Development of a bioavailable boron-containing PI-103 Bioisostere, PI-103BE

Article abstract of DOI:10.1016/j.bmcl.2020.127258

PI-103 (7) is a potent dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor, but its rapid in vivo metabolism hinders its further clinical development. To improve the bioavailability of PI-103, we designed and synthesized a PI-103 bioisostere, PI-103B

Full text of DOI:10.1016/j.bmcl.2020.127258

Journal Pre-proofs
Development of a Bioavailable Boron-Containing PI-103 Bioisostere,
PI-103BE
Lan Luo, Qiu Zhong, Shanchun Guo, Changde Zhang, Qiang Zhang, Shilong
Zheng, Ling He, Guangdi Wang
PII:
S0960-894X(20)30363-2
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127258
BMCL 127258
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
16 March 2020
7 May 2020
9 May 2020
Please cite this article as: Luo, L., Zhong, Q., Guo, S., Zhang, C., Zhang, Q., Zheng, S., He, L., Wang, G.,
Development of a Bioavailable Boron-Containing PI-103 Bioisostere, PI-103BE, Bioorganic & Medicinal
Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127258
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Graphical abstract
Novel PI-103 bioisostere, PI-103BE (9), displays higher bioavailability than PI 103 (7) in vivo.
O
O
N
N
O
N
N
O
N
N
N
N
O
OH
B
O
PI-103 (7)
PI-103BE (9)
AUC0-24h = 88.2 ng/mL*h
AUC0-24h = 0
Development of a Bioavailable Boron-Containing PI-103 Bioisostere, PI-103BE
a
b
b
b
b
b,
Lan Luo, Qiu Zhong, Shanchun Guo, Changde Zhang, Qiang Zhang, Shilong Zheng, * Ling
a,
b,
He, * Guangdi Wang *
a
Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, Sichuan
Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision
Industrial Technology, Department of Medicinal Chemistry, West China School of Pharmacy,
b
Sichuan University, Chengdu, Sichuan, 610041, China. RCMI Cancer Research Center and
Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA
ABSTRACT: PI-103 is a potent dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor, but
its rapid in vivo metabolism hinders its further clinical development. To improve the bioavailability
of PI-103, we designed and synthesized a PI-103 bioisostere, PI-103BE (9) in which the phenolic
hydroxyl group of PI-103 was replaced by a boronate, a structural modification known to enhance
bioavailability of molecules containing phenolic hydroxyl moieties. In cell culture, PI-103BE is
partially converted to its corresponding boronic acid (10) and to a lesser extent the active ingredient,
PI-103. This mixture contributes to the in vitro activity of 9 that shows reduced potency compared

to the parent compound. When administered to mice by oral gavage, 9 displays a significantly
improved pharmacokinetic profile compared to PI-103, which shows no oral bioavailability at the
same dose. Drug exposure of 9 as measured by the area under curve (AUC) value is 88.2 ng/mL*h
for 7 and 8879.9 ng/mL*h for 10. When given by intraperitoneal injection (IP), the prodrug
afforded an AUC of 32.3 ng/mL*h for 7 and 400.9 ng/mL*h for 10, compared to 9.7 ng/mL*h
from PI-103 injection. In plasma from both pharmacokinetic studies, 9 is fully converted to 10 and
7
, with the boronic acid metabolite (10) displaying antiproliferative activities comparable to 9, but
weaker than 7. The boronic bioisostere of PI-103 thus offers an improved bioavailability that could
be translated to in vivo efficacy of PI-103.
Keywords: PI-103 bioisosteres, boron-containing compound, synthesis, pharmacokinetics,
bioavailability.
INTRODUCTION
The PI3K/AKT/mTOR pathway is a validated therapeutic target for the treatment of several
1
2
malignancies including chronic lymphocytic leukemia (CLL), follicular lymphoma, and breast
cancer3, 4. As of January 2020, FDA has approved four PI3K inhibitors as oncology agents:
idelalisib (July 2014, NDA 206545) for leukemia and two types of lymphoma5
copanlisib (September
2017, NDA
209936)
for
relapsed
follicular
lymphoma,
duvelisib (September 2018, NDA 211155) for relapsed or refractory chronic lymphocytic
6
leukemia (CLL)/small lymphocytic lymphoma (SLL) (Figure 1), and alpelisib (BYL719), an
alpha-specific PI3K inhibitor for use in combination with endocrine therapy fulvestrant for
7
treatment of HR-positive and HER2/neu-negative breast cancer . More PI3K inhibitors are being
developed in different stages of clinical trials8-11
.
Pharmacological inhibition of PI3Ks
downregulates the expression of programmed death ligand-1 (PD-L1), as recent data from clinical
trials and preclinical mouse models indicate that the therapeutic inhibition of PI3Ks also enhances
antitumor immune-intrinsic properties 12, 13, suggesting additional clinical utility of PI3K inhibition.
1
4
PI-103 is a potent dual PI3K and mTOR inhibitor . Although the efficacy and tolerability of PI-
03 have been examined in preclinical models, PI-103 has not yet entered clinical trials mainly
1
because of its poor pharmacokinetic performance 15, 16. Recently, Zhu and Merino et al designed
a PI-103 prodrug (Figure 1) to improve metabolic stability of the compound. Their study showed

