibits PI3Kδ (IC₅₀= 14 nM) with high selectivity

over other class I PI3Ks (56∼83 fold). (S)-18 also achieves good

selectivity over other protein kinases in the kinome (S-score (35) =

0.015). In the cell, (S)-18 selectively and potently inhibits the

PI3Kδ-mediated phosphorylation of AKT T308 but not other class

I PI3K-mediated signaling. Additionally, (S)-18 exhibits no

apparent inhibitory eﬀect on CYP isoforms except for a moderate eﬀect on CYP2C9. Furthermore, it shows no apparent inhibitory

activity against hERG (IC₅₀> 10 μM). In vivo, (S)-18 displays favorable PK properties for inhaled delivery and improves lung

function in a rodent model of pulmonary inﬂammation. These results suggest that (S)-18 might be a new potential therapeutic

candidate for COPD.

ubiquitous,¹⁰and they both play essential roles in cell growth,

INTRODUCTION

■

survival, and proliferation.¹¹In addition, PI3Kα and PI3Kβ

The Global Burden of Disease study 2017 reports that chronic

knockout (KO) mice are embryonically lethal.¹²PI3Kγ is

obstructive pulmonary disease (COPD) is becoming a major

primarily present in leukocytes¹³and to a lesser extent in

global health problem and it has been ranked as the fourth

cardiomyocytes. Although PI3Kγ KO mice are viable and

cause of death.¹⁻⁴COPD, characterized by airﬂow limitation⁵

fertile, they have a reduced ability to cope with cardiac

and associated with abnormal inﬂammatory response of the

distress.¹⁴⁻¹⁶The expression and function of PI3Kδ are

lung, is a highly prevalent chronic progressive and not fully

predominantly restricted to immune cells. Many studies

reversible respiratory disease. The common symptoms of

indicated the importance of isoform-speciﬁc inhibitors for

COPD include emphysema, airway ﬁbrosis, chronic bronchitis,

clinical applications. Inhibition of PI3Kδ has been shown to be

etc., which can lead to innate immune response of

beneﬁcial to inﬂammatory diseases.^10,17−25Several small-

inﬂammatory cells. With disease progression, inﬂammatory

molecule PI3Kδ inhibitors such as 1 (GSK2269557),²³

2

associated cells such as macrophages, neutrophils, lympho-

cytes, and dendritic cells would be elevated in the micro-

environment. Increased inﬂammatory responses in turn

promote the development of COPD.⁶⁻⁸Currently, most of

the COPD therapies do not directly target the pathogenic

mechanisms of chronic inﬂammation.⁸Therefore, new

approaches such as targeting the inﬂammatory process have

attracted extensive interests for the new COPD therapy.

PI3Kδ belongs to the class I Phosphoinositide 3-kinases

(PI3Ks), which include the PI3Kα, PI3Kβ, and PI3Kγ

isoforms.⁹The expressions of PI3Kα and PI3Kβ are

(GSK2292767),²³and 3 (LAS191954)²⁶have been inves-

tigated either in the clinical trial or preclinical stages of COPD

(Figure 1). In this study, we adopted a fragment hybridization

Received: September 3, 2020

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A

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Figure 1. Chemical structures of representative PI3Kδ inhibitors for COPD.

Figure 2. (A) Schematic illustration of the discovery of (S)-18 (IHMT-PI3Kδ-372). (B) Overlay of 4 (green; PDB code: 4XE0²⁸) and 5 (wheat;

PDB code: 5M6U²⁴) bound to PI3Kδ in the speciﬁcity pocket (Trp760 and Met752), hydrophobic region II (Asp832, Thr833, and Asp836), and

hinge binding region (Glu826 and Val828). (C) Binding modes of 4 and 5 with PI3Kδ in the aﬃnity pocket (Ile825, Tyr813, Ile910, and Asp911).

strategy starting from two propeller-shaped PI3Kδ inhibitors 4

(CAL-101)^27,28and 5 (TGR-1202),²⁹which have been applied

to cancer. Through medicinal chemistry eﬀort, we have

discovered a novel PI3Kδ inhibitor compound (S)-18

(IHMT-PI3Kδ-372), which exhibited anti-COPD eﬃcacy in

vivo (Figure 2A).

PI3Kδ (Figure 2B,C). The quinazolinone moiety of 4 is

sandwich-folded into the induced hydrophobic speciﬁcity

pocket between Trp760 and Met752 with a hydrophobic

interaction with the kinase. It is worthy to note that this

speciﬁcity pocket is not present in the apo PI3Kδ kinase

structure in which Trp760 and Met752 are packed against each

other. The purine moiety of 4 locates in the hinge binding

region and forms hydrogen bonds with the backbone amide of

Val828 and the backbone carbonyl oxygen of Glu826 (PDB:

4XE0²⁸). The propeller-shaped selective PI3Kδ inhibitor 5

adopts a similar binding mode except that the ﬂuoro-

isopropoxyphenyl moiety interacts with the aﬃnity pocket,

which is not occupied by 4 (Figure 2B,C). Therefore, we tried

to combine the binding elements of 4 and 5 to design novel

PI3Kδ inhibitors and exploited the hydrophobic region II and

the aﬃnity pocket for the SAR study.

RESULTS AND DISCUSSION

■

Structure−Activity Relationship (SAR) Exploration. In

general, there are four distinct inhibitor binding regions in the

ATP-binding pocket of PI3Kδ,^27,30−32i.e., a hinge binding

region with hydrogen-bond interaction, an aﬃnity pocket deep

in the protein not occupied by ATP, a speciﬁcity pocket, and a

hydrophobic region II located at the entrance of the active site.

Compound 4, which adopts a special propeller shape, occupies

the hydrophobic region II, the speciﬁcity pocket, and the hinge

region, but not the aﬃnity pocket,^27,28which is potentially

essential for improving the potency and isoform selectivity to

We initially synthesized a series of compounds as racemic

mixtures to quickly explore the SAR of hydrophobic region II.

B

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c

Table 1. SAR Exploration of the R₁Moiety

a

b

c

All IC₅₀values were obtained by triplet testing. Determined from isoform-speciﬁc PI3K IC₅₀values. Data from ref 33.

The kinase inhibitory activity and isoform selectivity of the

new compounds were proﬁled using an ADP-Glo kinase assay.

Compound 6 was generated by appending a modiﬁed tail of 5

to the 5-ﬂuoro-3-phenylquinazolin-4(3H)-one fragment of 4.

C

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b

Table 2. SAR Exploration of the R₂Moiety

a

b

All IC₅₀values were obtained by triplet testing. Determined from isoform-speciﬁc PI3K IC₅₀values.

The results showed that 6 displayed decreased potency against

PI3Kδ and lost selectivity against PI3Kγ, while it maintained

selectivity over PI3Kα and PI3Kβ compared with 4. Mean-

while, it displayed comparable inhibitory activity against PI3Kδ

relative to 5 (Table 1). Unfortunately, introduction of larger

moieties such as biphenyl (7) at R₁resulted in loss of potency

against all of the class I PI3K isoforms. Replacement of the

phenyl group of 6 with the benzyl group (8) also decreased the

activity against PI3Kδ and other class I PI3K isoforms. The

smaller methylpyrazole group (9) at R₁retained the potency

against PI3Kδ and isoform selectivity in comparison with 6.

These results indicated that the hydrophobic region II could

not accommodate the larger hydrophobic groups such as the

biphenyl or benzyl group. Switching the phenyl group at R₁to

the aliphatic cyclohexyl group (10) resulted in signiﬁcant

activity loss against PI3Kδ. Replacement of aromatic

substituents with smaller cyclic aliphatic rings such as the

cyclopentyl group (11), the cyclobutyl group (12), and the

D

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cyclopropyl group (13) led to improved potencies against

PI3Kδ and retained the selectivity against other class I PI3K

members. This suggested that the hydrophobic region II could

accommodate the small cyclic aliphatic substituents. In

addition, introduction of the isobutyl group (14) at R₁did

not aﬀect the potency against PI3Kδ or the overall selectivity

over other class PI3K members. Replacement of the phenyl

group at R₁with the isopropyl group (15), the ethyl group

(16), and the methyl group (17) retained the activities against

PI3Kδ and the selectivity against PI3Kγ but lost the selectivity

over PI3Kα and PI3Kβ.

Table 3. Evaluation of the Enantiomers of 18 against PI3Kδ

Among the tested compounds, 13 displayed the highest

inhibitory activity against PI3Kδ and also retained isoform

selectivity. Therefore, we kept the cyclopropyl group at R₁and

further explored the SAR of other positions. Considering that

the ethyl group of 4 binds in a hydrophobic pocket formed by

Ile910, Met-900, and Met752,²²we also examined the ethyl

analogue and found that 18 retained the potency against

PI3Kδ and selectivity over PI3Kα and PI3Kβ (Table 2).

Additionally, it exhibited increased selectivity over PI3Kγ.

Therefore, we went forward to investigate the SAR of the R₂

moiety in 18, which interacts with the aﬃnity pocket of PI3Kδ.

Removal of the ﬂuoromethoxyphenyl moiety in 18 (19) led to

complete loss of activity against class I PI3K members,

suggesting that the hydrophobic interactions between these

compounds and the aﬃnity pocket of PI3Kδ are critical to the

potencies against PI3Kδ. Replacing the ﬂuoromethoxyphenyl

moiety with a phenyl group (20) led to signiﬁcant activity loss

against PI3Kδ, indicating that the substituents in the phenyl

moiety are necessary to form the hydrophobic interactions

with PI3Kδ. Removal of the ﬂuorine atom (21) in the R₂

moiety of 18 or replacement of the ﬂuorine atom by the

methoxyl group (22) or the methyl group (23) resulted in

slightly decreased activities against PI3Kδ and selectivity over

other class I PI3K members. Replacing the methoxy group in

18 with the isopropoxy group (24) led to signiﬁcant activity

loss against PI3Kδ, indicating that the methoxy group played

an important role in the interactions with PI3Kδ. Mono-

substituted phenyl moieties at R₂such as 25 and 26 also led to

decreased potencies against PI3Kδ. Further increasing the size

and hydrophobicity of the R₂moiety (27−28) did not improve

the potency to PI3Kδ or the isoform selectivity. Interestingly,

replacement of the ﬂuoromethoxyphenyl moiety with a smaller

methylpyrozole moiety (29) retained both the potency against

PI3Kδ and isoform selectivity. However, installation of the

thienyl moiety at R₂(30) led to dramatic activity loss against

PI3Kδ.

Since the racemic compound 18 exhibited optimum potency

against PI3Kδ and isoform selectivity, we then evaluated the

two stereoisomers of 18 (Table 3). The S-enantiomer ((S)-18)

was slightly more active than the R-enantiomer ((R)-18). In

addition, (S)-18 showed better selectivity over PI3Kβ and

PI3Kγ than (R)-18 and thus it was selected for further

examination.

Selectivity Evaluation of Compound (S)-18. We next

used the DiscoverX KINOMEscan technology to further

characterize the kinome-wide selectivity proﬁle of (S)-18. The

results showed that it exhibited high selectivity with an S-score

(35) = 0.015 among 468 kinases/mutant tested at 1 μM

concentration (Figure 3 and Supporting Table 1). Besides

strong binding to PI3Kδ, (S)-18 also displayed binding to

PI3Kβ, γ, and PI4Kβ in the PIK family as well as Chk2 kinase.

class I PI3K isoform selectivity

a

a b

,

PI3Kδ

(ratio)

compd

(IC₅₀: nM)

α/δ

β/δ

γ/δ

(R)-18

(S)-18

19 7.0

14 1.2

124

83

41

56

74

81

a

b

All IC₅₀values were obtained by triplet testing. Determined from

isoform-speciﬁc PI3K IC₅₀values.

Since KINOMEscan is a binding-based assay that may not

really reﬂect the actual inhibitory activity of the kinase

inhibitor, we also examined the cellular potencies of (S)-18

against all four class I PI3K isoforms (α, β, γ, δ) using a series

of PI3K isoform-speciﬁc in vitro cell-based assays to better

evaluate the isoform selectivity (Figure 4). Upon anti-IgM

stimulation, the PI3Kδ-mediated AKT T308 phosphorylation

in Raji cells was inhibited by (S)-18 with an EC₅₀value of 67

nM. In addition, (S)-18 did not show apparent inhibitory

eﬀects on AKT phosphorylation controlled by PI3Kα (NIH-

3T3, PDGF-BB stimulation), PI3Kβ (NIH-3T3, LPA stim-

ulation), and PI3Kγ (RAW 264.7, human C5a stimulation) up

to 3 μM.

To further understand the binding mode of (S)-18, the

inhibitor was docked into the PI3Kδ structure (PDB:

5M6U²⁴) that contains a propeller compound via molecular

modeling (Figure 5). The model revealed that (S)-18 binds to

the ATP-binding site of the kinase domain of PI3Kδ with a

propeller shape. Two hydrogen bonds are formed between the

amine pyrazolopyrimidine moiety and residues Glu826 and

Val828 in the hinge binding region (Figure 5A). The

ﬂuoroquinazolinone moiety is sandwiched into the induced

speciﬁcity pocket between Trp760 and Met752. The cyclo-

propyl group is located at hydrophobic region II. The

methoxyphenyl moiety interacts with the aﬃnity pocket

formed by Ile825, Tyr813, Ile910, and Asp911 (Figure 5B).

The oxygen atom of the methoxyphenyl forms a hydrogen

bond with the backbone of Ile910. The contribution of these

interactions is consistent with the observed potency diﬀerence

between 19 and (S)-18.

Primary Safety Evaluation of Compound (S)-18. To

evaluate the potential drug−drug interactions, (S)-18 was

tested for inhibition of the most important hepatic metabolic

enzymes (Table 4). (S)-18 showed moderate inhibition of

CYP2C9 (IC₅₀= 2.7 μM) and no apparent inhibition against

CYP1A2, CYP2B6, CYP2C19, and CYP3A4 (IC₅₀s > 10 μM).

The potential heart toxicity of (S)-18 was also examined with

the electrophysiology functional hERG assay with a patch

clamp (Figure 6). The results revealed a very weak inhibitory

activity of (S)-18 (IC₅₀= 13.8 μM), which was considered in a

safe range (>10 μM).

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Figure 3. Kinome-wide selectivity proﬁling of (S)-18 with the DiscoverX KINOMEscan technology. Measurements were performed at a

concentration of 1 μM (S)-18. TREEspot interaction maps for (S)-18 against 468 kinases. S-score is a quantitative measure of compound

selectivity. It is calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding

mutant variants. S is the number of hits/number of assays.

Figure 4. Inhibitory eﬃcacies of (S)-18 against class I PI3K isoforms in cell-based assays.

Pharmacokinetic Proﬁles of Compound (S)-18. We

next evaluated the in vivo pharmacokinetic proﬁles of (S)-18 in

rats. At an oral dose of 10 mg/kg, (S)-18 showed high in vivo

clearance of 375.9 mL/min/kg and poor oral bioavailability of

5.2% (Table 5), indicating that it might not be a good

candidate for oral delivery. Given the fact that COPD is a

respiratory disease, we then investigated inhalation as an

alternative route of administration. In rats, inhalation of 5 mg/

kg dose of (S)-18 displayed a half-life of 2.3 h, low exposure of

66 ng/mL, and high clearance of 348.5 mL/min/kg in plasma

(Table 5) but high exposure of 5599 ng/g (6 h after

inhalation) in lung tissue (Figure 7). These data indicated that

(S)-18 is suitable for inhaled delivery.^23,24In addition, we also

evaluated the metabolic stability of (S)-18 in liver microsomes

among diﬀerent species. The results showed that (S)-18 is

stable in human, rat, and mouse liver microsomes, while it has

moderate stability in monkey and dog liver microsomes (Table

6).

In Vivo Eﬃcacy Evaluation of Compound (S)-18. The

in vivo eﬃcacy of (S)-18 was evaluated in an animal model.

The cigarette-smoke- and LPS-induced rodent model mimics

the characteristic features of COPD, including lung function

decline and increased lung inﬂammation. Aerosolized (S)-18

was dosed at 1 mg/kg/day (low dose), 3 mg/kg/day (medium

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Figure 5. Molecular modeling of (S)-18 (yellow) with PI3Kδ kinase (PDB: 5M6U). The key binding amino acid residues from the protein were

labeled as follows: carbon in green, nitrogen in blue, and oxygen in red. Hydrogen bonds are indicated by red dashed lines to the key amino acid

residues. (A) Cartoon view of the speciﬁcity pocket formed by Trp760, Met752, and the hinge binding region. (B) Cartoon view of the aﬃnity

pocket formed by Ile825, Tyr813, Ile910, and Asp911 to accommodate the methoxyphenyl moiety.

Table 4. Inhibition of (S)-18 against Five Cytochrome

P450s

CYP enzyme

CYP1A2 CYP2B6 CYP2C9 CYP2C19 CYP3A4

>10 >10 2.7 >10 >10

(S)-18 (IC_50:μM)

Figure 7. Concentration of (S)-18 in the lung tissue of rats via an

inhaled route.

Table 6. Metabolic Stability of (S)-18 in Liver Microsomes

a

species

t_1/2(min)

CL_int(μL/min/mg)

CL^hep(mL/min/kg)

HLM

MkLM

DLM

RLM

187

18.6

36.5

94.9

365

7.40

74.6

38.0

14.6

3.80

5.04

30.6

19.7

20.1

12.9

Figure 6. Patch-clamp functional hERG assay of (S)-18.

dose), and 5 mg/kg/day (high dose) to Sprague-Dawley (SD)

rats via the inhalation route for 28 consecutive days (Figure 8).

