Synthesis and biological evaluation of 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine derivatives as novel PI3Kδ inhibitors

Article abstract of DOI:10.1016/j.cclet.2015.05.041

An efficient synthesis of novel 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine scaffold has been designed and deveopled. A series of 5-phenylurea derivatives was synthesized using this method. Their cytotoxic activities against breast cancer cell line BT-474 were evaluated by CCK-8 assay. Most of them showed potent anti-proliferative activities, of which compound 20 and 21 exhibited IC₅₀s of 1.565 μmol/L and 1.311 μmol/L, respectively. Furthermore, compound 20 and 21 also showed potent inhibitory activities against PI3Kδ with IC₅₀s of 0.286 μmol/L and 0.452 μmol/L, respectively. These results indicate that these 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine derivatives are novel antitumor agents through the inhibition of PI3Kδ.

Full text of DOI:10.1016/j.cclet.2015.05.041

Accepted Manuscript
Title: Synthesis and biological evaluation of
3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine derivatives as
novel PI3Kδ inhibitors
Author: Jia-Lin Guo Yun-Yong Liu Ya-Zhong Pei
PII:
S1001-8417(15)00232-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2015.05.041
CCLET 3345
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
1-2-2015
1-4-2015
20-5-2015
Please cite this article as: <doi>http://dx.doi.org/10.1016/j.cclet.2015.05.041</doi>
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Graphical Abstract
Synthesis and biological evaluation of 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine derivatives as novel PI3Kδ inhibitors
Jia-Lin Guoa, Yun-Yong Liub, Ya-Zhong Peia,*
a The Center for Combinatorial Chemistry and Drug Discovery of Jilin University, School of Pharmaceutical Sciences, Jilin
University, Changchun 130021, China
b Nanjing Gator MediTech Company, Ltd., Nanjing 211300, China
O
N
IC50 (µmol/L)
O
N
Compd.
R
PI3K d
BT-474
N
N
O
20
21
-CH2CH2F
-CH3
0.286
0.452
1.565
1.311
R
N
H
N
H
N
Bn
An efficient synthesis of 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine derivatives has been designed and developed. A series of novel PI3Kδ
inhibitors with potent antitumor activities have been identified.
Page 1 of 8

Original article
Synthesis and biological evaluation of 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine
derivatives as novel PI3Kδ inhibitors
Jia-Lin Guoa, Yun-Yong Liub, Ya-Zhong Peia,
a The Center for Combinatorial Chemistry and Drug Discovery of Jilin University, School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China
b Nanjing Gator MediTech Company, Ltd., Nanjing 211300,. China
ARTICLE INFO
ABSTRACT
Article history:
An efficient synthesis of novel 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine scaffold has been
designed and deveopled. A series of 5-phenylurea derivatives was synthesized using this
method. Their cytotoxic activities against breast cancer cell line BT-474 were evaluated by
CCK-8 assay. Most of them showed potent anti-proliferative activities, of which compound 20
and 21 exhibited IC50s of 1.565 µmol/L and 1.311 µmol/L, respectively. Furthermore,
Received 1 February 2015
Received in revised form 1 April 2015
Accepted 20 May 2015
Available online
δ
compound 20 and 21 also showed potent inhibitory activities against PI3K with IC50s of 0.286
µmol/L and 0.452 µmol/L, respectively. These results indicate that these 3-(piperidin-4-
Keywords:
yl)isoxazolo[4,5-d]pyrimidine derivatives are novel antitumor agents through the inhibition of
Synthesis
δ
PI3K .
Isoxazolopyrimidine
PI3Kδ inhibitors
Cytotoxicity
1. Introduction
Phosphoinositide-3-kinases (PI3Ks) are members of lipid kinase family that phosphorylate inositol lipids at the 3-hydroxy group [1].