the enhanced selectivity of the prodrug against Kasumi-1 AM cells over normal cells, but no
1
7
bioavailability data were provided .
N
NH
N
N
N
Cl
O
N
N
O
N
NH
N
N
N
N
H
H
O
O
NH
N
N
N
O
F
O
N
N
H N
2
N
duvelisib (IPI-145)
idelalisib
copanlisib
O
NH2
N
N
O
N
NH
N
O
O
NH2
PI-103 prodrug
Figure 1. PI3K inhibitors in clinical use and PI-103 prodrug
The oxidation of boronic acids and their esters to alcohols is a biorthogonal reaction that can occur
1
8
inside living systems without interfering with native biochemical processes . This approach was
first applied to the development of imaging probes 19 and was later successfully used to design the
2
0
21
bioisosteres or prodrugs of phenolic compounds including 4-hydroxytamoxifen , endoxifen ,
fulvestrant22,23, GW7604 , combretastatin A-4 , metalloproteinase inhibitor , SN-38
24
25
26
camptothecin derivative) , and estrone28,29. We have found the replacement of the phenolic
2
7
(
hydroxyl group with a boronic acid or a boronate significantly improves the bioavailability of the
drugs and led to targeted uptake and accumulation in solid tumors21, 24, 30-33. Chang et al also
reported that an imaging agent attached to aryl boronic acid moiety can spread throughout the body
1
9
of living mice to reach deep tumor tissues due to the unique biocompatibility of boronic acid . It
was also reported that the insulin derivatives modified with aliphatic phenylboronic acid
3
4
conjugates could provide long-term glucose-mediated insulin activity . All of these results
encouraged us to modify the hydroxyl group of PI-103 with a boron-containing moiety to enhance
its bioavailability (Figure 2). Herein, we report the synthesis and biological evaluation of PI-
1
03BE (9), a PI-103 bioisostere.

RESULTS and DISCUSSION
Synthesis of PI-103BE (9). PI-103BE (9) was prepared as shown in Figure 2. Condensation
reaction of ethyl 3-aminofuro[2,3-b]pyridine-2-carboxylate (1) with 3-methoxybenzoyl chloride
(2) provided the corresponding amide 3 in dichloromethane. The treatment of 3 with aqueous
ammonium hydroxide gave 4 and subsequent cyclization of 4 with aqueous sodium hydroxide
afforded 5. The demethylation of 5 followed by acetylation formed 6. The chlorination of 6 and
subsequent substitution with morpholine resulted in the formation of PI-103 (7)35,36. 7 was
converted to the aryl triflates 8 as a colorless solid by trifluoromethanesulfonic anhydride. The
formation of 9 (PI-103BE) was carried out via a boronation reaction using bis(pinacolato)diboron
o
37
as the boron reagent, KOAc as the base, and PdCl (dppf) as the catalyst in dioxane at 80 C .
2
O
O
N
N
N
N
O
O
N
N
N
N
O
OH
B
O
PI-103 (7)
PI-103BE (9)
N
O
O
N
O
O
O
N
O
O
O
NH2
a
b
O
+
NH
NH
Cl
O
O
O
NH2
1
2
OCH3
OCH3
O
4
3
N
O
O
N
O
N
O
O
N
N
c
d
e
NH
NH
N
N
O
N
O
O
OH
6
7
(PI-103)
5
O
O
N
N
N
O
N
N
N
O
f
g
N
N
O
B
OTf
O
9
(PI-103BE)
8