The results showed that the lung function parameters such as

forced expiratory volume in the ﬁrst second (FEV1), forced

vital capacity (FVC), and peak expiratory ﬂow (PEF) were

improved dose-dependently (Figure 8A and Supporting Table

2). Blood-gas analysis showed that the partial pressure of

arterial oxygen (PaO₂) and the arterial oxygen saturation

(SaO₂) were also improved in the drug-treated groups

compared with the vehicle group, and meanwhile the partial

pressure of arterial carbon dioxide (PaCO₂) decreased (Figure

8B and Supporting Table 3). Cell counting of bronchoalveolar

lavage ﬂuid (BALF) demonstrated that the abnormally high

level of leukocytes including the alveolar macrophages,

neutrophils, and lymphocytes were reduced by drug treatment

MLM

a

Species: HLM, human liver microsomes; MkLM, monkey liver

microsomes; DLM, dog liver microsomes; RLM, rat liver microsomes;

MLM, mouse liver microsomes.

(Figure 8C and Supporting Table 4). Meanwhile, the

inﬂammatory cytokines such as IL-6, IL-8, TNF-α, and IL-1β

were also downregulated in both BALF (Figure 8D and

Supporting Table 5) and plasma (Figure 8E and Supporting

Table 6). In addition, histopathological analysis of the rat lung

tissue demonstrated that (S)-18 decreased the inﬂammatory

cell inﬁltration in a dose-dependent manner (HE staining in

Figure 8F). Furthermore, apoptosis detection showed that the

number of TUNEL-positive cells (stained brown or tan)

a

Table 5. Pharmacokinetic Proﬁles of (S)-18 in Rats

b

dose (mg/kg)

administration route

t_1/2(h)

C_max(ng/mL)

AUC_0−t(h*ng/mL)

Cl (mL/min/kg)

F (%)

1

10

5

i.v.

p.o.

inhal.

1.7 0.8

2.3 0.3

2.3 0.9

942.3 34.0

122.0 130.9

65.5 23.2

904 264

521.1 241.2

240.2 93.5

18.1 5.9

375.9 190.3

348.5 137.1

5.2 2.4

a

b

Mean SD, n = 3. Route of administration: i.v., intravenous administration; p.o., oral administration; inhal., inhaled administration.

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Figure 8. In vivo eﬃcacy evaluation of (S)-18 in a cigarette-smoke-induced and LPS-induced lung inﬂammation rodent model. (A) Lung function

assessment. (B) Blood-gas analysis. (C) BALF cell counts. (D) Measurement of the inﬂammatory cytokines in BALF. (E) Measurement of the

inﬂammatory cytokines in plasma. Mean SD, n = 10. *p < 0.05, **p < 0.01 vs the vehicle group; ## for p < 0.01 vs the normal group. (F)

Histopathological examination of the lung tissue by HE staining and apoptosis detection by the TUNEL assay.

gradually decreased in the low-, medium-, and high-dose (S)-

18 groups compared with the vehicle group (TUNEL assay in

Figure 8F). Overall, the in vivo results indicated that (S)-18

improved lung function and reduced the inﬂammatory patterns

characteristic of COPD (Figure 8).

or butyric anhydride followed by reduction of the nitro group

with zinc in acetic acid and spontaneous cyclization aﬀorded

the quinazolinones 39a−e. Bromination of 39a−e with NBS

and AIBN provided the intermediates 40a−e. Subsequent

nucleophilic substitution reaction oﬀered the target com-

pounds 8, 10−12, and 18−30 as racemic mixtures.

Compound 13 was synthesized from 33 through the amide

coupling reaction, acylation, nucleophilic substitution, and

cyclization in four steps as racemic mixtures (Scheme 3). (R)-

2-Chlorobutanoic acid and (S)-2-chlorobutanoic acid (44)

were treated with oxalyl dichloride and then coupled with 41

to give the corresponding diamides 45a−b (Scheme 4). The

quinazolinones 46a−b were obtained via cyclization of 45a−b,

and subsequent nucleophilic substitution with 32a aﬀorded the

two stereoisomers (R)-18 and (S)-18.

CHEMISTRY

■

The synthetic routes are depicted in Schemes 1−4. The

intermediates 32a−l were synthesized by the Suzuki−Miyaura

coupling reaction (Scheme 1). Benzoic acid 33 was reacted

with diﬀerent amines under standard 1-[bis(dimethylamino)-

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-

ﬂuorophosphate (HATU)-mediated coupling condition to

yield the amides 34a−g, followed by treatment with acyl

chloride to aﬀord the diamides 35a−g. Hexamethyldisilazane

(HMDS)/ZnCl₂-mediated cyclization of 35a−g in acetonitrile

provided the quinazolinone analogues 36a−g,³⁴which were

then converted to the target compounds 6−7, 9, and 14−17

by nucleophilic substitution as racemic mixtures.

CONCLUSIONS

■

In summary, we have discovered a novel potent and isoform-

selective PI3Kδ inhibitor compound (S)-18 through a

fragment hybridization strategy. It exhibited an IC₅₀value of

14 nM against PI3Kδ with high selectivity over other class I

PI3Ks (56−83 fold). By comparison, (S)-18 was slightly less

potent than 4 but more potent than 5, and meanwhile it

As shown in Scheme 2, benzoic acid 37 was treated with

oxalyl dichloride followed by coupling with a set of amines to

yield the corresponding amides 38a−e (Scheme 2).

Propionylation or butyrylation of 38a−e with excess propionic

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a

Scheme 1. Synthetic Route to Racemic Compounds 6−7, 9, and 14−17

a

Reagents and conditions: (a) boronic acid or ester, Pd(PPh₃)₄, K₂CO₃, dioxane, H₂O, 135 °C, 14 h; (b) amine, HATU, N,N-

diisopropylethylamine (DIPEA), tetrahydrofuran (THF), rt, 6 h; (c) acyl chloride, DIPEA, THF, 0 °C to rt, 6 h; (d) HMDS, ZnCl₂, MeCN, reﬂux,

5 h; (e) 32a, K₂CO₃, DMF, 60 °C, 8 h.

a

Scheme 2. Synthetic Route to Racemic Compounds 8, 10−12, and 18−30

a

Reagents and conditions: (a) (COCl)₂, DMF, THF, 0 °C to rt, 5 h; (b) amine, DIPEA, THF, 0 °C to rt, 6 h; (c) acid anhydride, microwave, 140

°C, 3 h; (d) Zn, AcOH, rt, 8 h; (e) NBS, AIBN, CCl₄, reﬂux, 10 h; (f) 32a−l, K₂CO₃, DMF, 60 °C, 8 h.

I

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a

Scheme 3. Synthetic Route to Racemic Compound 13

a

Reagents and conditions: (a) cyclopropanamine, HATU, DIPEA, THF, rt, 6 h; (b) 2-chloropropanoyl chloride, DIPEA, THF, 0 °C to rt, 6 h; (c)

32a, K₂CO₃, DMF, 60 °C, 8 h; (d) HMDS, ZnCl₂, MeCN, reﬂux, 5 h.

a

Scheme 4. Synthetic Route to Compounds (R)-18 and (S)-18

a

Reagents and conditions: (a) (COCl)₂, DMF, THF, 0 °C to rt, 5 h; (b) 41, DIPEA, THF, 0 °C to rt, 6 h; (c) HMDS, ZnCl₂, MeCN, reﬂux, 5 h;

(d) 32a, K₂CO₃, DMF, 60 °C, 8 h.

displayed higher selectivity against PI3Kγ. (S)-18 also

exhibited high selectivity over other protein kinases, which

was revealed in the KINOMEscan proﬁling (S-score (35) =

0.015). Furthermore, the CYP enzymatic assay indicated a low

potential for induction of the drug−drug interactions, and the

patch-clamp electrophysiology functional assay implied a low

risk of hERG-mediated cardiac toxicity.

Notably, the pharmacokinetic proﬁles of (S)-18 by oral

delivery, such as high plasma clearance and low oral

bioavailability, indicated that it is a poor oral drug candidate

compared with most of the reported PI3Kδ inhibitors.

However, administration of (S)-18 by the inhaled route

showed high exposure in rat lung tissue upon 6 h, and

meanwhile high plasma clearance and low exposure in blood

plasma were retained. This is actually an advantage if the

inhibitor is aiming for lung disease because it has been

reported that oral administration of PI3Kδ inhibitor 4

exhibited adverse eﬀects as an anticancer drug in the clinic,³⁵

whereas delivering drugs directly to the site of action, e.g.,

inhaled drug delivery for respiratory disease, may result in a

rapid onset of desired eﬀects, the use of smaller doses, and with

a potential reduction or avoidance of systemic side eﬀects.²⁴

Therefore, although not designed purposely, the pharmacoki-

netic proﬁle of (S)-18 happened to make it a good candidate

for inhaled delivery. The present favorable in vitro biochemical

properties combined with the PK properties and in vivo anti-

COPD eﬃcacy indicated that (S)-18 might be a suitable

potential inhaled drug candidate for clinical investigation of

COPD.

EXPERIMENTAL SECTION

■

Chemistry. All reagents and solvents were purchased from

commercial sources and were used as received unless speciﬁed

otherwise or prepared as described in the literature. All moisture-

sensitive reactions were carried out using dry solvents under ultrapure

argon protection. Glassware was dried in an oven at 140 °C for at

least 12 h prior to use and then assembled quickly while hot, sealed

with rubber septa, and allowed to cool under a stream of argon.

Reactions were stirred magnetically using Teﬂon-coated magnetic

stirring bars. Commercially available disposable syringes were used for

transferring the reagents and solvents. High-resolution mass

spectrometry (HRMS) and high-performance liquid chromatography

(HPLC) analyses were performed on an Agilent 6224 TOF using an

ESI source coupled to an Agilent 1260 Inﬁnity HPLC system

operating in the reverse mode with an Agilent HC-C18 column (4.6

mm × 250 mm, 5 μm) using a water/acetonitrile (each with 0.3% (v/

1

v) formic acid) gradient at a ﬂow rate at 0.8 mL/min. H and ¹³C

spectra were recorded with a Bruker 500 MHz NMR spectrometer

and referenced to deuterated methanol (CD₃OD), deuterated

J

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dimethyl sulfoxide (DMSO-d₆), or deuterated chloroform (CDCl₃).

Chemical shifts are expressed in ppm. In the NMR tabulation, s

indicates singlet; d, doublet; t, triplet; q, quartet, and m, multiplet.

Flash column chromatography was conducted using silica gel

(Silicycle 40−64 μm). The purities of all ﬁnal compounds were

determined to be ≥95% by HPLC.

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-5-ﬂuoro-3-phenylquinazolin-4(3H)-one (6).

To a solution of 36a (100 mg, 0.29 mmol, 1.0 equiv) in anhydrous

DMF (3 mL) was added 32a (114 mg, 0.44 mmol, 1.5 equiv) and

K₂CO₃(80 mg, 0.58 mmol, 2.0 equiv). Then, the reaction mixture

was heated to 60 °C for 8 h. The resulting mixture was concentrated

to dryness to give the crude product. The residue was diluted with

water and extracted with EtOAc. The combined organic layers were

washed with water and brine and dried over anhydrous Na₂SO₄.

Evaporation of the solvent aﬀorded the crude product, which was

puriﬁed by ﬂash chromatography (eluting with 0−3% MeOH in

dimethyl chloride (DCM)) to give 6 as a white solid (81 mg, 53%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.93−7.87 (m, 2H), 7.64 (d, J =

8.2 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.46−7.40 (m, 2H), 7.37 (qd, J

= 6.2, 4.4 Hz, 2H), 7.29 (t, J = 8.7 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H),

6.85 (td, J = 7.8, 1.4 Hz, 1H), 6.35 (d, J = 8.6 Hz, 1H), 5.92 (q, J =

6.6 Hz, 1H), 3.89 (s, 3H), 1.74 (d, J = 6.7 Hz, 3H). ¹³C NMR (126

MHz, DMSO-d₆) δ 160.96 (d, J_C‑F= 264.0 Hz), 158.86 (d, J_C‑F= 4.0

Hz), 158.14, 155.71, 154.72, 153.85, 152.02 (d, J_C‑F= 244.6 Hz),

148.87, 148.09 (d, J_C‑F= 10.5 Hz), 143.73, 136.09 (d, J_C‑F= 10.6 Hz),

135.59, 129.99, 128.67, 128.41, 128.13, 127.74, 125.82 (d, J_C‑F= 6.9

Hz), 125.34 (d, J_C‑F= 3.0 Hz), 124.26 (d, J_C‑F= 3.4 Hz), 116.35 (d,

1H), 7.41 (d, J = 12.1 Hz, 1H), 7.36 (t, J = 9.2 Hz, 2H), 7.28 (t, J =

8.6 Hz, 1H), 6.88 (s, 2H), 6.10 (q, J = 6.3 Hz, 1H), 3.89 (s, 3H), 3.56

(s, 3H), 1.75 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD) δ

161.75 (d, J_C‑F= 265.9 Hz), 160.49 (d, J_C‑F= 4.3 Hz), 158.44, 155.71,

154.60, 153.90, 153.15 (d, J_C‑F= 248. 4 Hz), 149.33 (d, J_C‑F= 10.4

Hz), 149.04, 144.92, 137.09, 135.95 (d, J_C‑F= 10.4 Hz), 128.89,

125.78 (d, J_C‑F= 6.7 Hz), 125.11 (d, J_C‑F= 3.4 Hz), 124.70 (d, J_C‑F

=

4.1 Hz), 116.82 (d, J_C‑F= 19.4 Hz), 116.79, 114.92 (d, J_C‑F= 20.9

Hz), 114.63 (d, J_C‑F= 1.6 Hz), 110.98 (d, J_C‑F= 6.0 Hz), 98.52, 56.77,

54.81, 39.45, 18.96. HRMS (ESI, m/z): [M + H]⁺calcd for

C₂₆H₂₂F₂N₉O₂, 530.1865; found, 530.1866. HPLC retention time

11.59 min, >95% pure.

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-3-cyclohexyl-5-ﬂuoroquinazolin-4(3H)-one

1

(10). Yield 56%. H NMR (500 MHz, DMSO-d₆) δ 8.37 (s, 1H),

7.85−7.77 (m, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.32−7.23 (m, 4H),

6.51 (q, J = 6.3 Hz, 1H), 3.89 (t, J = 9.8 Hz, 1H), 3.85 (s, 3H), 2.45

(dt, J = 21.4, 7.3 Hz, 1H), 2.18 (td, J = 12.1, 9.2 Hz, 1H), 1.79 (d, J =

6.4 Hz, 3H), 1.77−1.66 (m, 2H), 1.37 (d, J = 12.5 Hz, 1H), 1.23−

1.14 (m, 2H), 0.92 (q, J = 13.2 Hz, 1H), 0.12 (d, J = 11.1 Hz, 1H),

0.00 (q, J = 12.9 Hz, 1H). ¹³C NMR (126 MHz, CD₃OD) δ 161.57

(d, J_C‑F= 265.3 Hz), 160.98 (d, J_C‑F= 3.9 Hz), 158.49, 156.42, 153.91,

153.77, 153.02 (d, J_C‑F= 248.1 Hz), 149.22 (d, J_C‑F= 10.8 Hz),

148.70, 145.11, 134.79 (d, J _C‑F= 10.4 Hz), 125.69 (d, J_C‑F= 7.0 Hz),

125.04 (d, J_C‑F= 3.4 Hz), 124.23 (d, J_C‑F= 4.2 Hz), 116.80 (d, J_C‑F

=

19.4 Hz), 114.49 (d, J_C‑F= 1.8 Hz), 114.27 (d, J_C‑F= 21.2 Hz), 112.32

(d, J_C‑F= 4.6 Hz), 98.75, 60.44, 56.78, 54.82, 29.06, 28.34, 26.72,

26.11, 25.38, 19.98. HRMS (ESI, m/z): [M + H]⁺calcd for

C₂₈H₂₈F₂N₇O₂, 532.2273; found, 532.2270. HPLC retention time

16.37 min, >97% pure.

J_C‑F= 19.0 Hz), 114.83 (d, J_C‑F= 1.8 Hz), 114.46 (d, J_C‑F= 20.4 Hz),

110.89 (d, J_C‑F= 5.5 Hz), 97.63, 56.57, 53.97, 18.95. HRMS (ESI, m/

z): [M + H]⁺calcd for C₂₈H₂₂F₂N₇O₂, 526.1803; found, 526.1798.

HPLC retention time 5.75 min, >98% pure.

Compounds 7−12, 14−30, (R)-18, (S)-18, and 43 were prepared

following the synthetic procedure of 6.

3-([1,1′-Biphenyl]-4-yl)-2-(1-(4-amino-3-(3-ﬂuoro-4-methoxy-

phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-ﬂuoroquinazo-

lin-4(3H)-one (7). Yield 49%. ¹H NMR (500 MHz, DMSO-d₆) δ 7.92

(dd, J = 13.7, 8.1 Hz, 1H), 7.80 (s, 1H), 7.68−7.64 (m, 3H), 7.49−

7.35 (m, 8H), 7.26 (t, J = 8.7 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.30

(d, J = 7.4 Hz, 1H), 6.01 (q, J = 6.4 Hz, 1H), 3.88 (s, 3H), 1.76 (d, J =

6.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 161.41 (d, J_C‑F= 266.7

Hz), 159.60 (d, J_C‑F= 4.2 Hz), 157.37, 155.47, 153.96, 153.52, 152.64

(d, J_C‑F= 248.9 Hz), 148.79, 148.69 (d, J_C‑F= 10.7 Hz), 143.86,

142.29, 139.89, 134.89 (d, J_C‑F= 10.2 Hz), 134.17, 128.80, 128.75,

127.97, 127.73, 127.67, 127.41, 127.23, 125.70 (d, J_C‑F= 6.7 Hz),

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-3-cyclopentyl-5-ﬂuoroquinazolin-4(3H)-one

1

(11). Yield 55%. H NMR (500 MHz, DMSO-d₆) δ 8.31 (s, 1H),

7.85−7.77 (m, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.36−7.25 (m, 4H),

6.58 (q, J = 6.5 Hz, 1H), 4.65−4.55 (m, 1H), 3.87 (s, 3H), 2.19−2.09

(m, 1H), 1.84 (t, J = 11.0 Hz, 5H), 1.73−1.62 (m, 2H), 1.40 (dt, J =

19.7, 7.1 Hz, 1H), 1.10−1.01 (m, 1H), 0.22 (dd, J = 15.9, 7.5 Hz,

1H). ¹³C NMR (126 MHz, CD₃OD) δ 161.01 (d, J_C‑F= 264.9 Hz),

159.78 (d, J_C‑F= 3.9 Hz), 157.94, 155.92, 153.75, 153.54, 152.48 (d,

J_C‑F= 248.3 Hz), 148.68 (d, J_C‑F= 10.7 Hz), 148.23, 144.54, 134.25

(d, J_C‑F= 10.5 Hz), 125.22 (d, J_C‑F= 6.8 Hz), 124.55 (d, J_C‑F= 3.3

Hz), 123.75 (d, J_C‑F= 3.9 Hz), 116.31 (d, J_C‑F= 19.4 Hz), 113.95 (d,

J_C‑F= 1.8 Hz), 113.70 (d, J_C‑F= 21.2 Hz), 111.60 (d, J_C‑F= 4.8 Hz),

98.27, 58.99, 56.26, 54.59, 28.52, 27.94, 25.84, 25.73, 19.23. HRMS

(ESI, m/z): [M + H]⁺calcd for C₂₇H₂₆F₂N₇O₂, 518.2116; found,

518.2118. HPLC retention time 15.63 min, >96% pure.

124.49 (d, J_C‑F= 3.4 Hz), 124.20 (d, J_C‑F= 3.9 Hz), 116.35 (d, J_C‑F

=

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

19.3 Hz), 114.22 (d, J_C‑F= 20.6 Hz), 114.01 (d, J_C‑F= 1.6 Hz), 111.10

(d, J_C‑F= 5.6 Hz), 98.05, 56.41, 54.09, 18.67. HRMS (ESI, m/z): [M

+ H]⁺calcd for C₃₄H₂₆F₂N₇O₂, 602.2116; found, 602.2113. HPLC

retention time 15.17 min, >97% pure.