They play key regulatory roles in many cellular processes, including cell growth, survival, proliferation, motility, metabolism, and
differentiation [2-4]. They are the central components of the PI3K/Akt/mTOR signalling pathway and activated by receptor tyrosine
kinases (RTKs) and G-protein coupled receptors (GPCRs). The activated PI3Ks transduce signals from various growth factors and
cytokines into intracellular messages by generating phospholipids. As the key second messengers, these phospholipids activate the
serine/threonine protein kinase Akt and other downstream signaling pathways, promoting cell growth and proliferation [2]. PI3Ks can be
divided into three classes according to their structures, substrates and functions, including class I (IA and IB), II, and III. The class I are
heterodimers composed of a regulatory subunit and a catalytic subunit. The class IA consists of p85 and p110 (α,
class IB is p101 and p110 . The class II consists of a monomeric catalytic subunit, including three isoforms (PI3KC2
PI3KC2 ). The class III consists of a single heterodimer of a catalytic (Vps34) and a regulatory (Vsp15) subunit [5,6]. Aberrations of
PI3K signaling are found in various tumors, including breast, prostate, colon, liver, and pancreatic cancers. The most frequently observed
abnormalities are the loss or attenuation of PTEN function and mutations of the gene that encodes p110 [7]. The overexpression of
p110 , and has also been implicated in various forms of human tumors [4]. In addition, PI3K pathway activations also lead to
resistance to current cytotoxic agents as well as targeted anticancer therapies [8]. Idelalisib (CAL-101 or GS-1101), a PI3K inhibitor, is
β
and
δ
), whereas the
γ
α
, PI3KC2 and
β
γ
α
β
,
δ
γ
δ
the first in its class that has gained the approvals of the FDA and EMA for the treatment of relapsed chronic lymphocytic leukemia (CLL)
in July 2014. A number of other PI3K inhibitors are currently being studied in clinics, including GDC-0941, XL-147, BKM-120 and
BEZ235 (Fig. 1) (http://ClinicalTrials.gov, [4]). These data indicate that regulating the activities of PI3Ks is an effective strategy for
anticancer therapy.
S
N
N
O
N
O
N
F
O
N
NC
N
S
CF3
N
N
O
N
N
NH
N
N
N
NH
N
N
N
HN
N
N
N
N
NH
O S O
O
N
H2N
N
O
N
N
S
NH
O
BEZ235
CAL-101
GDC-0941
XL147
BKM-120
———
Corresponding author.
E-mail address: peiyz@jlu.edu.cn (Y.-Z. Pei).
Page 2 of 8

Fig. 1. Reported PI3K inhibitors.
PI3K inhibitors from several structural classes, such as GDC-0941, BKM-120, and PKI-402 [9] contain a common 2,4-disubstituted
pyrimidine fragment with 4-morpholino group at the 4-position. Their binding data indicated that 2- and 4-substituents could pick up key
hydrogen bond interactions with PI3Ks, whereas the pyrimidine ring mainly serve as a scaffold [9-11]. Based on this hypothesis, we
designed the novel 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidines (Fig. 2). This bicyclic template allows us to maintain the 2,4-
disubstituented pyrimidine structure while investigating the effect of the fused 3-(piperidin-4-yl)isoxazole. This report describes the
syntheses of a series of novel 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine derivatives. Their anti-proliferative activities were evaluated
in a cellular CCK-8 assay against breast cancer cell line BT-474. The lead compounds, 20 and 21, were found to be potent inhibitors of
PI3Kδ.
O
PKI-402
IC
N
N
50
PI3K
a
nmol/L
1
7
N
N
N
O
N
b
g
O
N
O
N
16
14
N
N
N
d
H
H
GDC-0941
IC
S
N
NH
50
N
PI3K
nmol/L
3
N
a
N
O
O
N
b
g
33
75
3
N
O
N
N
N
S
d
O
O
N
N
O
N
O
O
N
1
R
R
N
N
N
N
BKM-120
IC
H
H
H
H
N
2
50
12-23
R
PI3K
a
nmol/L
52
CF
N
3
N
N
b
g
166
O
262
116
H N
N
2
d
Fig. 2. Design considerations.
2. Experimental
2.1 Syntheses
The syntheses of compounds 2-21 are outlined in Scheme 1. Details of reaction conditions and characterization data related to
compounds 2-11 are provided in the Supplementary data [12-19].
To a solution of N1-(2-(dimethylamino)ethyl)-N1-methylbenzene-1,4-diamine (116 mg, 0.6 mmol) and TEA (182 mg, 1.8 mmol) in
anhydrous DCM (4 mL) at -18 oC was added dropwise a solution of triphosgene (89 mg, 0.3 mmol) in anhydrous DCM (2 mL). After 15
min., a solution of compound 11 (94 mg, 0.2 mmol) in anhydrous DCM (2 mL) was added. The mixture was stirred overnight and
allowed to warm to room temperature. The reaction mixture was diluted with water (5 mL) and extracted with DCM (5 mL × 3). The
combined organic layers were subsequently washed with H2O (8 mL×3) and brine (8 mL×3), dried over anhydrous Na2SO4 and
concentrated under vacuum. The crude product was purified on a silica gel column (DCM/MeOH/TEA = 60/1/1, v/v/v) to give compound
12 (50 mg, 36% yield) as a yellow solid.