Figure 2. Design and synthesis of PI-103BE (9).
Reagents and conditions: (a)Et N, DCM, 77%; (b)28% aq NH , MeOH, 87%; (c)2 N NaOH,
3
3
MeOH, reflux, 88%; (d)(i) 48% HBr, AcOH, reflux, (ii) Ac O, AcONa, reflux, 65% for two
2
steps; (e) (i) POCl , reflux, (ii) morpholine, reflux, 32% for two steps; (f) Tf O, py, DCM, 84%;
3
2
(
g)PdCl (dppf), Bis(pinacolato)diboron, KOAc, dioxane, 80℃, 76%.
2
In Vitro Antiproliferative Properties
We first evaluated the antiproliferation activity of the bioisostere PI-103BE (9) against human lung
carcinoma A549, human breast adenocarcinoma MDA-MB-231, human cervical cancer HeLa,
human prostate carcinoma 22Rv1, human prostate adenocarcinoma PC-3, and human ovarian
adenocarcinoma SKOV-3 cell lines. The results in Table 1 show that bioisostere 9 with IC values
5
0
of 0.57 µM, 0.44 µM, 3.82 µM, 0.66 µM, 0.72 µM, and 1.33 µM is 3 ~ 24 times less potent than
PI-103 (7) against these six cell lines, respectively. To test if the reduced activity of 9 is a result
of partial conversion into the active ingredient (7), we tested the drug stability in cell culture by
determination the concentrations of PI-103BE (9), PI-103BA (10), and PI-103 at various time
points of treatment. It was found that PI-103BE underwent rapid hydrolysis in culture medium to
become PI-103BA (10), the boronic acid of the bioisostere (>95%) (Table 2). A small fraction of
7
at <4% was also detected in culture medium (Table 2), indicating that the activity of the
bioisostere in vitro may be accounted for by the combination of reduced potency of 9 and the
incomplete conversion to PI-103 (7).
Table 1 In vitro antiproliferative activity of PI-103BE (9) on a panel of cancer cell lines
±
: standard error (SEM) of 3 replicate experiments
Compounds
IC (µM)
5
0
A549
0.18 ± 0.04
MDA-MB-231
0.14 ± 0.01
HeLa
22Rv1
PC-3
SKOV-3
PI-103 (7)
0.16 ± 0.05 0.10 ± 0.03 0.17 ± 0.03 0.11 ± 0.03
PI-103BE (9) 0.57 ± 0.06
0.44 ± 0.04
3.82 ± 0.18 0.66 ± 0.03 0.72 ± 0.02 1.33 ± 0.11
Table 2. Stability of PI-103BE (9) in cell culture medium
Concentration (M) and fraction of total drug content (%) in cell culture media
Compound
0.5 hour
1 hour
3 hour
8 hour
24 hour
PI-103BE (9)
0.04 (1.61%)
0.01 (0.32%)
0.00 (0%)
0.00 (0%)
0.00 (0%)

PI-103BA (10) 2.39 (97.95%) 3.08 (97.47%) 3.52 (96.70%) 3.55 (96.73%) 3.53 (96.19%)
PI-103 (7) 0.05 (2.02%) 0.08 (2.52%) 0.12 (3.30%) 0.12 (3.27%) 0.14 (3.81%)
Pharmacokinetics and in vivo metabolites of PI-103BE (9) in mice plasma
O
O
N
N
N
N
O
O
N
N
N
N
O
OH
OH
B
B
O
PI-103BE (9)
PI-103BA (10)
O
N
N
N
O
N
OH
PI-103 (7)
Figure 3. Metabolism of PI-103BE (9) to active metabolite PI-103 (7)
To determine the bioavailability of PI-103BE (9), we conducted pharmacokinetic studies of 9 in
mice with PI-103 (7) as a control. Mice were administered a single dose of 10 mg/kg of 9 or 7 by
intraperitoneal injection and blood samples were collected at 0.5, 1, 3, 8, and 24 hours post drug
administration. Measured plasma concentrations of the administered drug and metabolites are
plotted over a period of 24 hours in Figure 4 and summarized in Table 3. Peak concentration of 7
in the blood of 9-treated mice reached 14.9 ng/mL at 0.5 hours after IP administration, then
decreased over time, and at 24 h, maintained a concentration of PI-103 at 0.4 ng/mL (Fig 4A). The
major form of the drug was PI-103BA, the boronic acid while no PI-103BE was detected in plasma,
consistent with the stability profile of the bioisostere in culture media. In comparison, the peak
concentration of PI-103, reached at 1 hour, was at 1.4 ng/mL, 10 times lower than achieved by the
i.p. administration of PI-103BE (Fig 4B).
Table 3. Pharmacokinetics parameters of PI-103BE (9) and PI-103 (7) in mice after
intraperitoneal injection (IP)