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-3-benzyl-5-ﬂuoroquinazolin-4(3H)-one (8).

Yield 54%. ¹H NMR (500 MHz, CDCl₃) δ 8.28 (s, 1H), 7.64 (td, J =

8.1, 5.4 Hz, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.23−7.15 (m, 2H), 7.12−

7.06 (m, 1H), 7.00−6.91 (m, 4H), 6.72 (s, 2H), 6.24 (q, J = 6.6 Hz,

1H), 5.42 (d, J = 16.0 Hz, 1H), 5.25 (d, J = 16.1 Hz, 1H), 3.84 (s,

3H), 1.82 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 160.21

(d, J_C‑F= 266.4 Hz), 158.57 (d, J_C‑F= 4.1 Hz), 156.58, 154.71, 152.95,

152.84, 151.45 (d, J_C‑F= 248.2 Hz), 147.67, 147.52 (d, J_C‑F= 10.4

Hz), 143.12, 133.64 (d, J_C‑F= 10.3 Hz), 127.01, 125.67, 124.52 (d,

d]pyrimidin-1-yl)ethyl)-3-cyclobutyl-5-ﬂuoroquinazolin-4(3H)-one

1

(12). Yield 54%. H NMR (500 MHz, CDCl₃) δ 8.31 (s, 1H), 7.55−

7.50 (m, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.28−7.20 (m, 2H), 7.00 (dd,

J = 10.1, 8.7 Hz, 1H), 6.95 (t, J = 8.4 Hz, 1H), 6.26 (q, J = 6.6 Hz,

1H), 4.58 (p, J = 8.6 Hz, 1H), 3.81 (s, 3H), 3.14 (p, J = 9.8 Hz, 1H),

2.72 (p, J = 9.9 Hz, 1H), 2.16 (dd, J = 18.9, 8.6 Hz, 1H), 1.89 (d, J =

6.6 Hz, 3H), 1.80 (q, J = 10.5 Hz, 1H), 1.48−1.35 (m, 1H), 1.08−

0.99 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 161.04 (d, J_C‑F= 266.5

Hz), 160.67 (d, J_C‑F= 3.9 Hz), 158.11, 156.12, 153.92, 153.63, 152.50

(d, J_C‑F= 248.4 Hz), 148.63 (d, J_C‑F= 10.4 Hz), 148.18, 144.24,

134.13 (d, J_C‑F= 10.3 Hz), 125.50 (d, J_C‑F= 6.7 Hz), 124.55 (d, J_C‑F

=

3.2 Hz), 123.68 (d, J_C‑F= 4.1 Hz), 116.35 (d, J_C‑F= 19.3 Hz), 113.92

(d, J_C‑F= 1.6 Hz), 113.70 (d, J_C‑F= 21.1 Hz), 111.76 (d, J_C‑F= 4.8

Hz), 98.33, 56.32, 54.49, 52.27, 26.85, 26.81, 19.11, 14.48. HRMS

(ESI, m/z): [M + H]⁺calcd for C₂₆H₂₄F₂N₇O₂, 504.1960; found,

504.1963. HPLC retention time 15.13 min, >99% pure.

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-

one (13). To a solution of 43 (50 mg, 0.1 mmol, 1.0 equiv) in

anhydrous MeCN (3 mL) were added HMDS (0.07 mL, 0.33 mmol,

3.3 equiv) and ZnCl₂(45 mg, 0.33 mmol, 3.3 equiv). Then, the

reaction mixture was heated at reﬂux for 5 h. The resulting mixture

was concentrated to dryness to give the crude product. The residue

was diluted with water and extracted with DCM. The combined

J

_C‑F= 6.9 Hz), 124.17, 123.46 (d, J_C‑F= 3.5 Hz), 123.13 (d, J_C‑F= 4.0

Hz), 115.43 (d, J_C‑F= 18.9 Hz), 112.98 (d, J_C‑F= 20.6 Hz), 112.75 (d,

J_C‑F= 1.8 Hz), 109.61 (d, J_C‑F= 5.5 Hz), 97.33, 55.32, 52.57, 44.83,

18.03. HRMS (ESI, m/z): [M + H]⁺calcd for C₂₉H₂₄F₂N₇O₂,

540.1960; found, 540.1965. HPLC retention time 14.17 min, >98%

pure.

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-5-ﬂuoro-3-(1-methyl-1H-pyrazol-4-yl)-

1

quinazolin-4(3H)-one (9). Yield 57%. H NMR (500 MHz, DMSO-

d₆) δ 8.13 (s, 1H), 7.89 (dd, J = 13.6, 8.1 Hz, 1H), 7.62 (d, J = 8.2 Hz,

K

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organic layers were washed with water and brine and dried over

anhydrous Na₂SO₄. Evaporation of the solvent provided the crude

product, which was puriﬁed by ﬂash chromatography (eluting with 0−

10% EtOAc in hexane) to give 13 as a white solid (21 mg, yield 44%).

¹H NMR (500 MHz, CD₃OD) δ 8.23 (d, J = 5.6 Hz, 1H), 7.56 (td, J

= 8.2, 5.4 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.32−7.23 (m, 2H),

7.04−6.98 (m, 2H), 6.71 (q, J = 6.8 Hz, 1H), 3.85 (s, 3H), 2.22 (dq, J

= 7.1, 4.2 Hz, 1H), 1.93 (d, J = 6.8 Hz, 3H), 1.24 (ddd, J = 11.9, 8.0,

5.9 Hz, 1H), 1.20−1.15 (m, 1H), 0.98−0.85 (m, 2H). ¹³C NMR (126

MHz, CD₃OD) δ 161.25 (d, J_C‑F= 3.9 Hz), 161.23 (d, J_C‑F= 266.1

Hz), 158.34, 156.59, 156.19, 154.71, 152.95 (d, J_C‑F= 248.2 Hz),

149.06 (d, J_C‑F= 10.7 Hz), 148.72, 144.59, 134.87 (d, J_C‑F= 10.3 Hz),

6.6 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD) δ 161.45 (d, J_C‑F= 264.0

Hz), 160.64 (d, J_C‑F= 3.8 Hz), 158.77, 156.49, 154.50, 154.38, 153.06

(d, J_C‑F= 247.0 Hz), 149.28 (d, J_C‑F= 10.5 Hz), 149.13, 145.36,

135.29 (d, J_C‑F= 10.5 Hz), 125.75 (d, J_C‑F= 6.9 Hz), 125.22 (d, J_C‑F

=

3.5 Hz), 124.44 (d, J_C‑F= 4.2 Hz), 116.90 (d, J_C‑F= 19.4 Hz), 114.53

(d, J_C‑F= 2.2 Hz), 114.35 (d, J_C‑F= 21.0 Hz), 110.77 (d, J_C‑F= 5.6

Hz), 98.88, 56.72, 54.98, 30.35, 18.97. HRMS (ESI, m/z): [M + H]⁺

calcd for C₂₃H₂₀F₂N₇O₂, 464.1647; found, 464.1644. HPLC retention

time 13.07 min, >99% pure.

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-

one (18). Yield 65%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.30 (s, 1H),

7.78 (dd, J = 13.6, 8.1 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.40 (t, J =

9.2 Hz, 2H), 7.34−7.25 (m, 2H), 6.53 (dd, J = 8.9, 5.0 Hz, 1H), 3.90

(s, 3H), 2.52−2.38(m, 2H), 2.21−2.13 (m, 1H), 1.22−1.14 (m, 3H),

0.95 (t, J = 7.3 Hz, 3H), 0.84−0.81 (m, 1H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 160.43 (d, J_C‑F= 263.3 Hz), 159.76 (d, J_C‑F= 3.8 Hz),

158.74, 156.94, 156.44, 155.77, 151.96 (d, J_C‑F= 244.4 Hz), 148.32,

148.11 (d, J_C‑F= 10.4 Hz), 143.98, 135.38 (d, J_C‑F= 10.6 Hz), 125.79

(d, J_C‑F= 6.8 Hz), 125.38 (d, J_C‑F= 2.8 Hz), 123.81 (d, J_C‑F= 3.4 Hz),

116.37 (d, J_C‑F= 19.1 Hz), 114.81, 114.01 (d, J_C‑F= 20.6 Hz), 110.90

(d, J_C‑F= 5.7 Hz), 97.63, 59.28, 56.54, 26.75, 25.94, 11.26, 10.83,

10.09. HRMS (ESI, m/z): [M + H]⁺calcd for C₂₆H₂₄F₂N₇O₂,

504.1960; found, 504.1965. HPLC retention time 14.14 min, >99%

pure.

125.92 (d, J_C‑F= 7.1 Hz), 125.08 (d, J_C‑F= 3.4 Hz), 124.13 (d, J_C‑F

=

3.9 Hz), 116.86 (d, J_C‑F= 19.4 Hz), 114.38 (d, J_C‑F= 1.9 Hz), 114.24

(d, J_C‑F= 21.0 Hz), 111.47 (d, J_C‑F= 6.0 Hz), 98.75, 56.76, 54.31,

26.86, 18.92, 11.30, 10.39. HRMS (ESI, m/z): [M + H]⁺calcd for

C₂₅H₂₂F₂N₇O₂, 490.1803; found, 490.1804. HPLC retention time

13.39 min, >97% pure.

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-5-ﬂuoro-3-isobutylquinazolin-4(3H)-one

(14). Yield 66%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.30 (s, 1H), 7.81

(dd, J = 13.7, 8.2 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.37 (t, J = 9.9

Hz, 2H), 7.33−7.27 (m, 2H), 6.32 (q, J = 6.5 Hz, 1H), 3.93 (dd, J =

14.0, 7.8 Hz, 1H), 3.88 (s, 3H), 3.74 (dd, J = 13.7, 7.3 Hz, 1H), 1.99

(dt, J = 13.6, 6.7 Hz, 1H), 1.87 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.6

Hz, 3H), 0.68 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ

161.17 (d, J_C‑F= 265.7 Hz), 159.69 (d, J_C‑F= 4.3 Hz), 157.96, 156.08,

(R)-2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo-

[3,4-d]pyrimidin-1-yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-

1

4(3H)-one ((R)-18). Yield 52%. H NMR (500 MHz, DMSO-d₆) δ

154.30, 153.92, 152.56 (d, J_C‑F= 248.3 Hz), 148.63, 148.61 (d, J_C‑F

=

8.31 (s, 1H), 7.78 (td, J = 8.1, 5.7 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H),

7.43−7.37(m, 2H), 7.34−7.25 (m, 2H), 6.54 (dd, J = 9.0, 5.0 Hz,

1H), 3.90 (s, 3H), 2.53−2.37 (m, 2H), 2.21−2.13 (m, 1H), 1.25−

1.10 (m, 3H), 0.95 (t, J = 7.3 Hz, 3H), 0.87−0.78 (m, 1H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 160.43 (d, J_C‑F= 261.1 Hz), 159.76 (d, J_C‑F

= 3.8 Hz), 158.74, 156.94, 156.44, 155.77, 151.97 (d, J_C‑F= 245.3

Hz), 148.32, 148.11 (d, J_C‑F= 10.3 Hz), 143.99, 135.37 (d, J_C‑F= 10.5

Hz), 125.80 (d, J_C‑F= 7.1 Hz), 125.38 (d, J_C‑F= 3.0 Hz), 123.81 (d,

10.2 Hz), 144.14, 134.30 (d, J_C‑F= 10.3 Hz), 125.74 (d, J_C‑F= 7.1

Hz), 124.58 (d, J_C‑F= 3.4 Hz), 123.95 (d, J_C‑F= 4.1 Hz), 116.50 (d,

J_C‑F= 19.3 Hz), 113.89 (d, J_C‑F= 1.7 Hz), 113.69 (d, J_C‑F= 20.9 Hz),

110.79 (d, J_C‑F= 5.2 Hz), 98.56, 56.35, 53.53, 49.03, 28.58, 20.05,

19.99, 18.99. HRMS (ESI, m/z): [M + H]⁺calcd for C₂₆H₂₆F₂N₇O₂,

506.2116; found, 506.2120. HPLC retention time 15.33 min, >99%

pure.

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

J_C‑F= 3.4 Hz), 116.37 (d, J_C‑F= 19.0 Hz), 114.80, 114.01 (d, J_C‑F

=

d]pyrimidin-1-yl)ethyl)-5-ﬂuoro-3-isopropylquinazolin-4(3H)-one

1

20.6 Hz), 110.91 (d, J_C‑F= 5.8 Hz), 97.64, 59.28, 56.53, 26.75, 25.94,

11.25, 10.83, 10.09. HRMS (ESI, m/z): [M + H]⁺calcd for

C₂₆H₂₄F₂N₇O₂, 504.1960; found, 504.1963. HPLC retention time

14.13 min, >99% pure.

(15). Yield 61%. H NMR (500 MHz, DMSO-d₆) δ 8.32 (s, 1H),

7.85−7.75 (m, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.37−7.24 (m, 4H),

6.50 (q, J = 6.5 Hz, 1H), 4.48 (dt, J = 13.3, 6.6 Hz, 1H), 3.87 (s, 3H),

1.82 (d, J = 6.4 Hz, 3H), 1.51 (d, J = 6.6 Hz, 3H), 0.71 (d, J = 6.6 Hz,

3H). ¹³C NMR (126 MHz, CDCl₃) δ 161.15 (d, J_C‑F= 263.8 Hz),

160.08 (d, J_C‑F= 1.8 Hz), 157.93, 156.27, 153.94, 153.44, 152.58 (d,

(S)-2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo-

[3,4-d]pyrimidin-1-yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-

1

4(3H)-one ((S)-18). Yield 65%. H NMR (500 MHz, DMSO-d₆) δ

J_C‑F= 248.18 Hz), 148.70 (d, J_C‑F= 10.7 Hz), 148.35, 144.23, 134.07

8.30 (s, 1H), 7.78 (td, J = 8.1, 5.7 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H),

7.42−7.38 (m, 2H), 7.34−7.25 (m, 2H), 6.53 (dd, J = 9.0, 5.0 Hz,

1H), 3.90 (s, 3H), 2.53−2.37 (m, 2H), 2.20−2.13 (m, 1H), 1.25−

1.10 (m, 3H), 0.95 (t, J = 7.3 Hz, 3H), 0.86−0.79 (m, 1H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 160.43 (d, J_C‑F= 260.1 Hz), 159.76 (d, J_C‑F

= 3.8 Hz), 158.74, 156.94, 156.44, 155.77, 151.96 (d, J_C‑F= 248.1

Hz), 148.32, 148.11 (d, J_C‑F= 10.4 Hz), 143.99, 135.38 (d, J_C‑F= 10.4

Hz), 125.79 (d, J_C‑F= 6.8 Hz), 125.38 (d, J_C‑F= 2.9 Hz), 123.81 (d,

(d, J_C‑F= 10.4 Hz), 125.54 (d, J_C‑F= 7.0 Hz), 124.55 (d, J_C‑F= 3.4

Hz), 123.77 (d, J_C‑F= 4.0 Hz), 116.41 (d, J_C‑F= 19.3 Hz), 113.96 (d,

J_C‑F= 1.7 Hz), 113.68 (d, J_C‑F= 21.2 Hz), 111.96 (d, J_C‑F= 4.7 Hz),

98.44, 56.38, 54.29, 51.39, 19.72, 19.39, 18.50. HRMS (ESI, m/z):

[M + H]⁺calcd for C₂₅H₂₄F₂N₇O₂, 492.1960; found, 492.1962.

HPLC retention time 14.68 min, >99% pure.

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-3-ethyl-5-ﬂuoroquinazolin-4(3H)-one (16).

1

J_C‑F= 3.5 Hz), 116.37 (d, J_C‑F= 19.0 Hz), 114.81, 114.01 (d, J_C‑F

=

Yield 64%. H NMR (500 MHz, DMSO-d₆) δ 8.32 (s, 1H), 7.81

20.5 Hz), 110.90 (d, J_C‑F= 5.8 Hz), 97.63, 59.28, 56.54, 26.75, 25.94,

11.26, 10.83, 10.09. HRMS (ESI, m/z): [M + H]⁺calcd for

C₂₆H₂₄F₂N₇O₂, 504.1960; found, 504.1965. HPLC retention time

14.13 min, >97% pure.