General procedure for compounds 13-21: To a solution of compound 11 (94 mg, 0.2 mmol) and TEA (61 mg, 0.6 mmol) in anhydrous
o
DCM (2 mL) at -18 C was added dropwise a solution of triphosgene (30 mg, 0.1 mmol) in anhydrous DCM (2 mL). After 15 min., a
solution of amine (5 eq.) in anhydrous DCM (2 mL) or amine hydrochloride (5 eq.) and TEA (6 eq.) was added. The mixture was stirred
overnight and allowed to warm to room temperature. The reaction mixture was diluted with water (5 mL) and extracted with DCM (5 mL
× 3). The combined organic layers were subsequently washed with H2O (6 mL×3) and brine (6 mL×3), dried over anhydrous Na2SO4 and
concentrated under vacuum. The crude product was purified on a silica gel column or preparative thin-layer chromatography to give
compounds 13-21 [20]. All data of compounds 12-21 are summarized in the Supplementary data.
Page 3 of 8

O
O
COOEt
NO2
CONH2
O
O
N
CONH2
O
CONH2
NO2
COOH
N
N
NO2
NO2
HO
N
N
a
b
c
e
d
f
NO2
NO2
N
Boc
1
N
Boc
2
N
Boc
3
N
Boc
N
Boc
HN
N
Bn
6
7
5
4
g
O
N
O
N
O
N
O
O
CONH2
NH2
O
O
O
N
O
N
NH
N
N
N
N
N
N
k
j
l
h
N
H
O
N
Cl
N
N
O
R1
NH2
N
H
N
H
N
Bn
N
Bn
N
Bn
N
Bn
N
Bn
11
9
8
12-21
10
Scheme 1. Syntheses of compounds 2–21. Reagents and conditions: (a) (1) CDI, anhydrous THF, N2, r.t., 1-2 h; (2) DBU, nitromethane, r.t., 36 h, 96%. (b)
o
hydroxylamine hydrochloride, NaHCO3, EtOH, 50 C, overnight, 95%. (c) (1) ethyl 2-chloro-2-oxoacetate, anhydrous ether, r.t., 24 h; (2) TEA, 0 °C-r.t., 60 h,
58%. (d) NH3/MeOH, r.t., 3 h, 96%. (e) TFA, DCM, r.t., 2 h, 100% (TFA salt). (f) BnBr, DIPEA, anhydrous MeCN, 0 °C-r.t., 6 h, 81%. (g) Zn, NH4Cl,
o
EtOH/water = 2/1, 0 C-r.t., 4 h, 78%. (h) triphosgene, anhydrous THF, reflux, 1 h, 90% (hydrochloride salt). (i) (1) POCl3, reflux, 24 h; (2) morpholine, TEA,
o
DCM, -18 oC, 10 min., 62%. (j) 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, Pd(Pcy3)2Cl2, CsF, NMP/water = 9/1, Ar, 100 C, 48 h, 34%. (k) (1) TEA,
triphosgene, anhydrous DCM, -18 oC, 15 min; (2) amine or amine hydrochloride and TEA, -18 oC-r.t., overnight, 30%-56%.
The syntheses of compounds 22 and 23 are outlined in Scheme 2. To a solution of compound 11 (471 mg, 1 mmol) and TEA (126 mg,
1.25 mmol) in anhydrous DCM (4 mL) was slowly added a solution of methylcarbamic chloride (103 mg, 1.1 mmol) in anhydrous DCM
(3 mL). The mixture was then refluxed for 3 days. The reaction mixture was diluted with water (5 mL) and extracted with DCM (8
mL×3). The combined organic layers were subsequently washed with H2O (10 mL×3) and brine (10 mL×3), dried over anhydrous
Na2SO4 and concentrated in vacuo. The crude product was purified on a silica gel column (DCM/MeOH = 20/1, v/v) to give compound
21 (447 mg, 71% yield) [21].