2-(1-(4-Amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl)-3-cyclo-

propyl-5-ﬂuoroquinazolin-4(3H)-one (19). Yield 58%. ¹H NMR

(500 MHz, DMSO-d₆) δ 8.20 (s, 1H), 8.13 (s, 1H), 7.79 (dd, J =

13.7, 8.2 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.28 (dd, J = 10.3, 8.7 Hz,

1H), 6.44 (dd, J = 9.0, 5.0 Hz, 1H), 2.48−2.40 (m, 1H), 2.39−2.29

(m, 1H), 2.00−1.93 (m, 1H), 1.18−1.04 (m, 3H), 0.86 (t, J = 7.3 Hz,

3H), 0.77−0.70 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 160.43

(d, J_C‑F= 263.8 Hz), 159.74 (d, J_C‑F= 4.1 Hz), 158.63, 157.01, 156.58,

(dd, J = 13.7, 8.2 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.41−7.33 (m,

2H), 7.29 (dd, J = 19.3, 10.4 Hz, 2H), 6.40 (q, J = 6.5 Hz, 1H), 4.07−

3.91 (m, 2H), 3.88 (s, 3H), 1.85 (d, J = 6.6 Hz, 3H), 0.68 (t, J = 6.9

Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 161.08 (d, J_C‑F= 265.5 Hz),

159.27 (d, J_C‑F= 4.3 Hz), 157.93, 156.19, 154.25, 153.54, 152.58 (d,

J_C‑F= 248.5 Hz) 148.70, 148.62, 144.17, 134.26 (d, J_C‑F= 10.3 Hz),

125.66 (d, J_C‑F= 7.0 Hz), 124.56 (d, J_C‑F= 3.4 Hz), 123.95 (d, J_C‑F

=

4.1 Hz), 116.50 (d, J_C‑F= 19.4 Hz), 113.91 (d, J_C‑F= 1.7 Hz), 113.64

(d, J_C‑F= 20.8 Hz), 110.83 (d, J_C‑F= 5.5 Hz), 98.60, 56.37, 53.68,

38.77, 19.11, 13.23. HRMS (ESI, m/z): [M + H]⁺calcd for

C₂₄H₂₂F₂N₇O₂, 478.1803; found, 478.1799. HPLC retention time

13.96 min, >98% pure.

2-(1-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-5-ﬂuoro-3-methylquinazolin-4(3H)-one

(17). Yield 60%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.30 (s, 1H), 7.82

(dd, J = 13.7, 8.1 Hz, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.41−7.25 (m,

4H), 6.41 (q, J = 6.5 Hz, 1H), 3.87 (s, 3H), 3.20 (s, 3H), 1.83 (d, J =

154.61, 148.34, 135.43 (d, J_C‑F= 10.5 Hz), 133.54, 123.79 (d, J_C‑F

=

3.6 Hz), 114.04 (d, J_C‑F= 20.6 Hz), 110.90 (d, J_C‑F= 5.6 Hz), 100.16,

59.06, 26.62, 26.05, 11.11, 10.78, 10.01. HRMS (ESI, m/z): [M + H]⁺

calcd for C₁₉H₁₉FN₇O, 380.1635; found, 380.1631. HPLC retention

time 9.66 min, >98% pure.

L

https://dx.doi.org/10.1021/acs.jmedchem.0c01544

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2-(1-(4-Amino-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-

2.3 Hz), 116.72 (d, J_C‑F= 20.0 Hz), 113.74 (d, J_C‑F= 20.9 Hz), 110.92

(d, J_C‑F= 5.8 Hz), 98.09, 72.46, 59.12, 26.50, 25.82, 21.86, 21.84,

11.20, 10.87, 10.12. HRMS (ESI, m/z): [M + H]⁺calcd for

C₂₈H₂₈F₂N₇O₂, 532.2273; found, 532.2275. HPLC retention time

15.49 min, >98% pure.

propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-one (20). Yield

1

60%. H NMR (500 MHz, DMSO-d₆) δ 8.28 (s, 1H), 7.78 (td, J =

8.1, 5.5 Hz, 1H), 7.59 (d, J = 7.1 Hz, 2H), 7.54−7.43 (m, 4H), 7.28

(dd, J = 10.5, 8.5 Hz, 1H), 6.52 (dd, J = 9.1, 4.9 Hz, 1H), 2.49−2.36

(m, 2H), 2.14−2.09 (m, 1H), 1.20−1.08 (m, 3H), 0.93 (t, J = 7.3 Hz,

3H), 0.82−0.76 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 160.84 (d,

J_C‑F= 265.41 Hz), 160.63 (d, J_C‑F= 3.9 Hz), 158.03, 155.90, 155.71,

155.45, 148.38, 145.35, 134.17 (d, J_C‑F= 10.3 Hz), 132.92, 129.26,

129.20, 128.61, 123.76 (d, J_C‑F= 4.0 Hz), 113.65 (d, J_C‑F= 20.8 Hz),

111.16 (d, J_C‑F= 5.8 Hz), 98.30, 59.20, 26.49, 25.92, 11.35, 11.04,

10.23. HRMS (ESI, m/z): [M + H]⁺calcd for C₂₅H₂₃FN₇O,

456.1948; found, 456.1945. HPLC retention time 13.83 min, >99%

pure.

2-(1-(4-Amino-3-(4-methoxyphenyl)-1H-pyrazolo[3,4-d]-

pyrimidin-1-yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-one

(21). Yield 63%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.29 (s, 1H), 7.78

(dd, J = 13.6, 8.1 Hz, 1H), 7.53 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.2

Hz, 1H), 7.28 (dd, J = 10.3, 8.7 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H),

6.52 (dd, J = 9.0, 5.0 Hz, 1H), 3.81 (s, 3H), 2.52−2.37 (m, 2H),

2.14−2.08 (m, 1H), 1.21−1.09 (m, 3H), 0.94 (t, J = 7.3 Hz, 3H),

0.82−0.75 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 160.43 (d,

J_C‑F= 263.2 Hz), 160.16, 159.77 (d, J_C‑F= 3.7 Hz), 158.77, 157.00,

156.39, 155.72, 148.33, 144.98, 135.41 (d, J_C‑F= 10.4 Hz), 130.13,

125.42, 123.81 (d, J_C‑F= 3.2 Hz), 115.05, 114.02 (d, J_C‑F= 20.6 Hz),

110.90 (d, J_C‑F= 5.6 Hz), 97.59, 59.21, 55.69, 26.74, 25.97, 11.26,

10.81, 10.06. HRMS (ESI, m/z): [M + H]⁺calcd for C₂₆H₂₅FN₇O₂,

486.2054; found, 486.2056. HPLC retention time 13.71 min, >98%

pure.

4-(4-Amino-1-(1-(3-cyclopropyl-5-ﬂuoro-4-oxo-3,4-dihydroqui-

nazolin-2-yl)propyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-methyl-

benzamide (25). Yield 64%. ¹H NMR (500 MHz, DMSO-d₆) δ

8.62−8.55 (m, 1H), 8.33 (s, 1H), 8.00 (d, J = 8.3 Hz, 2H), 7.78 (td, J

= 8.2, 5.7 Hz, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.2 Hz, 1H),

7.28 (dd, J = 10.4, 8.6 Hz, 1H), 6.57 (dd, J = 9.0, 5.0 Hz, 1H), 2.83

(d, J = 4.4 Hz, 3H), 2.53−2.39 (m, 2H), 2.23−2.17 (m, 1H), 1.26−

1.11 (m, 3H), 0.95 (t, J = 7.3 Hz, 3H), 0.87−0.81 (m, 1H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 166.58, 160.43 (d, J_C‑F= 263.5 Hz), 159.76

(d, J_C‑F= 3.8 Hz), 158.66, 156.92, 156.41, 155.89, 148.30, 144.48,

135.47, 135.41 (d, J_C‑F= 10.6 Hz), 134.88, 128.69, 128.33, 123.82 (d,

J_C‑F= 3.5 Hz), 114.04 (d, J_C‑F= 20.5 Hz), 110.91 (d, J_C‑F= 5.6 Hz),

97.76, 59.38, 26.76, 25.94, 11.28, 10.86, 10.13. HRMS (ESI, m/z):

[M + H]⁺calcd for C₂₇H₂₆FN₈O₂, 513.2163; found, 513.2162. HPLC

retention time 10.68 min, >96% pure.

N-(4-(4-Amino-1-(1-(3-cyclopropyl-5-ﬂuoro-4-oxo-3,4-dihydro-

quinazolin-2-yl)propyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)-

acetamide (26). Yield 62%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.24

(s, 1H), 8.29 (s, 1H), 7.82−7.73 (m, 3H), 7.53 (d, J = 8.4 Hz, 2H),

7.45 (d, J = 8.2 Hz, 1H), 7.28 (dd, J = 10.4, 8.6 Hz, 1H), 6.52 (dd, J =

9.0, 5.0 Hz, 1H), 2.51−2.35 (m, 2H), 2.15−2.11 (m, 1H), 2.08 (s,

3H), 1.23−1.10 (m, 3H), 0.93 (t, J = 7.3 Hz, 3H), 0.83−0.75 (m,

1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 169.02, 160.43 (d, J_C‑F

=

263.0 Hz), 159.78 (d, J_C‑F= 3.8 Hz), 158.68, 156.98, 156.33, 155.73,

148.31, 145.01, 140.41, 135.43 (d, J_C‑F= 10.3 Hz), 129.25, 127.50,

123.82 (d, J_C‑F= 3.8 Hz), 119.72, 114.04 (d, J_C‑F= 20.6 Hz), 110.91

(d, J_C‑F= 5.7 Hz), 97.59, 59.23, 26.76, 25.96, 24.52, 11.26, 10.85,

10.09. HRMS (ESI, m/z): [M + H]⁺calcd for C₂₇H₂₆FN₈O₂,

513.2163; found, 513.2158. HPLC retention time 10.71 min, >99%

pure.

2-(1-(4-Amino-3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]-

pyrimidin-1-yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-one

(22). Yield 57%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.29 (s, 1H), 7.78

(dd, J = 13.6, 8.1 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.28 (dd, J =

10.3, 8.7 Hz, 1H), 7.14−7.08 (m, 3H), 6.52 (dd, J = 8.9, 5.0 Hz, 1H),

3.81 (s, 3H), 3.80 (s, 3H), 2.52−2.35 (m, 2H), 2.13−2.07 (m, 1H),

1.22−1.06 (m, 3H), 0.95 (t, J = 7.3 Hz, 3H), 0.86−0.77 (m, 1H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 160.43 (d, J_C‑F= 263.2 Hz), 159.81

(d, J_C‑F= 3.8 Hz), 158.75, 157.02, 156.39, 155.70, 149.80, 149.41,

2-(1-(4-Amino-3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazolo[3,4-d]-

pyrimidin-1-yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-one

1

(27). Yield 55%. H NMR (500 MHz, DMSO-d₆) δ 8.26 (s, 1H),

7.81−7.74 (m, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.28 (dd, J = 10.5, 8.5

Hz, 1H), 7.08−7.01 (m, 3H), 6.49 (dd, J = 9.0, 5.0 Hz, 1H), 6.08 (s,

2H), 2.49−2.33 (m, 2H), 2.12−2.05 (m, 1H), 1.19−1.06 (m, 3H),

0.92 (t, J = 7.3 Hz, 3H), 0.82−0.74 (m, 1H). ¹³C NMR (126 MHz,

CDCl₃) δ 160.84 (d, J_C‑F= 265.0 Hz), 160.62 (d, J_C‑F= 3.9 Hz),

157.97, 155.95, 155.71, 155.38, 148.56, 148.44, 148.37, 145.01,

134.18 (d, J_C‑F= 10.3 Hz), 126.76, 123.74 (d, J_C‑F= 3.8 Hz), 122.39,

113.66 (d, J_C‑F= 20.9 Hz), 111.15 (d, J_C‑F= 5.8 Hz), 109.09, 108.94,

101.51, 98.18, 59.09, 26.49, 25.89, 11.36, 11.02, 10.24. HRMS (ESI,

m/z): [M + H]⁺calcd for C₂₆H₂₃FN₇O₃, 500.1846; found, 500.1849.

HPLC retention time 13.65 min, >99% pure.

148.32, 145.21, 135.40 (d, J_C‑F= 10.4 Hz), 125.51, 123.80 (d, J_C‑F

=

3.6 Hz), 121.23, 114.01 (d, J_C‑F= 20.4 Hz), 112.66, 112.30, 110.88 (d,

J_C‑F= 5.8 Hz), 97.65, 59.28, 56.01, 55.82, 26.75, 25.96, 11.28, 10.81,

10.05. HRMS (ESI, m/z): [M + H]⁺calcd for C₂₇H₂₇FN₇O₃,

516.2159; found, 516.2161. HPLC retention time 12.51 min, >98%

pure.

2-(1-(4-Amino-3-(4-methoxy-3-methylphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-

one (23). Yield 52%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.29 (s, 1H),

7.78 (td, J = 8.1, 5.7 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.40 (d, J =

8.4 Hz, 1H), 7.38 (s, 1H), 7.28 (dd, J = 10.4, 8.6 Hz, 1H), 7.08 (d, J =

8.4 Hz, 1H), 6.52 (dd, J = 9.0, 5.0 Hz, 1H), 3.84 (s, 3H), 2.52−2.37

(m, 2H), 2.20 (s, 3H), 2.14−2.08 (m, 1H), 1.23−1.10 (m, 3H), 0.93

(t, J = 7.3 Hz, 3H), 0.85−0.78 (m, 1H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 160.43 (d, J_C‑F= 264.4 Hz), 159.77 (d, J_C‑F= 3.9 Hz),

158.74, 158.31, 157.01, 156.38, 155.68, 148.32, 145.21, 135.40 (d, J_C‑F

= 10.6 Hz), 130.80, 127.70, 126.87, 124.99, 123.81 (d, J_C‑F= 3.5 Hz),

114.01 (d, J_C‑F= 20.6 Hz), 111.23, 110.90 (d, J_C‑F= 5.6 Hz), 97.58,

59.19, 55.84, 26.75, 25.98, 16.51, 11.25, 10.84, 10.06. HRMS (ESI, m/

z): [M + H]⁺calcd for C₂₇H₂₇FN₇O₂, 500.2210; found, 500.2207.

HPLC retention time 14.33 min, >96% pure.

2-(1-(4-Amino-3-(quinolin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-

yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-one (28). Yield

1

46%. H NMR (500 MHz, DMSO-d₆) δ 9.05 (d, J = 1.9 Hz, 1H),

8.56 (d, J = 1.4 Hz, 1H), 8.32 (s, 1H), 8.09 (dd, J = 11.2, 8.6 Hz, 2H),

7.82 (t, J = 7.6 Hz, 1H), 7.77 (dd, J = 13.7, 8.2 Hz, 1H), 7.67 (t, J =

7.5 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.27 (dd, J = 10.6, 8.5 Hz, 1H),

6.58 (dd, J = 9.1, 5.0 Hz, 1H), 2.58−2.53 (m, 1H), 2.49−2.42 (m,

1H), 2.27−2.19 (m, 1H), 1.24−1.12 (m, 3H), 0.96 (t, J = 7.3 Hz,

3H), 0.83 (dd, J = 10.8, 5.5 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ

160.83 (d, J_C‑F= 265.6 Hz), 160.56 (d, J_C‑F= 4.1 Hz), 157.95, 156.18,

155.79, 155.67, 149.84, 148.32, 147.92, 142.09, 135.57, 134.25 (d, J_C‑F

= 10.3 Hz), 130.45, 129.39, 128.12, 127.59, 127.46, 126.08, 123.72

(d, J_C‑F= 3.9 Hz), 113.73 (d, J_C‑F= 21.0 Hz), 111.12 (d, J_C‑F= 5.6

Hz), 98.75, 59.27, 26.55, 25.89, 11.40, 11.10, 10.34. HRMS (ESI, m/

z): [M + H]⁺calcd for C₂₈H₂₄FN₈O, 507.2057; found, 507.2058.

HPLC retention time 13.25 min, >99% pure.

2-(1-(4-Amino-3-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]-

pyrimidin-1-yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-one

(29). Yield 57%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.24 (s, 1H), 8.02

(s, 1H), 7.78 (dd, J = 13.7, 8.2 Hz, 1H), 7.65 (s, 1H), 7.45 (d, J = 8.2

Hz, 1H), 7.28 (dd, J = 10.3, 8.7 Hz, 1H), 6.46 (dd, J = 9.1, 5.0 Hz,

1H), 3.87 (s, 3H), 2.48−2.31 (m, 2H), 2.08−2.04 (m, 1H), 1.20−

2-(1-(4-Amino-3-(3-ﬂuoro-4-isopropoxyphenyl)-1H-pyrazolo-

[3,4-d]pyrimidin-1-yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-

1

4(3H)-one (24). Yield 48%. H NMR (500 MHz, DMSO-d₆) δ 8.27

(s, 1H), 7.81−7.74 (m, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.36 (d, J =

12.9 Hz, 1H), 7.33−7.25 (m, 3H), 6.50 (dd, J = 9.0, 4.9 Hz, 1H), 4.68

(dt, J = 12.0, 6.0 Hz, 1H), 2.49−2.33 (m, 2H), 2.15−2.06 (m, 1H),

1.31 (d, J = 6.0 Hz, 6H), 1.20−1.06 (m, 3H), 0.92 (t, J = 7.3 Hz, 3H),

0.81−0.73 (m, 1H). ¹³C NMR (126 MHz, CD₃OD) δ 160.90 (d, J_C‑F

= 3.8 Hz), 160.72 (d, J_C‑F= 264.1 Hz), 157.83, 155.67, 155.52,

155.14, 153.57 (d, J_C‑F= 247.7 Hz), 148.24, 146.82 (d, J_C‑F= 10.3

Hz), 144.33, 134.41 (d, J_C‑F= 10.2 Hz), 125.64 (d, J_C‑F= 7.0 Hz),

124.52 (d, J_C‑F= 3.4 Hz), 123.69 (d, J_C‑F= 4.0 Hz), 117.61 (d, J_C‑F

=

M

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1.06 (m, 3H), 0.93−0.86 (m, 3H), 0.81−0.72 (m, 1H). ¹³C NMR

(126 MHz, CDCl₃) δ 158.9 (d, J_C‑F= 265.3 Hz), 158.74 (d, J_C‑F= 3.9

Hz), 156.32, 154.06, 153.72, 153.29, 146.43, 136.41, 135.64, 132.28

(d, J_C‑F= 10.3 Hz), 128.10, 121.84 (d, J_C‑F= 4.1 Hz), 112.43, 111.76

(d, J_C‑F= 20.8 Hz), 109.26 (d, J_C‑F= 5.7 Hz), 96.77, 57.09, 37.30,

24.55, 24.05, 9.48, 9.05, 8.28. HRMS (ESI, m/z): [M + H]⁺calcd for

C₂₃H₂₃FN₉O, 460.2010; found, 460.2008. HPLC retention time 10.22

min, >98% pure.

4-(4-Amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-methylbenza-

1

mide (32g). Yield 71%. H NMR (500 MHz, DMSO-d₆) δ 8.60 (s,

1H), 8.27 (s, 1H), 8.04 (d, J = 7.5 Hz, 2H), 7.80 (d, J = 7.5 Hz, 2H),

6.85 (s, 2H), 2.86 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

166.78, 158.53, 156.95, 156.14, 144.12, 136.43, 134.54, 128.55,

128.28, 97.56, 26.79. HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₃H₁₃N₆O, 269.1151; found, 269.1150.

N-(4-(4-Amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)-

1

acetamide (32h). Yield 77%. H NMR (500 MHz, DMSO-d₆) δ

2-(1-(4-Amino-3-(thiophen-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-

13.56 (s, 1H), 10.16 (s, 1H), 8.25 (s, 1H), 7.79 (d, J = 8.2 Hz, 2H),

7.63 (d, J = 8.2 Hz, 2H), 2.10 (d, J = 13.6 Hz, 3H). HRMS (ESI, m/

z): [M + H]⁺calcd for C₁₃H₁₃N₆O, 269.1151; found, 269.1153.

3-(Benzo[d][1,3]dioxol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-

amine (32i). Yield 63%. ¹H NMR (500 MHz, DMSO-d₆) δ 13.53 (s,

1H), 8.23 (s, 1H), 7.19 (s, 1H), 7.15 (d, J = 7.8 Hz, 1H), 7.08 (d, J =

7.9 Hz, 1H), 6.12 (s, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 158.52,

156.43, 156.22, 148.30, 148.10, 144.64, 127.67, 122.63, 109.29,

109.03, 101.80, 97.38. HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₂H₁₀N₅O₂, 256.0834; found, 256.0831.

yl)propyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-one (30). Yield

1

65%. H NMR (500 MHz, DMSO-d₆) δ 8.27 (s, 1H), 7.81 (dd, J

= 2.8, 1.2 Hz, 1H), 7.78 (dt, J = 13.6, 6.9 Hz, 1H), 7.70 (dd, J = 4.9,

2.9 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.32 (dd, J = 4.9, 1.2 Hz, 1H),

7.27 (dd, J = 10.3, 8.5 Hz, 1H), 6.50 (dd, J = 9.0, 5.0 Hz, 1H), 2.48−

2.36 (m, 2H), 2.15−2.09 (m, 1H), 1.21−1.07 (m, 3H), 0.92 (t, J =

7.3 Hz, 3H), 0.83−0.75 (m, 1H). ¹³C NMR (126 MHz, CD₃OD) δ

160.88 (d, J_C‑F= 4.2 Hz), 160.72 (d, J_C‑F= 265.3 Hz) 157.93, 155.70,

155.50, 154.92, 148.24, 140.99, 134.39 (d, J_C‑F= 10.5 Hz), 133.39,

127.62, 127.59, 124.89, 123.67 (d, J_C‑F= 3.9 Hz), 113.73 (d, J_C‑F

=

3-(Quinolin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (32j).

20.8 Hz), 110.95 (d, J_C‑F= 5.8 Hz), 98.42, 59.13, 26.49, 25.84, 11.21,

10.86, 10.11. HRMS (ESI, m/z): [M + H]⁺calcd for C₂₃H₂₁FN₇OS,

462.1512; found, 462.1513. HPLC retention time 13.50 min, >96%

pure.

1

Yield 70%. H NMR (500 MHz, DMSO-d₆) δ 9.22 (d, J = 1.4 Hz,

1H), 8.61 (s, 1H), 8.28 (s, 1H), 8.11 (d, J = 8.3 Hz, 2H), 7.83 (t, J =

7.6 Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.06 (s, 2H). ¹³C NMR (126

MHz, DMSO-d₆) δ 158.74, 157.12, 156.27, 150.50, 147.56, 142.13,

135.52, 130.26, 129.23, 129.15, 128.04, 127.45, 127.16, 97.94. HRMS

(ESI, m/z): [M + H]⁺calcd for C₁₄H₁₁N₆, 263.1045; found,

263.1047.

3-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-

amine (32k). Yield 46%¹H NMR (500 MHz, DMSO-d₆) δ 13.42 (s,

1H), 8.20 (s, 1H), 8.08 (s, 1H), 7.76 (s, 1H), 6.86 (s, 2H), 3.93 (s,

3H). HRMS (ESI, m/z): [M + H]⁺calcd for C9H10N7, 216.0998;

found, 216.0997.

3-(3-Fluoro-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-

amine (32a). To a solution of 31 (2.00 g, 7.7 mmol, 1.0 equiv) in

dioxane (30 mL)/H₂O (10 mL) were added (3-ﬂuoro-4-

methoxyphenyl)boronic acid (3.9 g, 23.1 mmol, 3.0 equiv),

Pd(PPh₃)₄(924 mg, 0.8 mmol, 0.1 equiv), and K₂CO₃(2.1 g, 15.4

mmol, 2.0 equiv) at room temperature under argon. The reaction

mixture was degassed by argon, and then it was heated to 135 °C for

14 h. The resulting mixture was concentrated to dryness. The residue

was diluted with water and ﬁltered, and the precipitate was washed

1

3-(Thiophen-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (32l).

with DCM to give 32a as an oﬀ-white solid (1.6 g, 79%). H NMR

1

Yield 56%. H NMR (500 MHz, DMSO-d₆) δ 13.55 (s, 1H), 8.22

(500 MHz, DMSO-d₆) δ 8.22 (s, 1H), 7.49−7.42 (m, 2H), 7.33 (t, J

= 8.7 Hz, 1H), 3.92 (s, 3H). HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₂H₁₁FN₅O, 260.0948; found, 260.0953.

Compounds 32b−l were prepared following the synthetic

procedure of 32a.

(s, 1H), 7.84 (d, J = 1.5 Hz, 1H), 7.75 (dd, J = 4.9, 2.9 Hz, 1H), 7.45

(dd, J = 4.9, 1.2 Hz, 1H), 6.83 (s, 2H). HRMS (ESI, m/z): [M + H]⁺

calcd for C₉H₈N₅S, 218.0500; found, 218.0503.

2-Amino-6-ﬂuoro-N-phenylbenzamide (34a). To a solution of 33

(1.0 g, 6.4 mmol, 1.0 equiv) in anhydrous THF (20 mL) were added

aniline (0.6 mL, 6.4 mmol, 1.0 equiv), HATU (2.9 g, 7.7 mmol, 1.2

equiv), and DIPEA (3.4 mL, 19.2 mmol, 3.0 equiv). Then, the

reaction mixture was stirred at room temperature for 6 h. The

resulting mixture was concentrated to dryness to give the crude

product. The residue was diluted with water and extracted with

EtOAc. The combined organic layers were washed with water and

brine and dried over anhydrous Na₂SO₄. Evaporation of the solvent

provided the crude product, which was puriﬁed by ﬂash

chromatography (eluting with 0−25% EtOAc in hexane) to give

34a as a white solid (1.1 g, yield 73%). ¹H NMR (500 MHz, DMSO-

d₆) δ 10.26 (s, 1H), 7.73 (d, J = 7.8 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H),

7.12 (dt, J = 15.0, 7.7 Hz, 2H), 6.57 (d, J = 8.2 Hz, 1H), 6.40 (t, J =

9.1 Hz, 1H), 5.76 (s, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 163.51,

160.66 (d, J_C‑F= 243.0 Hz), 149.78 (d, J_C‑F= 6.2 Hz), 139.42, 131.82

(d, J_C‑F= 11.1 Hz), 129.13, 124.16, 120.28, 111.87 (d, J_C‑F= 2.0 Hz),

109.06 (d, J_C‑F= 19.3 Hz), 102.42 (d, J_C‑F= 22.8 Hz). HRMS (ESI,

m/z): [M + H]⁺calcd for C₁₃H₁₂FN₂O, 231.0934; found, 231.0937.

Compounds 34b−g and 41 were prepared following the synthetic

procedure of 34a.

3-Phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (32b). Yield

1

84%. H NMR (500 MHz, DMSO-d₆) δ 8.25 (s, 1H), 7.70 (d, J =

7.4 Hz, 2H), 7.58−7.55 (m, 2H), 7.49 (t, J = 7.3 Hz, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 158.52, 156.71, 156.17, 144.79,

133.96, 129.55, 128.91, 128.70, 97.46. HRMS (ESI, m/z): [M + H]⁺

calcd for C₁₁H₁₀N₅, 212.0936; found, 212.0937.

3-(4-Methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

1

(32c). Yield 77%. H NMR (500 MHz, DMSO-d₆) δ 8.22 (s, 1H),

7.61 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H), 3.84 (s, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 159.90, 158.55, 156.73, 156.03,

144.56, 130.01, 126.35, 114.99, 97.44, 55.68. HRMS (ESI, m/z): [M

+ H]⁺calcd for C₁₂H₁₂N₅O, 242.1042; found, 242.1046.

3-(3,4-Dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

1

(32d). Yield 80%. H NMR (500 MHz, DMSO-d₆) δ 8.21 (s, 1H),

7.27−7.17 (m, 2H), 7.12 (d, J = 8.2 Hz, 1H), 3.83 (s, 6H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 158.55, 156.78, 155.99, 149.51,149.46,

144.73, 126.54, 120.98, 112.63, 112.24, 97.45, 56.02, 55.89. HRMS

(ESI, m/z): [M + H]⁺calcd for C₁₃H₁₄N₅O₂, 272.1147; found,

272.1142.

3-(4-Methoxy-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-

1

amine (32e). Yield 79%. H NMR (500 MHz, DMSO-d₆) δ 8.22 (s,

N-([1,1′-Biphenyl]-4-yl)-2-amino-6-ﬂuorobenzamide (34b). Yield

1

1H), 7.47−7.46 (m, 2H), 7.10 (d, J = 8.0 Hz, 1H), 3.86 (s, 3H), 2.24

(s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 158.56, 158.10, 156.47,

156.17, 144.80, 130.82, 127.51, 126.80, 125.78, 111.19, 97.40, 55.84,

16.59. HRMS (ESI, m/z): [M + H]⁺calcd for C₁₃H₁₄N₅O, 256.1198;

found, 256.1201.

3-(3-Fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-

4-amine (32f). Yield 72%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.19 (s,

1H), 7.45 (t, J = 10.2 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.31 (t, J =

8.4 Hz, 1H), 6.73 (s, 2H), 4.70 (dd, J = 11.6, 5.7 Hz, 1H), 1.32 (t, J =

13.5 Hz, 6H). HRMS (ESI, m/z): [M + H]⁺calcd for C₁₄H₁₅FN₅O,

288.1261; found, 288.1263.

75%. H NMR (500 MHz, DMSO-d₆) δ 10.41 (s, 1H), 7.85 (d, J =

8.4 Hz, 2H), 7.68−7.66 (m, 4H), 7.47−7.44 (m, 2H), 7.34 (t, J = 7.4

Hz, 1H), 7.15 (dd, J = 14.9, 8.1 Hz, 1H), 6.60 (d, J = 8.2 Hz, 1H),

6.46−6.38 (m, 1H), 5.83 (s, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ

163.54, 160.68 (d, J_C‑F= 243.0 Hz), 149.84 (d, J_C‑F= 6.2 Hz), 140.19,

138.94, 135.80, 131.89 (d, J_C‑F= 11.1 Hz), 129.39, 127.55, 127.34,

126.77, 120.61, 111.89, 108.99 (d, J_C‑F= 19.3 Hz), 102.39 (d, J_C‑F

=

22.8 Hz). HRMS (ESI, m/z): [M + H]⁺calcd for C₁₉H₁₆FN₂O,

307.1247; found, 307.1244.

2-Amino-6-ﬂuoro-N-(1-methyl-1H-pyrazol-4-yl)benzamide

1

(34c). Yield 72%. H NMR (500 MHz, DMSO-d₆) δ 10.29 (s, 1H),

N

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7.99 (s, 1H), 7.52 (s, 1H), 7.18−7.05 (m, 1H), 6.56 (d, J = 8.2 Hz,

1H), 6.39 (dd, J = 9.8, 8.7 Hz, 1H), 5.87 (s, 2H), 3.82 (s, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 161.66, 160.65 (d, J_C‑F= 242.9 Hz),

150.14 (d, J_C‑F= 5.9 Hz), 131.84 (d, J_C‑F= 11.2 Hz), 130.51, 122.04,

121.84, 111.89 (d, J_C‑F= 1.6 Hz), 107.91 (d, J_C‑F= 19.1 Hz), 102.31

(d, J_C‑F= 23.1 Hz), 39.12. HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₁H₁₂FN₄O, 235.0995; found, 235.0999.

10.67 (s, 1H), 10.22 (s, 1H), 7.99 (s, 1H), 7.76 (d, J = 8.2 Hz, 1H),

7.56−7.47 (m, 2H), 7.17 (t, J = 8.9 Hz, 1H), 4.81 (q, J = 6.8 Hz, 1H),

3.83 (s, 3H), 1.59 (d, J = 6.8 Hz, 3H). HRMS (ESI, m/z): [M + H]⁺

calcd for C₁₄H₁₅ClFN₄O₂, 325.0868; found, 325.0870.

2-(2-Chloropropanamido)-6-ﬂuoro-N-isobutylbenzamide (35d).

Yield 81%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.29 (s, 1H), 8.71 (t, J

= 5.4 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.48 (dd, J = 15.2, 7.6 Hz,

1H), 7.11 (t, J = 9.0 Hz, 1H), 4.84 (q, J = 6.7 Hz, 1H), 3.17−3.03 (m,

2H), 1.83 (dp, J = 13.3, 6.6 Hz, 1H), 1.62 (d, J = 6.8 Hz, 3H), 0.90

(d, J = 6.7 Hz, 6H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.32,

163.06, 159.33 (d, J_C‑F= 245.9 Hz), 137.38 (d, J_C‑F= 5.2 Hz), 131.68

(d, J_C‑F= 9.9 Hz), 118.80 (d, J_C‑F= 1.9 Hz), 117.15 (d, J_C‑F= 19.7

Hz), 112.35 (d, J_C‑F= 22.6 Hz), 55.51, 47.09, 28.39, 22.00, 20.54.

HRMS (ESI, m/z): [M + H]⁺calcd for C₁₄H₁₉ClFN₂O₂, 301.1119;

found, 301.1121.

2-Amino-6-ﬂuoro-N-isobutylbenzamide (34d). Yield 84%. ¹H

NMR (500 MHz, DMSO-d₆) δ 8.19 (s, 1H), 7.06 (dd, J = 14.8, 8.0

Hz, 1H), 6.50 (d, J = 8.2 Hz, 1H), 6.32 (dd, J = 10.0, 8.6 Hz, 1H),

5.79 (s, 2H), 3.06 (t, J = 6.4 Hz, 2H), 1.81 (dp, J = 13.4, 6.7 Hz, 1H),

0.89 (d, J = 6.7 Hz, 6H). ¹³C NMR (126 MHz, DMSO-d₆) δ 164.67,

160.75 (d, J_C‑F= 242.8 Hz), 150.04 (d, J_C‑F= 6.4 Hz), 131.32 (d, J_C‑F

= 11.2 Hz), 111.71 (d, J_C‑F= 2.1 Hz), 108.42 (d, J_C‑F= 18.9 Hz),

102.33 (d, J_C‑F= 23.5 Hz), 46.78, 28.46, 20.57. HRMS (ESI, m/z):

[M + H]⁺calcd for C₁₁H₁₆FN₂O, 211.1247; found, 211.1244.

2-Amino-6-ﬂuoro-N-isopropylbenzamide (34e). Yield 83%. ¹H

NMR (500 MHz, DMSO-d₆) δ8.04 (d, J = 7.2 Hz, 1H), 7.05 (dd, J =

14.8, 8.1 Hz, 1H), 6.49 (d, J = 8.2 Hz, 1H), 6.35−6.26 (m, 1H), 5.74

(s, 2H), 4.11−4.01 (m, 1H), 1.13 (d, J = 6.6 Hz, 6H). ¹³C NMR (126

MHz, DMSO-d₆) δ 163.69, 160.66 (d, J_C‑F= 242.8 Hz), 149.88 (d,

J_C‑F= 6.4 Hz), 131.23 (d, J_C‑F= 11.2 Hz), 111.64 (d, J_C‑F= 2.2 Hz),

108.70 (d, J_C‑F= 19.2 Hz), 102.32 (d, J_C‑F= 23.2 Hz), 41.15, 22.69.

HRMS (ESI, m/z): [M + H]⁺calcd for C₁₀H₁₄FN₂O, 197.1090;

found, 197.1087.

2-(2-Chloropropanamido)-6-ﬂuoro-N-isopropylbenzamide

1

(35e). Yield 74%. H NMR (500 MHz, DMSO-d₆) δ 10.17 (s, 1H),

8.57 (d, J = 7.5 Hz, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.46 (dd, J = 15.3,

7.6 Hz, 1H), 7.10 (t, J = 8.9 Hz, 1H), 4.86 (q, J = 6.7 Hz, 1H), 4.13−

4.02 (m, 1H), 1.62 (d, J = 6.8 Hz, 3H), 1.14 (d, J = 6.6 Hz, 6H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 168.29, 161.96, 159.24 (d, J_C‑F

=

246.0 Hz), 137.07 (d, J_C‑F= 5.4 Hz), 131.48 (d, J_C‑F= 9.6 Hz), 119.07

(d, J_C‑F= 2.5 Hz), 117.85 (d, J_C‑F= 19.9 Hz), 112.43 (d, J_C‑F= 22.4

Hz), 55.44, 41.67, 22.54, 22.47, 21.99. HRMS (ESI, m/z): [M + H]⁺

calcd for C₁₃H₁₇ClFN₂O₂, 287.0963; found, 287.0960.