To a solution of compound 21 (1.342 g, 2.54 mmol) in DCE (40 mL) was added ACE-Cl (2.910 g, 20.35 mmol) and stirred overnight
at room temperature. The mixture was concentrated to dryness. The residue was refluxed in MeOH (20 mL) for 2 h and concentrated
under vacuum to give the crude 1-methyl-3-(4-(7-morpholino-3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidin-5-yl)phenyl)urea
hydrochloride (1.218 g) [22]. This crude intermediate was suspended in DCM (50 mL) at 0 oC. TEA (936 mg, 9.25 mmol) and a solution
of (Boc)2O (618 mg, 2.83 mmol) in anhydrous DCM (10 mL) was then added. The mixture was stirred for 3 h, diluted with water (20 mL)
and extracted with DCM (20 mL×3). The combined organic layers were washed with H2O (25 mL×3) and brine (25 mL×3), dried over
anhydrous Na2SO4, and concentrated in vacuo. The crude product was purified on a silica gel column (DCM/MeOH = 50/1, v/v) to give
tert-butyl-4-(5-(4-(3-methylureido)phenyl)-7-morpholinoisoxazolo[4,5-d]pyrimidin-3-yl)piperidine-1-carboxylate (1.037 g, 76% yield)
as a white solid. The solution of the above intermediate (330 mg, 0.61 mmol) in DCM (1 mL) and TFA (3 mL) was stirred for 2 h at
room temperature. The mixture was concentrated to dryness in vacuo to give compound 22 as its di-TFA salt (405 mg, 99% yield) as a
yellow solid, which was used in the next step without further purification. Note: Compound 22 was very difficult to purify “as is”. The
crystallization of its hydrochloride salt did not furnish a product with satisfactory yield and purity. The t-Boc-protection/deprotection
steps were for the ease of purification only.
o
To a suspension of compound 22 di-TFA salt (100 mg, 0.15 mmol) and DIPEA (71 mg, 0.55 mmol) in MeCN (3 mL) at -18 C was
added iodoethane (29 mg, 0.18 mmol). The mixture was stirred for 1 h at the same temperature followed by stirring at room temperature
o
for 5 h. TLC showed that reaction was not complete. The mixture was stirred at 65 C for 11 h and followed by refluxing for 8 h. The
reaction mixture was concentrated in vacuo and diluted with water (3 mL). The aqueous layer was adjusted to pH 14 using 6 mol/L
NaOH(aq.) and extracted with DCM/MeOH = 20/1 (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4. The
crude product was purified on a silica gel column (DCM/MeOH/TEA = 55/1/1, v/v/v) to give compound 23 (48 mg, 69% yield) as a
yellow solid [16,23]. All analytical data of compounds 22 and 23 were summarized in the Supplementary data. A small amount of
quaternary salt was also observed during the reaction by LC-MS. It was easily removed during the work-up and purification.
O
N
O
N
O
N
O
N
O
O
N
O
a
O
b
c
N
N
N
N
N
N
N
N
O
N
O
N
O
N
N
H
N
H
N
N
N
N
NH2
H
H
H
H
N
H
N
N
N
Bn
Bn
11
21
23
22
Page 4 of 8

Scheme 2. Syntheses of compounds 22 and 23. Reagents and conditions: (a) methylcarbamic chloride, TEA, anhydrous DCM, reflux, 3 d, 71%. (b) (1) ACE-Cl,
DCE, r.t., overnight; (2) MeOH, reflux, 2 h; (3) (Boc)2O, TEA, DCM, 0 oC, 3 h, 76%; (4) TFA, DCM, r.t., 2 h, 99% (di-TFA salt). (c) iodoethane, DIPEA, MeCN,
-18 °C-reflux, 69%.
2.2 Biological evaluation
The anti-proliferative activities of compounds 11–23 were assessed against BT-474 cells (human breast ductal carcinoma) by CCK-8
assay. BT-474 cells were plated in 96-well flat-bottomed microtiter plates (cells suspended in 100 µL culture medium per well) and
incubated at 37 oC for 24 h under a 5% CO2 and 100% relative humidity atmosphere. A 25 µL aliquot of culture medium containing the
tested compound was added to the wells. The plates were incubated for an additional 72 h. After removal of culture medium, fresh
culture medium containing 10% CCK-8 was added and incubated at 37 oC for 2-4 h. The absorbance was measured on a SpectraMax M5
Microplate Reader at 450 nm. The percentage of inhibition at each compound concentration was calculated according to formula: The
percentage of inhibition = (Asample-Anegative)/(Ablank-Anegative)×100%. Compounds were studied for dose-response relationship at 100 µmol/L,
25 µmol/L, 6.25 µmol/L, 1.563 µmol/L, 0.391 µmol/L, 0.098 µmol/L, 0.024 µmol/L, 0.006 µmol/L, 0.0015 µmol/L, 0.0004 µmol/L.
Their IC50s were calculated using GraphPad Prism 5.
PI3K
used as a positive control. Typically, 5 µL of ATP solution (40 µmol/L) was added into a mixture of 10 µL of PIP2 solution (20 µmol/L)
containing PI3K enzyme (80 ng) and 5 µL of test compound solution. The negative control and blank control were composed of the
same mixed solutions except replacing test compound with DMSO. The blank control did not contain PI3K enzyme. After a 30 min.
δ
kinase activity was determined using a PI3-Kinase (human) HTRFTM Assay kit in 384 well opaque black plates. CAL-101 was
δ
δ
incubation at room temperature, the reaction was stopped by the addition of 5 µL of stop buffer (stop A/stop B = 3/1) followed by
detection buffer (5 µL, DMC/DMA/DMB = 18/1/1). After 1 h incubation at room temperature, emission signal was measured on
EnVision® Multilabel Reader. Emission Ratio (ER) of each well was calculated according to the formula: Emission Ratio (ER) = 665 nm
Emission signal/620 nm Emission signal. The percentage of inhibition at each compound concentration was calculated according to
formula: The percentage of inhibition = (ERsample-ERnegative)/(ERblank-ERnegative)×100%. Compounds were studied for dose-response
relationship at 100 µmol/L, 25 µmol/L, 6.25 µmol/L, 1.563 µmol/L, 0.391 µmol/L, 0.098 µmol/L, 0.024 µmol/L, 0.006 µmol/L, 0.0015
µmol/L, 0.0004 µmol/L. Their IC50s were calculated using GraphPad Prism 5.
3. Results and discussion
3.1 Chemistry
A facile synthesis of novel 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine scaffold was developed, starting from N-Boc-piperidine-4-
carboxylic acid (Scheme 1). Using this route, fifteen derivatives were synthesized for their anti-proliferative activity evaluation and initial
structure activity relationship development.
We at first attempted a two-step process for the synthesis of intermediate 26 as shown in Scheme 3A, which has the potential of
avoiding the exchange of N-substitution on piperidine from Boc- to benzyl. Condensation of aldehyde 24 and nitromethane in the
presence of KF worked well, giving the alcohol 25 in 96% yield. Oxidation of 25, under both Dess-Martin periodinane and Swern
conditions, gave nitro olefin 27 in nearly quantitative yield (86% and 81%, respectively) instead of the α-nitroketone 26. These N-benzyl
piperidine derivatives were also difficult to purify due to their good aqueous solubility. For the ease of intermediate isolation, we
therefore decided to use the N-Boc-piperidines during the early part of the syntheses. Details of reaction conditions and characterization
data related to compounds 24, 25 and 27 are summarized in the Supplementary data.
O
NO2
HO
N
Bn 26
CHO
NO2
a
b or c
A)
NO2
N
Bn
24
N
Bn
25
N
Bn
27
f or g
O
OPh
B)
d
e
1
2
N
Boc
28
Scheme 3. Syntheses of α-nitroketone 2. Reagents and conditions: (a) nitromethane, KF, 2-propanol, r.t., overnight, 96%. (b) Dess-Martin periodinane, anhydrous
DCM, r.t., 35 min., 86%. (c) (1) oxalyl chloride, DCM, -78 oC, DMSO, 15 min.; (2) compound 25, DCM, -78 oC, 1 h; (3) TEA, -78 oC, 15 min., r.t., 1 h, 81%. (d)
(1) (COCl)2, DMF, anhydrous DCM, 0 oC, 2 h; (2) PhOH, TEA, anhydrous DCM, 0 oC, 3 h, 43%. (e) nitromethane, t-BuOK, DMSO, r.t., 8 h, 77%. (f) (1) CDI,
anhydrous THF, N2, reflux, 1 h; (2) nitromethane, t-BuOK, reflux, 24 h, 75%. (g) CDI, anhydrous THF, N2, r.t., 1-2 h; (2) nitromethane, DBU, N2, r.t., 36 h, 96%.