1

2-(2-Chloropropanamido)-N-ethyl-6-ﬂuorobenzamide (35f).

2-Amino-N-ethyl-6-ﬂuorobenzamide (34f). Yield 79%. H NMR

1

Yield 70%. H NMR (500 MHz, DMSO-d₆) δ 10.34 (s, 1H), 8.67

(500 MHz, DMSO-d₆) δ 8.19 (s, 1H), 7.06 (q, J = 7.5 Hz, 1H), 6.50

(d, J = 8.2 Hz, 1H), 6.34−6.29 (m, 1H), 5.87 (s, 2H), 3.25 (p, J = 6.7

Hz, 2H), 1.09 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

164.48, 160.76 (d, J_C‑F= 242.8 Hz), 150.14 (d, J_C‑F= 6.4 Hz), 131.40

(d, J_C‑F= 11.3 Hz), 111.76 (d, J_C‑F= 2.0 Hz), 108.04 (d, J_C‑F= 18.6

Hz), 102.21 (d, J_C‑F= 23.3 Hz), 34.20, 15.09. HRMS (ESI, m/z): [M

+ H]⁺calcd for C₉H₁₂FN₂O, 183.0934; found, 183.0938.

(s, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.48 (dd, J = 15.3, 7.6 Hz, 1H), 7.11

(t, J = 9.0 Hz, 1H), 4.85 (q, J = 6.7 Hz, 1H), 3.35−3.22 (m, 2H), 1.62

(d, J = 6.8 Hz, 3H), 1.12 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 168.31, 162.79, 160.32, 158.36, 137.42, 137.38, 131.73,

131.66, 118.92, 118.90, 117.18, 117.03, 112.44, 112.26, 55.49, 34.59,

21.97, 14.86. ¹³C NMR (126 MHz, DMSO-d₆) δ 168.31, 162.79,

159.34 (d, J_C‑F= 246.2 Hz), 137.40 (d, J_C‑F= 5.2 Hz), 131.70 (d, J_C‑F

= 9.7 Hz), 118.91 (d, J_C‑F= 2.5 Hz), 117.11 (d, J_C‑F= 19.4 Hz),

112.35 (d, J_C‑F= 22.5 Hz), 55.50, 34.59, 21.98, 14.86. HRMS (ESI,

m/z): [M + H]⁺calcd for C₁₂H₁₅ClFN₂O₂, 273.0806; found,

273.0811.

2-Amino-6-ﬂuoro-N-methylbenzamide (34g). Yield 76%. ¹H

NMR (500 MHz, DMSO-d₆) δ 8.12 (s, 1H), 7.07 (dd, J = 15.3,

7.5 Hz, 1H), 6.51 (d, J = 8.2 Hz, 1H), 6.35−6.30 (m, 1H), 5.96 (s,

2H), 2.75 (d, J = 4.6 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

165.28, 160.87 (d, J_C‑F= 243.0 Hz), 150.29 (d, J_C‑F= 6.4 Hz), 131.51

(d, J_C‑F= 11.6 Hz), 111.85 (d, J_C‑F= 2.2 Hz), 107.59 (d, J_C‑F= 18.8

Hz), 102.15 (d, J_C‑F= 23.7 Hz), 26.46. HRMS (ESI, m/z): [M + H]⁺

calcd for C₈H₁₀FN₂O, 169.0777; found, 169.0776.

2-(2-Chloropropanamido)-6-ﬂuoro-N-methylbenzamide (35g).

1

Yield 73%. H NMR (500 MHz, DMSO-d₆) δ 10.45 (s, 1H), 8.58

(d, J = 3.8 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.48 (dd, J = 15.3, 7.6

Hz, 1H), 7.11 (t, J = 9.1 Hz, 1H), 4.84 (q, J = 6.7 Hz, 1H), 2.79 (d, J

= 4.5 Hz, 3H), 1.61 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 168.34, 163.55, 159.43 (d, J_C‑F= 246.2 Hz), 137.63 (d,

J_C‑F= 5.1 Hz), 131.86 (d, J_C‑F= 10.0 Hz), 118.81 (d, J_C‑F= 2.5 Hz),

116.56 (d, J_C‑F= 19.3 Hz), 112.30 (d, J_C‑F= 22.7 Hz), 55.50, 26.70,

21.95. HRMS (ESI, m/z): [M + H]⁺calcd for C₁₁H₁₃ClFN₂O₂,

259.0650; found, 259.0653.

2-(2-Bromopropanamido)-6-ﬂuoro-N-phenylbenzamide (35a).

To a solution of 34a (737 mg, 3.2 mmol, 1.0 equiv) in anhydrous

THF (20 mL) was added DIPEA (1.7 mL, 9.6 mmol, 3.0 equiv), and

then 2-bromopropanoyl chloride (0.38 mL, 3.8 mmol, 1.2 equiv) was

slowly added to the mixture at 0 °C. The reaction mixture was stirred

at 0 °C for 1 h and then warmed up to room temperature for 6 h. The

resulting mixture was concentrated to dryness to give the crude

product. The residue was diluted with water and extracted with

EtOAc. The combined organic layers were washed with water and

brine and dried over anhydrous Na₂SO₄. Evaporation of the solvent

provided the crude product, which was puriﬁed by ﬂash

chromatography (eluting with 0−15% EtOAc in hexane) to give

Compounds 36a−g and 46a−b were prepared following the

synthetic procedure of 13.

2-(1-Bromoethyl)-5-ﬂuoro-3-phenylquinazolin-4(3H)-one (36a).

Yield 54%. ¹H NMR (500 MHz, CDCl₃) δ 7.67−7.63 (m, 1H), 7.54−

7.51 (m, 2H), 7.49−7.46 (m, 3H), 7.11−7.07 (m, 2H), 4.45 (q, J =

6.8 Hz, 1H), 1.95 (d, J = 6.7 Hz, 3H). HRMS (ESI, m/z): [M + H]⁺

calcd for C₁₆H₁₃BrFN₂O, 347.0195; found, 347.0192/349.0173.

3-([1,1′-Biphenyl]-4-yl)-2-(1-chloroethyl)-5-ﬂuoroquinazolin-

4(3H)-one (36b). Yield 52%. ¹H NMR (500 MHz, DMSO-d₆) δ

7.93−7.88 (m, 3H), 7.79 (d, J = 7.3 Hz, 2H), 7.64 (t, J = 9.3 Hz, 2H),

7.58−7.56 (m, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.44 (t, J = 7.4 Hz, 1H),

7.39 (dd, J = 10.4, 8.6 Hz, 1H), 4.68 (q, J = 6.5 Hz, 1H), 1.82 (d, J =

6.5 Hz, 3H). HRMS (ESI, m/z): [M + H]⁺calcd for C₂₂H₁₇ClFN₂O,

379.1013; found, 379.1014.

2-(1-Chloroethyl)-5-ﬂuoro-3-(1-methyl-1H-pyrazol-4-yl)-

quinazolin-4(3H)-one (36c). Yield 39%. ¹H NMR (500 MHz,

DMSO-d₆) δ 8.00 (s, 1H), 7.88 (dd, J = 13.6, 8.0 Hz, 1H), 7.61−

7.55 (m, 2H), 7.39−7.32 (m, 1H), 4.88 (q, J = 6.3 Hz, 1H), 3.94 (s,

3H), 1.79 (d, J = 6.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

160.87 (d, J_C‑F= 264.0 Hz), 158.59 (d, J_C‑F= 4.1 Hz), 156.88, 148.67,

138.13, 136.21 (d, J_C‑F= 10.5 Hz), 130.65, 124.10 (d, J_C‑F= 3.8 Hz),

116.18, 114.70 (d, J_C‑F= 20.5 Hz), 110.53 (d, J_C‑F= 5.7 Hz), 53.95,

1

35a as a yellow solid (920 mg, yield 77%). H NMR (500 MHz,

DMSO-d₆) δ 10.59 (s, 1H), 10.06 (s, 1H), 7.72 (d, J = 7.8 Hz, 2H),

7.63 (d, J = 8.2 Hz, 1H), 7.55−7.48 (m, 1H), 7.38−7.32 (m, 2H),

7.18 (t, J = 8.8 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 4.84 (q, J = 6.7 Hz,

1H), 1.69 (d, J = 6.7 Hz, 3H). HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₆H₁₅BrFN₂O₂, 365.0301; found, 365.0304/367.0284.

Compounds 35b−g and 42 were prepared following the synthetic

procedure of 35a.

N-([1,1′-Biphenyl]-4-yl)-2-(2-chloropropanamido)-6-ﬂuoroben-

1

zamide (35b). Yield 82%. H NMR (500 MHz, DMSO-d₆) δ 10.71

(s, 1H), 10.18 (s, 1H), 7.81 (d, J = 8.6 Hz, 2H), 7.70−7.64 (m, 5H),

7.54 (dd, J = 14.7, 8.2 Hz, 1H), 7.47−7.44(m, 2H), 7.34 (t, J = 7.4

Hz, 1H), 7.20 (t, J = 8.8 Hz, 1H), 4.82 (q, J = 6.7 Hz, 1H), 1.57 (d, J

= 6.8 Hz, 3H). HRMS (ESI, m/z): [M + H]⁺calcd for

C₂₂H₁₉ClFN₂O₂, 397.1119; found, 397.1122.

2-(2-Chloropropanamido)-6-ﬂuoro-N-(1-methyl-1H-pyrazol-4-

1

yl)benzamide (35c). Yield 69%. H NMR (500 MHz, DMSO-d₆) δ

O

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22.20. HRMS (ESI, m/z): [M + H]⁺calcd for C₁₄H₁₃ClFN₄O,

307.0762; found, 307.0764.

(ESI, m/z): [M + H]⁺calcd for C₁₃H₁₆FN₂O₃, 267.1145; found,

267.1146.

N-Cyclopentyl-2-ﬂuoro-6-nitrobenzamide (38c). Yield 75%. ¹H

NMR (500 MHz, CDCl₃) δ 7.92 (dd, J = 8.2, 0.9 Hz, 1H), 7.54 (td, J

= 8.3, 5.5 Hz, 1H), 7.43 (td, J = 8.3, 1.0 Hz, 1H), 4.49−4.39 (m, 1H),

2.14−2.06 (m, 2H), 1.74−1.65 (m, 4H), 1.61−1.54 (m, 2H). HRMS

(ESI, m/z): [M + H]⁺calcd for C₁₂H₁₄FN₂O₃, 253.0988; found,

253.0990.

2-(1-Chloroethyl)-5-ﬂuoro-3-isobutylquinazolin-4(3H)-one

(36d). Yield 57%. ¹H NMR (500 MHz, DMSO-d₆) δ 7.84 (td, J = 8.2,

5.6 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.34 (dd, J = 10.7, 8.5 Hz, 1H),

5.52 (q, J = 6.3 Hz, 1H), 4.24 (dd, J = 14.1, 7.3 Hz, 1H), 3.83 (dd, J =

14.2, 8.0 Hz, 1H), 2.19−2.08 (m, 1H), 1.92 (d, J = 6.4 Hz, 3H),

0.95−0.91 (m, 6H). HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₄H₁₇ClFN₂O, 283.1013; found, 283.1009.

2-(1-Chloroethyl)-5-ﬂuoro-3-isopropylquinazolin-4(3H)-one

(36e). Yield 55%. ¹H NMR (500 MHz, DMSO-d₆) δ 7.80 (td, J = 8.1,

5.5 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.30 (dd, J = 10.8, 8.4 Hz, 1H),

5.62 (q, J = 5.9 Hz, 1H), 4.86−4.72 (m, 1H), 1.90 (d, J = 6.3 Hz,

3H), 1.63−1.59 (m, 6H). HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₃H₁₅ClFN₂O, 269.0857; found, 269.0858.

N-Cyclobutyl-2-ﬂuoro-6-nitrobenzamide (38d). Yield 81%. ¹H

NMR (500 MHz, DMSO-d₆) δ8.99 (d, J = 7.2 Hz, 1H), 8.02 (d, J =

7.9 Hz, 1H), 7.79−7.70 (m, 2H), 4.38−4.28 (m, 1H), 2.30−2.21 (m,

2H), 2.00−1.91 (m, 2H), 1.73−1.66 (m, 2H). HRMS (ESI, m/z): [M

+ H]⁺calcd for C₁₁H₁₂FN₂O₃, 239.0832; found, 239.0828.

1

N-Cyclopropyl-2-ﬂuoro-6-nitrobenzamide (38e). Yield 77%. H

NMR (500 MHz, DMSO-d₆) δ 8.80 (d, J = 2.2 Hz, 1H), 8.01 (d, J =

7.8 Hz, 1H), 7.79−7.70 (m, 2H), 2.79 (tq, J = 7.4, 3.8 Hz, 1H), 0.74−

0.71 (m, 2H), 0.52−0.47 (m, 2H). HRMS (ESI, m/z): [M + H]⁺

calcd for C₁₀H₁₀FN₂O₃, 225.0675; found, 225.0672.

2-(1-Chloroethyl)-3-ethyl-5-ﬂuoroquinazolin-4(3H)-one (36f).

1

Yield 59%. H NMR (500 MHz, DMSO-d₆) δ 7.83 (td, J = 8.2, 5.7

Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.33 (dd, J = 10.8, 8.3 Hz, 1H),

5.53 (q, J = 6.3 Hz, 1H), 4.32 (dq, J = 14.2, 7.1 Hz, 1H), 4.05 (dq, J =

14.1, 7.0 Hz, 1H), 1.91 (d, J = 6.4 Hz, 3H), 1.32 (t, J = 7.0 Hz, 3H).

¹³C NMR (126 MHz, DMSO-d₆) δ 160.67 (d, J_C‑F= 263.9 Hz),

158.33 (d, J_C‑F= 4.3 Hz), 155.97, 148.66, 135.64 (d, J_C‑F= 10.5 Hz),

3-Benzyl-2-ethyl-5-ﬂuoroquinazolin-4(3H)-one (39a). A solution

of 38a (2.0 g, 7.3 mmol, 1.0 equiv) in propionic anhydride (14 mL)

was stirred at 140 °C in a microwave reactor for 3 h. The resulting

mixture was concentrated to dryness to give the crude product, which

was used in the next step without further puriﬁcation. To a solution of

the former crude product in anhydrous AcOH (20 mL) was added

zinc (2.4 g, 36.5 mmol, 5.0 equiv) at room temperature under argon.

Then, the reaction mixture was stirred at room temperature for 8 h.

The resulting mixture was concentrated to dryness. The residue was

diluted with water and extracted with DCM. The combined organic

layers were washed with water and brine and dried over anhydrous

Na₂SO₄. Evaporation of the solvent provided the crude product,

which was puriﬁed by ﬂash chromatography (eluting with 0−10%

123.94 (d, J_C‑F= 4.0 Hz), 114.30 (d, J_C‑F= 20.4 Hz), 110.44 (d, J_C‑F

=

5.7 Hz), 53.84, 38.81, 22.66, 14.47. HRMS (ESI, m/z): [M + H]⁺

calcd for C₁₂H₁₃ClFN₂O, 255.0700; found, 255.0687.

2-(1-Chloroethyl)-5-ﬂuoro-3-methylquinazolin-4(3H)-one (36g).

1

Yield 52%. H NMR (500 MHz, DMSO-d₆) δ 7.82 (td, J = 8.2, 5.6

Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.33 (dd, J = 10.9, 8.3 Hz, 1H),

5.55 (q, J = 6.4 Hz, 1H), 3.62 (s, 3H), 1.89 (d, J = 6.4 Hz, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 160.62 (d, J_C‑F= 263.8 Hz), 158.73

(d, J_C‑F= 4.4 Hz), 156.20, 148.59, 135.62 (d, J_C‑F= 10.7 Hz), 123.95

(d, J_C‑F= 3.9 Hz), 114.30 (d, J_C‑F= 20.7 Hz), 110.20 (d, J_C‑F= 5.6

Hz), 54.33, 29.93, 22.14. HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₁H₁₁ClFN₂O, 241.0544; found, 241.0543.

1

EtOAc in hexane) to give 39a as a white solid. Yield 48%. H NMR

(500 MHz, DMSO-d₆) δ 7.80 (td, J = 8.2, 5.7 Hz, 1H), 7.47 (d, J =

8.2 Hz, 1H), 7.37−7.34 (m, 2H), 7.32−7.24 (m, 2H), 7.21 (d, J = 7.3

Hz, 2H), 5.37 (s, 2H), 2.77 (q, J = 7.2 Hz, 2H), 1.19 (t, J = 7.2 Hz,

3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 160.89 (d, J_C‑F= 263.5 Hz),

N-Benzyl-2-ﬂuoro-6-nitrobenzamide (38a). To a solution of 37

(14 g, 75.6 mmol, 1.0 equiv) in anhydrous THF (50 mL) was added

DMF (0.5 mL), and then oxalyl dichloride (7.7 mL, 90.7 mmol, 1.2

equiv) was slowly added to the mixture at 0 °C. The reaction mixture

was stirred at 0 °C for 1 h and then warmed up to room temperature

for 5 h. The resulting mixture was concentrated to dryness to give the

crude product, which was used in the next step without further

puriﬁcation. To a solution of phenylmethanamine (8.3 mL, 75.6

mmol, 1.0 equiv) in anhydrous THF (50 mL) was added DIPEA

(26.3 mL, 151.2 mmol, 2.0 equiv), and then the previous crude

product in anhydrous THF was slowly added to the mixture at 0 °C

under argon. Then, the reaction mixture was stirred at room

temperature for 6 h. The resulting mixture was concentrated to

dryness. The residue was diluted with water and extracted with

EtOAc. The combined organic layers were washed with water and

brine and dried over anhydrous Na₂SO₄. Evaporation of the solvent

provided the crude product, which was puriﬁed by ﬂash

chromatography (eluting with 0−20% EtOAc in hexane) to give

38a as a yellow solid (17.4 g, yield 84%). ¹H NMR (500 MHz,

DMSO-d₆) δ 9.29 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.81−

7.70 (m, 2H), 7.39−7.33 (m, 4H), 7.31−7.25 (m, 1H), 4.51 (d, J =

5.9 Hz, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 161.08, 158.93 (d,

159.66, 158.96 (d, J_C‑F= 4.0 Hz), 149.53, 136.94, 135.53 (d, J_C‑F

=

10.6 Hz), 129.23, 127.71, 126.63, 123.47 (d, J_C‑F= 3.8 Hz), 113.23 (d,

J

_C‑F= 20.5 Hz), 109.93 (d, J_C‑F= 5.5 Hz), 45.64, 27.79, 10.99. HRMS

(ESI, m/z): [M + H]⁺calcd for C₁₇H₁₆FN₂O, 283.1247; found,

283.1252.