Page 5 of 8

As shown in Scheme 3B, key intermediate 2 can be obtained from N-Boc-piperidine-4-carboxylic acid (1) in a two-step process in
moderate overall yield. The condensation of phenol ester 28 with nitromethane gave 2 in high yield (77%). However, the conversion of 1
to 28 in the presence of oxalyl chloride was only 43%. Alternatively, 1 was first activated with CDI followed by addition of t-BuOK and
nitromethane in anhydrous THF to give 2 in high overall yield (75%) in a one-pot procedure. Using DBU instead of t-BuOK as base, 2
was obtained in excellent yield (96%). This optimized condition was used in the preparation of 3-(piperidin-4-yl)isoxazolo[4,5-
d]pyrimidine derivatives, as shown in Scheme 1. Details of reaction conditions and characterization data of compound 28 are
summarized in the Supplementary data.
Under reflux, the reaction of compound 2 with hydroxylamine hydrochloride in EtOH did not yield 3, but the t-Boc deprotection
product of 3. Most likely, the deprotection was due to the in situ generated hydrochloride during the condensation. When the reaction was
carried out in the presence of NaHCO3, 3 was obtained in 62% yield. We also found this reaction was highly temperature dependent. At
o
o
60 C and 50 C, 3 was isolated in 77% and 95% yield, respectively. Notably, intermediate 3 was an inseparable mixture of cis- and
trans-isomers (cis/trans = 6/1 by 1H NMR) and used “as is” in the next step. In a one-pot procedure, 3 was first condensed with 2-chloro-
2-oxoacetate in the presence of TEA. Complete disappearance of 3 was observed by TLC after 24 h, suggesting both cis- and trans-3
reacted with 2-chloro-2-oxoacetate. Only the intermediate derived from the cis-isomer cyclized to furnish isoxazole 4. No inter-
conversion between cis- and trans-isomers was observed at any stage of the reaction.
As shown in Scheme 4, intermediate 4 was reduced to 29. Direct cyclization of 29 with urea at high temperature failed to yield any
appreciable amount of 30 due to charring. Intermediate 4 was converted to amide 5 by aminolysis using NH3 in methanol. Intermediate 5
was reduced to 31 in high yield (90%). Reaction of 31 with triphosgene gave 32 as its hydrochloride salt in high yield (85%). Treatment
of 32 with POCl3 and N,N-dimethylaniline under reflux gave 33 cleanly, in which the N-Boc group was removed during the reaction
(confirmed by LC-MS). Compound 33 exhibited excellent aqueous solubility and could not be isolated during workup. Based on these
results, we decided to exchange the N-substituent on the piperidine from t-Boc- to benzyl (5 to 7 via 6) as shown in Scheme 1. Details of
reaction conditions and characterization data related to compounds 29, 31, and 32 are summarized in the Supplementary data.
O
COOEt
NO2
COOEt
NH2
O
N
O
N
O
N
NH
b
a
N
H
O
4
29
30
N
Boc
N
Boc
N
Boc
c
O
Cl
N
CONH2
NH2
CONH2
NO2
O
N
O
N
O
N
O
N
N
NH
e
a
d
Cl
N
H
O
5
32
31
33
N
Boc
N
N
Boc
N
H
H
Scheme 4. Constructing the fused pyrimidine. Reagents and conditions: (a) Zn, NH4Cl, EtOH/water = 1/1, 0 oC-r.t., 3 h, compound 29 (79% yield), compound 31
(90% yield). (b) urea, 210 oC. (c) NH3/MeOH, r.t., 3 h, 96%. (d) triphosgene, anhydrous dioxane, reflux, 1 h, 85% (hydrochloride salt). (e) N,N-dimethylaniline,
POCl3, reflux, 8 h.
The transformation of 10 to 11 using Suzuki coupling was a problematic step. We examined the commonly used catalysts, such as
Pd(PPh3)4, Pd(OAc)2, PdCl2(dppf), and Pd2(dba)3, but all failed to produce product 11. Pd(OAc)2 in combination with different phosphine
ligands, such as 2-(di-tert-butylphosphino)biphenyl, butyldi-1-adamantylphosphine, and 2-(dicyclohexylphosphino)biphenyl, also did not
yield any desired product. The starting material was recovered in all cases. Wang Shen [19] reported Pd(PCy3)2Cl2 was an effective
catalyst for the activation of the aryl chlorides in the Suzuki reaction. He speculated that the electron-rich nature of PCy3 might enhance
the oxidative insertion of palladium into the Ar-C1 bond and that the increased steric encumbrance of the ligand also might facilitate the
dissociation of the ligands from the palladium complex. Using Pd(PCy3)2Cl2 as the catalyst in the presence of CsF at 100 oC for 48 hours
(Scheme 1), 11 was obtained in 34% yield with 21% 10 recovered from the reaction mixture. Some isoxazole ring-opening product via
the reductive cleavage of the N-O bond was also observed. Further optimization, such as prolonging reaction time, higher reaction
temperature and various bases (K3PO4, K2CO3 and KF) did not improve the yield. Higher reaction temperature and/or longer reaction
time all led to decreased yield due to greater extent of the isoxazole ring-opening reaction.
3.2 Biological evaluation
Cytotoxicity of compounds 11-23 against BT-474 cells was evaluated by CCK-8 assay. As summarized in Table 1, compared to other
derivatives, 11 showed lower toxicity to BT-474 cells, indicating the urea moiety on the phenyl ring was required for activity. While
keeping R2 constant (R2 = benzyl), various substituent on the urea (R1) were examined. With large aromatic groups, 12, 13, and 14
showed substantial cytotoxicities, with IC50s in the low to mid single digit micromolar range. In comparison, most alkyl derivatives,
including the unsubstituted derivative 15, showed greater potency, with IC50s in the low single digit micromolar range, except the
branched alkyl (17, R1 = -CH(CH3)2, IC50 = 8.148 µmol/L). While keeping R1 = CH3, we also briefly investigated the effects of N-
Page 6 of 8

substituents on the piperidine ring. N-ethyl substitution (23) and unsubstituted piperidine (22) all yield potent inhibitors, with IC50s in the
low single digit micromolar range, indicating this site is amendable for further modifications.
Table 1. The cytotoxicity of compounds 11-23 in BT-474 cells.
O
N
O
N
N
N
O
R1
N
N
H
H
N
R2
Compd.
R1
R2
IC50 (µmol/L)
11
See Scheme 1
-Bn
22.12
N
N
12
13
14
-Bn
-Bn
-Bn
5.242
1.922
6.896
N
N
N
H
N
N
15
16
17
18
19
20
21
22
23
-H
-Bn
-Bn
-Bn
-Bn
-Bn
-Bn
-Bn
-H
-CH2CH3
1.724
1.895
8.148
2.759
2.229
1.565
1.311
1.347
2.343
-CH2CH2N(CH3)2
-CH(CH3)2
cyclopropyl
-CH2CH3
-CH2CH2F
-CH3
-CH3
-CH3
In combination with other data in hand, compounds 20 and 21 were further tested in biochemical assay to confirm their inhibition
against PI3K . As shown in Table 2, both 20 and 21 are potent PI3Kδ inhibitors with IC50s values of 0.286 µmol/L and 0.452 µmol/L,
δ
respectively. In the same assay, CAL-101 showed an IC50 of 0.036 µmol/L [24]. These data indicate their anti-proliferative activities are
most likely due to their inhibition of the PI3Kδ kinase.
Table 2 The inhibitory activities of compounds 20 and 21 against PI3K
δ
Compd.
20
PI3K
δ
, IC50 (µmol/L)
0.286
21
0.452
0.036
CAL-101
4. Conclusion
In summary, an efficient syntheses of novel 3-(piperidin-4-yl)isoxazolo[4,5-d]pyrimidine scaffold was designed and developed. Using
this method, a series of isoxazolo[4,5-d]pyrimidine derivatives has been prepared. Their cytotoxicity to BT-474 cells was evaluated by
CCK-8 assay. Most of the derivatives displayed inhibitory potency against the proliferation of BT-474 cells. Preliminary SAR
information from these compounds can be used to guide further exploration. Although the lead compounds were also shown to be potent
inhibitors of PI3Kδ kinase, indicating their anti-cancer activity could be through PI3K pathway, further studies are needed to optimize
their activities and to confirm their mechanism of action.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 81172914) and Tang Aoqing Professorship
research grant from Jilin University, China and Changchun Discovery Sciences, Ltd. The authors wish to thank Prof. Xu Bai for
insightful discussions on chemical synthesis.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
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