Compounds 39b−e were prepared following the synthetic

procedure of 39a.

3-Cyclohexyl-2-ethyl-5-ﬂuoroquinazolin-4(3H)-one (39b). Yield

1

24%. H NMR (500 MHz, DMSO-d₆) δ 7.72 (td, J = 8.1, 5.6 Hz,

1H), 7.38 (d, J = 8.2 Hz, 1H), 7.18 (dd, J = 10.8, 8.4 Hz, 1H), 4.20−

3.00 (m, 1H), 2.92 (q, J = 7.3 Hz, 2H), 2.58 (d, J = 11.0 Hz, 2H),

1.81 (d, J = 13.0 Hz, 2H), 1.71 (d, J = 11.2 Hz, 2H), 1.65 (d, J = 12.9

Hz, 1H), 1.39 (q, J = 13.1 Hz, 2H), 1.27 (t, J = 7.3 Hz, 3H), 1.24−

1.13 (m, 1H). HRMS (ESI, m/z): [M + H]⁺calcd for C₁₆H₂₀FN₂O,

275.1560; found, 275.1557.

3-Cyclopentyl-2-ethyl-5-ﬂuoroquinazolin-4(3H)-one (39c). Yield

1

28%. H NMR (500 MHz, DMSO-d₆) δ 7.72 (td, J = 8.2, 5.5 Hz,

1H), 7.39 (d, J = 8.1 Hz, 1H), 7.23−7.15 (m, 1H), 4.83−4.73 (m,

1H), 2.96 (q, J = 7.3 Hz, 2H), 2.23−2.13 (m, 2H), 2.03−1.95 (m,

2H), 1.93−1.86 (m, 2H), 1.66−1.56 (m, 2H), 1.29 (t, J = 7.3 Hz,

3H). HRMS (ESI, m/z): [M + H]⁺calcd for C₁₅H₁₈FN₂O, 261.1403;

found, 261.1406.

J_C‑F= 248.9 Hz), 147.13 (d, J_C‑F= 5.4 Hz), 138.95, 132.01 (d, J_C‑F

=

8.9 Hz), 128.81, 127.82, 127.49, 122.50 (d, J_C‑F= 22.5 Hz), 122.20 (d,

J

_C‑F= 24.2 Hz), 120.95 (d, J_C‑F= 2.9 Hz), 43.14. HRMS (ESI, m/z):

[M + H]⁺calcd for C₁₄H₁₂FN₂O₃, 275.0832; found, 275.0829.

Compounds 38b−e were prepared following the synthetic

procedure of 38a.

3-Cyclobutyl-2-ethyl-5-ﬂuoroquinazolin-4(3H)-one (39d). Yield

1

34%. H NMR (500 MHz, DMSO-d₆) δ 7.72 (td, J = 8.2, 5.6 Hz,

1H), 7.37 (d, J = 8.2 Hz, 1H), 7.24−7.15 (m, 1H), 4.87 (p, J = 8.6

Hz, 1H), 3.02−2.91 (m, 2H), 2.86 (q, J = 7.3 Hz, 2H), 2.37−2.27 (m,

2H), 1.93−1.83 (m, 1H), 1.82−1.70 (m, 1H), 1.24 (t, J = 7.3 Hz,

3H). HRMS (ESI, m/z): [M + H]⁺calcd for C₁₄H₁₆FN₂O, 247.1247;

found, 247.1245.

N-Cyclohexyl-2-ﬂuoro-6-nitrobenzamide (38b). Yield 81%. ¹H

NMR (500 MHz, DMSO-d₆) δ 8.63 (d, J = 7.8 Hz, 1H), 8.00 (d, J =

7.8 Hz, 1H), 7.77−7.68 (m, 2H), 3.74 (td, J = 14.1, 7.0 Hz, 1H),

1.89−1.80 (m, 2H), 1.72−1.69 (m, 2H), 1.57−1.55 (m, 1H), 1.36−

1.28 (m, 2H), 1.26−1.12 (m, 3H). ¹³C NMR (126 MHz, DMSO-d₆)

δ 159.91, 158.89 (d, J_C‑F= 248.6 Hz), 147.06 (d, J_C‑F= 5.5 Hz),

3-Cyclopropyl-5-ﬂuoro-2-propylquinazolin-4(3H)-one (39e).

1

Yield 41%. H NMR (500 MHz, DMSO-d₆) δ 7.71 (td, J = 8.2, 5.6

Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.18 (ddd, J = 11.1, 8.2, 0.8 Hz,

1H), 3.00−2.92 (m, 3H), 1.87−1.77 (m, 2H), 1.22−1.16 (m, 2H),

131.63 (d, J_C‑F= 8.8 Hz), 122.66 (d, J_C‑F= 24.5 Hz), 122.39 (d, J_C‑F

22.6 Hz), 120.77 (d, J_C‑F= 2.8 Hz), 48.58, 32.31, 25.63, 24.76. HRMS

=

P

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1.01 (t, J = 7.4 Hz, 3H), 0.86−0.81 (m, 2H). HRMS (ESI, m/z): [M

+ H]⁺calcd for C₁₄H₁₆FN₂O, 247.1247; found, 247.1251.

(d, J = 4.3 Hz, 1H), 8.26 (s, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.60 (dd, J

= 12.1, 1.8 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 14.8, 8.3

Hz, 1H), 7.34 (t, J = 8.7 Hz, 1H), 7.01 (t, J = 8.8 Hz, 1H), 5.65 (q, J =

7.2 Hz, 1H), 3.92 (s, 3H), 2.59 (tt, J = 7.9, 4.0 Hz, 1H), 1.83 (d, J =

7.2 Hz, 3H), 0.62−0.54 (m, 2H), 0.45−0.41 (m, 1H), 0.36−0.33(m,

1H). HRMS (ESI, m/z): [M + H]⁺calcd for C₂₅H₂₄F₂N₇O₃,

508.1909; found, 508.1912.

3-Benzyl-2-(1-bromoethyl)-5-ﬂuoroquinazolin-4(3H)-one (40a).

To a solution of 39a (1.0 g, 3.5 mmol, 1.0 equiv) in CCl₄(30 mL)

were added NBS (0.75 g, 4.2 mmol, 1.2 equiv) and AIBN (54 mg,

0.35 mmol, 0.1 equiv) at room temperature under argon. Then, the

reaction mixture was stirred at reﬂux for 10 h. The resulting mixture

was concentrated to dryness. The residue was diluted with water and

extracted with DCM. The combined organic layers were washed with

water and brine and dried over anhydrous Na₂SO₄. Evaporation of the

solvent provided the crude product, which was puriﬁed by ﬂash

chromatography (eluting with 0−8% EtOAc in hexane) to give 40a as

a white solid (740 mg, yield 57%). ¹H NMR (500 MHz, DMSO-d₆) δ

7.88 (td, J = 8.2, 5.6 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.39−7.34 (m,

3H), 7.28 (t, J = 7.3 Hz, 1H), 7.21 (d, J = 7.4 Hz, 2H), 5.67 (d, J =

16.7 Hz, 1H), 5.26 (q, J = 6.4 Hz, 1H), 5.19 (d, J = 16.7 Hz, 1H),

1.98 (d, J = 6.5 Hz, 3H). HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₇H₁₅BrFN₂O, 361.0352; found, 361.0357/363.0335.

(S)-2-(2-Chlorobutanamido)-N-cyclopropyl-6-ﬂuorobenzamide

(45a). To a solution of 44a (0.84 mL, 8.2 mmol, 1.0 equiv) in

anhydrous THF (20 mL) was added DMF (0.05 mL), and then oxalyl

dichloride (0.8 mL, 9.8 mmol, 1.2 equiv) was slowly added to the

mixture at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and

then warmed up to room temperature for 5 h. The resulting mixture

was concentrated to dryness to give the crude product, which was

used in the next step without further puriﬁcation. To a solution of 41

(0.57 mL, 8.2 mmol, 1.0 equiv) in anhydrous THF (20 mL) was

added DIPEA (4.3 mL, 24.6 mmol, 3.0 equiv), and then the crude

product of the former step (8.2 mmol, 1.0 eq) was slowly added to the

mixture at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and

then warmed up to room temperature for 6 h. The resulting mixture

was concentrated to dryness to give the crude product. The residue

was diluted with water and extracted with EtOAc. The combined

organic layers were washed with water and brine and dried over

anhydrous Na₂SO₄. Evaporation of the solvent provided the crude

product, which was puriﬁed by ﬂash chromatography (eluting with 0−

15% EtOAc in hexane) to give 45a as a yellow solid (1.6 g, yield

Compounds 40b−e were prepared following the synthetic

procedure of 40a.

2-(1-Bromoethyl)-3-cyclohexyl-5-ﬂuoroquinazolin-4(3H)-one

(40b). Yield 36%. ¹H NMR (500 MHz, DMSO-d₆) δ 7.79 (td, J = 8.1,

5.5 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.29 (dd, J = 10.7, 8.5 Hz, 1H),

5.66 (q, J = 6.2 Hz, 1H), 4.30−4.16 (m, 1H), 2.70−2.53 (m, 2H),

2.05 (d, J = 6.3 Hz, 3H), 1.86−1.77 (m, 4H), 1.70−1.65 (m, 1H),

1.50 (dt, J = 16.2, 8.4 Hz, 1H), 1.34 (q, J = 13.0 Hz, 1H), 1.19 (dd, J

= 25.6, 12.6 Hz, 1H). HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₆H₁₉BrFN₂O, 353.0665; found, 353.0668/355.0647.

2-(1-Bromoethyl)-3-cyclopentyl-5-ﬂuoroquinazolin-4(3H)-one

(40c). Yield 37%. ¹H NMR (500 MHz, CDCl₃) δ 7.66−7.59 (m, 1H),

7.46 (d, J = 8.2 Hz, 1H), 7.14−7.05 (m, 1H), 5.12−5.06 (m, 1H),

4.84 (dq, J = 17.0, 8.4 Hz, 1H), 2.46−2.31 (m, 2H), 2.20−2.08 (m,

5H), 2.08−2.00 (m, 1H), 1.93−1.84 (m, 1H), 1.66 (tdd, J = 14.4, 7.4,

4.9 Hz, 2H). HRMS (ESI, m/z): [M + H]⁺calcd for C₁₅H₁₇BrFN₂O,

339.0508; found, 339.0513/341.0484.

2-(1-Bromoethyl)-3-cyclobutyl-5-ﬂuoroquinazolin-4(3H)-one

(40d). Yield 40%. ¹H NMR (500 MHz, DMSO-d₆) δ 7.80 (td, J = 8.2,

5.5 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.30 (dd, J = 11.0, 8.2 Hz, 1H),

5.67 (q, J = 6.3 Hz, 1H), 5.04 (p, J = 8.6 Hz, 1H), 3.13−3.05 (m,

2H), 2.39−2.28 (m, 2H), 2.04 (d, J = 6.3 Hz, 3H), 1.99−1.90 (m,

1H), 1.84−1.74 (m, 1H). HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₄H₁₅BrFN₂O, 325.0352; found, 325.0351/327.0335.

1

67%). H NMR (500 MHz, DMSO-d₆) δ 10.20 (s, 1H), 8.68 (d, J =

3.7 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.46 (dt, J = 14.8, 7.4 Hz, 1H),

7.09 (t, J = 8.8 Hz, 1H), 4.69 (t, J = 6.6 Hz, 1H), 2.84 (tq, J = 7.7, 4.0

Hz, 1H), 2.08−1.98 (m, 1H), 1.94−1.85 (m, 1H), 0.98 (t, J = 7.3 Hz,

3H), 0.73−0.66 (m, 2H), 0.56−0.51 (m, 2H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 167.75, 164.14, 159.31 (d, J_C‑F= 246.0 Hz), 136.99 (d,

J_C‑F= 5.4 Hz), 131.54 (d, J_C‑F= 9.8 Hz), 119.49 (d, J_C‑F= 2.4 Hz),

117.96 (d, J_C‑F= 19.7 Hz), 112.51 (d, J_C‑F= 22.5 Hz), 61.51, 28.55,

23.29, 10.71, 6.20, 6.11. HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₄H₁₇ClFN₂O₂, 299.0963; found, 299.0962.

Compounds 45b were prepared following the synthetic procedure

of 45a.

(R)-2-(2-Chlorobutanamido)-N-cyclopropyl-6-ﬂuorobenzamide

1

(45b). Yield 65%. H NMR (500 MHz, DMSO-d₆) δ 10.18 (s, 1H),

8.68 (d, J = 3.2 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.46 (dt, J = 14.8,

7.4 Hz, 1H), 7.10 (t, J = 8.9 Hz, 1H), 4.69 (t, J = 6.6 Hz, 1H), 2.83

(tt, J = 7.7, 3.9 Hz, 1H), 2.08−1.96 (m, 1H), 1.93−1.85 (m, 1H),

0.98 (t, J = 7.3 Hz, 3H), 0.72−0.68 (m, 2H), 0.56−0.50 (m, 2H).

HRMS (ESI, m/z): [M + H]⁺calcd for C₁₄H₁₇ClFN₂O₂, 299.0963;

found, 299.0966.

(S)-2-(1-Chloropropyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-

one (46a). Yield 54%. ¹H NMR (500 MHz, DMSO-d₆) δ 7.80 (td, J =

8.2, 5.6 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.30 (dd, J = 10.4, 8.4 Hz,

1H), 5.62 (dd, J = 7.9, 6.2 Hz, 1H), 3.04 (qd, J = 7.0, 4.2 Hz, 1H),

2.38 (dp, J = 14.0, 7.2 Hz, 1H), 2.20 (dp, J = 14.8, 7.4 Hz, 1H), 1.33−

1.24 (m, 2H), 1.08 (t, J = 7.3 Hz, 3H), 1.00−0.96 (m, 1H), 0.94−

0.89 (m, 1H). HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₄H₁₅ClFN₂O, 281.0857; found, 281.0861.

(R)-2-(1-Chloropropyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-

one (46b). Yield 49%. ¹H NMR (500 MHz, DMSO-d₆) δ 7.80 (td, J =

8.2, 5.6 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.30 (dd, J = 10.3, 8.3 Hz,

1H), 5.62 (dd, J = 7.9, 6.2 Hz, 1H), 3.04 (qd, J = 7.1, 4.2 Hz, 1H),

2.38 (dp, J = 14.1, 7.2 Hz, 1H), 2.20 (dp, J = 14.8, 7.3 Hz, 1H), 1.34−

1.23 (m, 2H), 1.08 (t, J = 7.3 Hz, 3H), 1.00−0.96 (m, 1H), 0.95−

0.87 (m, 1H). HRMS (ESI, m/z): [M + H]⁺calcd for

C₁₄H₁₅ClFN₂O, 281.0857; found, 281.0855.

Cell Lines, Cell Culture, and Chemicals. The cell lines NIH-

3T3, RAW 264.7, and Raji were purchased from Cobioer Biosciences

Co., Ltd. (Nanjing, China). NIH-3T3 and RAW 264.7 cell lines were

cultured in DMEM media (Corning) with 10% fetal bovine serum

(FBS) and supplemented with 2% ^L-glutamine and 1% penicillin/

streptomycin. Raji cells were cultured in RPMI 1640 media (Corning)

with 10% fetal bovine serum (FBS), supplemented with 2% ^L-

2-(1-Bromopropyl)-3-cyclopropyl-5-ﬂuoroquinazolin-4(3H)-one

1

(40e). Yield 45%. H NMR (500 MHz, DMSO-d₆) δ 7.79 (dd, J =

13.6, 8.1 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.30 (dd, J = 10.7, 8.4 Hz,

1H), 5.67 (t, J = 7.1 Hz, 1H), 3.01−2.96 (m, 1H), 2.50−2.40 (m,

1H), 2.33−2.24 (m, 1H), 1.33−1.23 (m, 2H), 1.07 (t, J = 7.2 Hz,

3H),1.00−0.96 (m, 1H), 0.91−0.86 (m, 1H). HRMS (ESI, m/z): [M

+ H]⁺calcd for C₁₄H₁₅BrFN₂O, 325.0352; found, 325.0349/

327.0336.

1

2-Amino-N-cyclopropyl-6-ﬂuorobenzamide (41). Yield 84%. H

NMR (500 MHz, DMSO-d₆) δ 8.21 (s, 1H), 7.05 (dd, J = 14.8, 8.1

Hz, 1H), 6.50 (d, J = 8.2 Hz, 1H), 6.33−6.26 (m, 1H), 5.79 (s, 2H),

2.83 (tq, J = 7.7, 4.0 Hz, 1H), 0.70−0.64 (m, 2H), 0.54−0.49 (m,

2H). HRMS (ESI, m/z): [M + H]⁺calcd for C₁₀H₁₂FN₂O, 195.0934;

found, 195.0932.

2-(2-Chloropropanamido)-N-cyclopropyl-6-ﬂuorobenzamide

1

(42). Yield 76%. H NMR (500 MHz, DMSO-d₆) δ 10.22 (s, 1H),

8.68 (d, J = 3.5 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.46 (dd, J = 14.8,

8.2 Hz, 1H), 7.09 (t, J = 8.9 Hz, 1H), 4.85 (q, J = 6.8 Hz, 1H), 2.84

(tq, J = 7.7, 4.0 Hz, 1H), 1.62 (d, J = 6.8 Hz, 3H), 0.73−0.67 (m,

2H), 0.57−0.52 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.30,

164.15, 159.34 (d, J_C‑F= 246.1 Hz), 137.15 (d, J_C‑F= 5.4 Hz), 131.60

(d, J_C‑F= 9.7 Hz), 119.25 (d, J_C‑F= 2.0 Hz), 117.65 (d, J_C‑F= 19.6

Hz), 112.44 (d, J_C‑F= 22.4 Hz), 55.41, 23.30, 21.95, 6.22, 6.14.

HRMS (ESI, m/z): [M + H]⁺calcd for C₁₃H₁₅ClFN₂O₂, 285.0806;

found, 285.0811.

2-(2-(4-Amino-3-(3-ﬂuoro-4-methoxyphenyl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)propanamido)-N-cyclopropyl-6-ﬂuorobenzamide

(43). Yield 53%. ¹H NMR (500 MHz, DMSO-d₆) δ 9.73 (s, 1H), 8.56

Q

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glutamine and 1% penicillin/streptomycin. All cell lines were

maintained in humidiﬁed incubators at 37 °C with 5% CO₂.

Compounds 4 and 5 were purchased from MedChemExpress

(Shanghai, China).

dilution with ultrapure water (1:1, m/v) under continuous stirring.

(S)-18 was mixed with the HP-β-CD gel at a ratio of 1:7 and then

stirred in the dark at 4 °C for 6 h. After prefreezing at −80 °C for 2 h,

the mixture was freeze-dried overnight and redissolved in saline for

further animal study. The rats were randomly and equally divided into

three groups (3 animals/group) by diﬀerent routes of administration.

One group was injected with (S)-18 at a dosage of 1 mg/kg; the

second group was treated by oral administration of (S)-18 at a dosage

of 10 mg/kg; and the third group was treated by inhalation of

nebulized (S)-18 at a dosage of 5 mg/kg. Blood samples were

collected into heparinized tubes at 2, 5, 15, and 30 min and 1, 2, 4, 6,

9, 12, and 24 h after intravenous injection and at 5, 15, and 30 min

and 1, 2, 4, 6, 9, 12, 24, and 72 h after oral or inhaled administration.

Plasma (100 μL) was harvested by centrifuging the blood sample at 4

°C and 8000 rpm for 5 min and then stored at −80 °C until analysis.

An aliquot of 100 μL of each plasma sample was mixed with 20 μL of

the internal standard working solution (200 ng/mL of caﬀeine), and

then 400 μL of methanol was added for protein precipitation. After

vortexing for 5 min and centrifuging at 14 000 rpm for 10 min, 10 μL

of the supernatant was injected for liquid chromatography−mass

spectrometry (LC−MS)/MS analysis.

For lung tissue distribution study, the rats receiving (S)-18 at a

dose of 5 mg/kg by inhalation were sacriﬁced by bleeding the femoral

artery at 6, 12, 24, or 72 h (three rats per time point). After that, lung

tissue samples were excised and then washed using iced physiological

saline solution three times, blotted on ﬁlter paper, and stored at

−80 °C until analysis. Before analysis, the weighed tissues were

homogenized in iced physiological saline solution (1:4, w/v) to

prepare homogenates by Tissuelyser-24 (Jingxin Industrial Develop-

ment Co. Ltd, Shanghai, China). An aliquot of 100 μL of each lung

tissue sample was mixed with 20 μL of an internal standard working

solution (200 ng/mL of caﬀeine); then, 400 μL of methanol was

added for protein precipitation. After vortexing for 5 min and

centrifuging at 14 000 rpm for 10 min, 10 μL of the supernatant was

injected for LC−MS/MS analysis.

The in vitro metabolic stability of (S)-18 was performed using

human, monkey, dog, rat, and mice liver microsomes, respectively, in

triplicate. The incubation mixtures (200 μL) consisted of human liver

microsomes (0.5 mg/mL), (S)-18 (1 mM), NADPH (1 mM), MgCl₂

(4 mM), and PBS. Reactions were initiated with the addition of

NADPH and kept in a water-bath at 37 °C. Aliquots were withdrawn

at 0, 5, 10, 15, 30, 60, and 90 min. The reaction was terminated with

acetonitrile containing caﬀeine (internal standard, 40 ng/mL) and

then centrifugated. The supernatant was subjected to HPLC-mass

spectrometry (MS/MS) analysis. The in vitro elimination half-life

(t_1/2), intrinsic hepatic clearance, and extrapolated hepatic clearance

were determined as described by Obach.³⁶

Drug Eﬃcacy Evaluation, Histopathological Analysis, and

Apoptosis Detection in a Rodent Model of COPD. The in vivo

assessment of drug eﬃcacy, histopathological analysis, and apoptosis

detection against COPD in rodent model were provided by Nanjing

Bosgene Biotechnology Co., Ltd. (Nanjing, China). All animal care

and experimental studies were carried out according to the National

Institutes of Health Guide for the Care and Use of Laboratory

Animals and approved by Laboratory Animal Ethics Committee of

BOSGENE Biotech (Nanjing, China). Five-week-old male Sprague-

Dawley rats purchased from Qinglongshan Animal Breeding Base

(SCXK2017−001) were used for this study and were fed under

controlled environmental conditions of a 12 h light/dark cycle and

maintained on standard food pellets and tap water. A week later, 10

animals were randomly selected as the normal group and exposed to

fresh air only. The rest of the animals were exposed to cigarette smoke

from day 2 to day 42 (6 days per week) and tracheal infusion of LPS

on day 1 and day 20, respectively. After 42 days, the animals were

selected and divided (10 animals per group) and administered with

diﬀerent doses of aerosolized compound (S)-18 by inhalation once a

day for 28 days. The vehicle group (10 animals) was treated with an

aerosolized equal volume of vehicle solution every day for 28 days.

After the end of the treatments, the rats were sacriﬁced by

anesthesia and autopsied. Lung tissue was removed, ﬁxed in 4%

PI3K Isoform ADP-Glo Assays. The ADP-Glo kinase assay

(Promega) was used to screen the compounds for their phosphoinosi-

tide kinase inhibition activities. Kinase reaction systems contain 3 μL

of PI3Kα (0.24 μg/mL), PI3Kβ (3 μg/mL), PI3Kδ (3.3 μg/mL), or

PI3Kγ (12 μg/mL) (Invitrogen), 3 μL of serially diluted compound,

and 6 μL of substrate PIP2:PS (0.1 mM) (Invitrogen) with 100 μM

ATP (200 μM ATP for PI3Kβ). Reactions were started immediately

by adding ATP and continued for an hour at 37 °C. After the

reaction, tubes were cooled for 5 min at room temperature, and 5 μL

of the reaction solution was transferred to a 384-well plate. Then, 5

μL of ADP-Glo reagent was added into each well to stop the reaction

and consume the remaining ADP within 40 minutes. At the end, 10

μL of the kinase detection reagent was added into each well and

incubated for 30 min to produce a luminescence signal, which was

measured with an automated plate reader (Perkin-Elmer Envision),

and each measurement was performed in triplicates.

PI3K Isoform Cellular Selectivity Assays. NIH-3T3, RAW

264.7, and Raji cells were treated with DMSO, serially diluted

compound (S)-18, and 1 μM compound 4 for 1 h, after which the

cells were treated with and without diﬀerent stimulating factors. All

cells were then washed with 1×PBS buﬀer and lysed in cell lysis buﬀer

at 4 °C for 30 min. The lysates were cleared by centrifugation, and the

protein concentrations were measured by BCA analysis (Beyotime,

China). Then, 50 μg of total protein lysates was mixed with 5×

loading buﬀer and heated in a metal bath for 10 min at 100 °C. The

cell lysates were analyzed by Western blotting with the following

antibodies purchased from Cell Signaling Technology (Danvers,

MA): P-AKT Thr308 (#2965S), P-AKT Ser473 (#4060S), and total

AKT (#4691S). The antibodies were used at 1:1000.

CYP Inhibition Assay. The CYP enzyme assays were performed

according to P450-Glo Assay instructions (Promega). Brieﬂy, assays

were assembled and performed in opaque white 96-well plates.

Compound (S)-18 at 10 diﬀerent ﬁnal concentrations descending 3-

fold from 10 μmol/L was added into the plates. After incubation for

20 or 30 min at 37 °C or room temperature (20−23 °C), 50 μL of the

luciferin detection reagent was added to each 50 μL CYP reaction to

stop the reactions and initiate luminescence, which was measured 20

min later using the Bio-Rad Microplate reader (Bio-Rad). Data were

normalized to control groups (DMSO), and IC₅₀values were

calculated using Prism 7.0 (GraphPad Software, San Diego, CA).

́

Patch-Clamp Functional hERG Assay. The human ether-a-go-

go-related gene (hERG) assay was provided by PharmaCore Labs

(Nantong, China). In short, the assay was performed with a stable

Kv11.1 transfected human embryonic kidney cell line (HEK293) by

whole-cell patch clamp carried out at room temperature. The Kv11.1

(hERG) ion channel blocker quinidine was used as the reference

compound, and the eﬀects of compound (S)-18 and quinidine were

normalized to the corresponding vehicle control. Whole-cell record-

ings were carried out with the patch-clamp device (HEKA

Instruments Inc., D-67466 Lambrecht, Pfalz, Germany) with

commercial ampliﬁers (EPC10, HEKA Elektronik Dr. Schulze

GmbH, Germany). Patch-clamp measurements were run on silicate-

coated chips with a hole of a deﬁned diameter. GI₅₀values were

calculated using Origin 8.5 (OriginLab Corporation, Northampton,

MA).

Pharmacokinetic Study. This study protocol was approved by

the Animal Ethics Committee of Hefei Institutes of Physical Science,

Chinese Academy of Sciences (Hefei, China). The pharmacokinetic

study was performed on male Sprague-Dawley (SD) rats. All of the

rats were allowed to acclimatize to the research facilities for one week

followed by fasting for 12 h prior to drug administration. (S)-18 was

formulated as a solution in DMSO/5% of glucose solution (70:30, v/

v) at a concentration of 2 mg/mL (intravenous) or a solution in

DMSO/0.5% of CMC-Na (4:96, v/v) at a concentration of 2 mg/mL

(oral). For inhaled formulation, gel-like HP-β-CD was ﬁrst prepared

by mixing with ultrapure water (1:2−1:3, m/v), followed by further

R

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Article

glutaraldehyde, and embedded in paraﬃn. Sections were then cut at 5

μm, stained with HE, and imaged using a Leica light microscope

(DM500, Germany) for histological analysis. Apoptosis detection in

the lung tissue was carried out using a Promega TUNEL staining kit

(Madison, Wisconsin) according to the manufacturer’s instructions.

Sections were prepared and then imaged using a Leica light

microscope (DM500, Germany) to identify the TUNEL-positive cells.

Molecular Docking. Docking simulations were performed using

the Induced Fit Docking (IFD) protocol in Schrodinger Suite 2017.

Prior to docking, the PI3K-delta receptor structure (PDB ID: 5M6U)

was prepared using the Protein Preparation Wizard with the OPLS3

force ﬁeld. The ligands were prepared using LigPrep.

P. R. China; Hefei Cancer Hospital, Chinese Academy of

Sciences, Hefei, Anhui 230031, P. R. China; orcid.org/

0000-0001-8722-694X

Zongru Jiang − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; Hefei Cancer Hospital, Chinese Academy of

Sciences, Hefei, Anhui 230031, P. R. China

Aoli Wang − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; Hefei Cancer Hospital, Chinese Academy of

Sciences, Hefei, Anhui 230031, P. R. China

Junjie Wang − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; University of Science and Technology of China,

Hefei, Anhui 230026, P. R. China

Cheng Chen − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; University of Science and Technology of China,

Hefei, Anhui 230026, P. R. China

Wenliang Wang − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; University of Science and Technology of China,

Hefei, Anhui 230026, P. R. China

Fengming Zou − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; Hefei Cancer Hospital, Chinese Academy of

Sciences, Hefei, Anhui 230031, P. R. China

Ziping Qi − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; Hefei Cancer Hospital, Chinese Academy of

Sciences, Hefei, Anhui 230031, P. R. China

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01544.

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sı

KINOMEScan proﬁling data of (S)-18, in vivo eﬃcacy

evaluation of (S)-18 in a COPD rat model, NMR

spectra of newly synthesized compounds, and HPLC

traces of ﬁnal compounds (PDF)

Molecular formula strings (CSV)

Docking pose for (S)-18 with PI3Kδ kinase (PDB)

AUTHOR INFORMATION

Corresponding Authors

■

Jing Liu − Anhui Province Key Laboratory of Medical Physics

and Technology; CAS Key Laboratory of High Magnetic

Field and Ion Beam Physical Biology; Institute of Health and

Medical Technology, Hefei Institutes of Physical Science,

Chinese Academy of Sciences, Hefei, Anhui 230031, P. R.

China; Hefei Cancer Hospital, Chinese Academy of Sciences,

Hefei, Anhui 230031, P. R. China; Precision Medicine

Research Laboratory of Anhui Province, Hefei, Anhui

230088, P. R. China; orcid.org/0000-0001-9513-3591;

Phone: 86-551-65593186; Email: jingliu@hm ﬂ .ac.cn

Qingsong Liu − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; University of Science and Technology of China,

Hefei, Anhui 230026, P. R. China; Hefei Cancer Hospital,

Chinese Academy of Sciences, Hefei, Anhui 230031, P. R.

China; Precision Medicine Research Laboratory of Anhui

Province, Hefei, Anhui 230088, P. R. China; Institute of

Physical Science and Information Technology, Anhui

University, Hefei, Anhui 230601, P. R. China; orcid.org/

0000-0002-7829-2547; Phone: 86-551-65596201;

Email: qsliu97@hm ﬂ .ac.cn

Authors

Feng Li − Anhui Province Key Laboratory of Medical Physics

and Technology; CAS Key Laboratory of High Magnetic

Field and Ion Beam Physical Biology; Institute of Health and

Medical Technology, Hefei Institutes of Physical Science,

Chinese Academy of Sciences, Hefei, Anhui 230031, P. R.

China; University of Science and Technology of China, Hefei,

Anhui 230026, P. R. China

Xiaofei Liang − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

Qingwang Liu − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; Hefei Cancer Hospital, Chinese Academy of

Sciences, Hefei, Anhui 230031, P. R. China

Zhenquan Hu − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

S

https://dx.doi.org/10.1021/acs.jmedchem.0c01544

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China

N,N-dimethylformamide; HATU, 1-[bis(dimethylamino)-

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-

ﬂuorophosphate; HE, hematoxylin-eosin; HMDS, hexamethyl-

disilazane; LPS, lipopolysaccharide; PI3K, phosphatidylinositol

3 kinase; SAR, structure−activity relationship; THF, tetrahy-

drofuran; TUNEL, terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labeling

Jiangyan Cao − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; University of Science and Technology of China,

Hefei, Anhui 230026, P. R. China

Hong Wu − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; Hefei Cancer Hospital, Chinese Academy of

Sciences, Hefei, Anhui 230031, P. R. China

Beilei Wang − Anhui Province Key Laboratory of Medical

Physics and Technology; CAS Key Laboratory of High

Magnetic Field and Ion Beam Physical Biology; Institute of

Health and Medical Technology, Hefei Institutes of Physical

Science, Chinese Academy of Sciences, Hefei, Anhui 230031,

P. R. China; Hefei Cancer Hospital, Chinese Academy of

Sciences, Hefei, Anhui 230031, P. R. China

Li Wang − Anhui Province Key Laboratory of Medical Physics

and Technology; CAS Key Laboratory of High Magnetic

Field and Ion Beam Physical Biology; Institute of Health and

Medical Technology, Hefei Institutes of Physical Science,

Chinese Academy of Sciences, Hefei, Anhui 230031, P. R.

China; Hefei Cancer Hospital, Chinese Academy of Sciences,

Hefei, Anhui 230031, P. R. China

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01544

Author Contributions

^#F.L., X.L., Z.J., and A.W. contributed equally to this work. The

manuscript was written through contributions of all authors.

All authors have given approval to the ﬁnal version of the

manuscript.

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Notes

The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

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This work was supported by the National Natural Science

Foundation of China (Grant Nos. 81872745, 81803366,

81703559, 81773777, and 81872748), the National Science

& Technology Major Project “Key New Drug Creation and

Manufacturing Program” of China (Grant No.

2018ZX09711002), the National Key Research and Develop-

ment Program of China (Grant No. 2016YFA0400900), the

Natural Science Foundation of Anhui Province (Grant Nos.

1808085MH268, 1808085MH274, and 1908085QH348), the

Postdoctoral Science Foundation of Anhui Province (Grant

No. 2019B326), the Frontier Science Key Research Program of

CAS (Grant No. QYZDB-SSW-SLH037), the CASHIPS

Director’s Fund (Grant No. BJPY2019A03), and the

University Synergy Innovation Program of Anhui Province

(Grant No. GXXT-2019−045). We are also grateful for the

support of Hefei leading talent for F.Z.

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ABBREVIATIONS USED

COPD, chronic obstructive pulmonary disease; DCM,

dimethyl chloride; DIPEA, N,N-diisopropylethylamine; DMF,

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J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of (S)-2-(1-(4-Amino-3-(3-fluoro-4-methoxyphenyl)-1 H-pyrazolo[3,4- d]pyrimidin-1-yl)propyl)-3-cyclopropyl-5-fluoroquinazolin-4(3 H)-one (IHMT-PI3Kδ-372) as a Potent and Selective PI3KδInhibitor for the Treatment of Chronic Obstructive Pulmonary Disease

DOI: 10.1021/acs.jmedchem.0c01544

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Article abstract of DOI:10.1021/acs.jmedchem.0c01544

Full text of DOI:10.1021/acs.jmedchem.0c01544

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