Time Point
h)
.5
PI-103BE (9)
PI-103BA (10) (ng/mL)
PI-103 (7)
PI-103 (ng/mL)
0.1 ± 0.0
1.4 ± 0.1
1.0 ± 0.1
0.3 ± 0.0
NF
(
PI-103 (ng/mL)
14.9 ± 0.5
5.4 ± 0.1
1.3 ± 0.1
1.0 ± 0.0
0
30.6 ± 1.4
44.5 ± 0.9
28.0 ± 1.0
17.2 ± 0.3
6.5 ± 0.2
1
3
8
2
4
0.4 ± 0.0
Cmax
Tmax
t1/2
14.9 ng/mL
0.5 hr
44.5 ng/mL
1.0 hr
1.4 ng/mL
3.0 hr
12.4 hr
10.2 hr
2.0 hr
AUC
32.3 ng/mL*h
400.9 ng/mL*h
9.7 ng/mL*h
Figure 4. Single dose pharmacokinetics of PI-103BE (A) and PI-103 (B) in mice.
We next examined the pharmacokinetic profile of PI-103BE via oral administration, again in
comparison with PI-103. After a single dose of 10 mg/kg by oral gavage of PI-103BE or PI-103,
blood samples were collected from mice and analyzed for concentration of PI-103 and 9 at 0.5, 1,
3
, 8, and 24 h time points after drug administration. Table 4 shows the pharmacokinetics
parameters of PI-103BE (9) and PI-103 (7) in mice after oral administration. In 9-treated mice the
peak plasma concentration of PI-103 was 8.2 ng/mL, reached at 1h after oral administration. In
contrast, no PI-103 was found in mice given a single oral dose of 10 mg/kg PI-103, indicating no
oral bioavailability. Importantly, the predominant form in 9-treated mice plasma is PI-103BA (10),

the corresponding free boronic acid of 9. The maximum concentration of 10 reached 709.2 ng/mL
at 1 hour, and its plasma level remained above 100 ng/mL at 24 h. These results confirm that PI-
1
03 is poorly bioavailable in vivo, especially by oral administration, whereas PI-103BE (9) has
significantly enhanced bioavailability in both its boronic acid form and the active ingredient form
of PI-103 as a metabolite of the bioisostere.
Table 4. Pharmacokinetics parameters of PI-103BE (9) and PI-103 (7) in mice after oral
administration
PI-103BE (9)
PI-103 (7) (ng/mL)
ND
8.2 ± 0.7
6.5 ± 0.4
4.1 ± 0.3
0.7 ± 0.1
8.2 ng/mL
1 hr
PI-103 (7)
PI-103 (7) (ng/mL)
Time Point (hrs)
PI-103BA (10) (ng/mL)
402.8 ± 22.4
709.2 ± 11.9
516.2 ± 13.9
354. 2 ± 3.2
106.4 ± 2.2
709.2 ng/mL
1 hr
0
1
3
8
2
.5
ND
ND
ND
ND
ND
0
ND
ND
0
4
Cmax
Tmax
t1/2
AUC0-24h
6.5 hr
9.2 hr
88.2 ng/mL*h
8879.9 ng/mL*h
±
: standard error (SEM) of 5 mice; ND: not detected
Activity of the major metabolite PI-103BA (10)

MDA-MB-231
A549
100
100
PI-103
50
50
PI-103
PI-103BE (9)
PI-103BA (10)
PI-103BE (9)
PI-103BA (10)
0
0
-9
-8
-7
-6
-5
-9
-8
-7
-6
-5
log[Concentration], M
log[Concentration], M
HeLa
22RV1
1
00
1
00
50
0
PI-103
PI-103
5
0
PI-103BE (9)
PI-103BA (10)
PI-103BE (9)
PI-103BA (10)
0
-9
-8
-7
-6
-5
-9
-8
-7
-6
-5
log[Concentration], M
log[Concentration], M
Figure 5. Antiproliferative activity of PI-103BA (10) in A549, MDA-MB-231, HeLa, and
2RV1 human carcinoma cell lines.
Both in vitro stability and in vivo pharmacokinetic studies confirm that the boronic acid PI-103BA
10) is the major metabolite of prodrug 9, which may contribute to the overall therapeutic efficacy
2
(
of the prodrug. We therefore sought to determine the anticancer activity of the boronic acid
metabolite of 9. To prepare the compound 10, we performed the oxidation of 9 by sodium
periodate, which led to the hydrolysis of 9 to yield the boronic acid 10 (PI-103BA) in THF-H O.
2
The antiproliferative activity of 10 was then evaluated in four cancer cell lines, A549, MDA-MB-
2
31, HeLa, and 22RV1. As shown in Figure 5, compound 10 displays dose dependent
antiproliferative activities against all four cancer cell lines with potency comparable to that the
prodrug 9, but less than PI-103 (7).
CONCLUSION

Based on the unique properties of the boronic acid and its ester as a bioavailability-enhancing
molecular functional group, we replaced the hydroxyl group of the PI3k inhibitor PI-103 (7) with
the boronic acid pinacol ester moiety to design a PI-103 bioisostere, PI-103BE (9). PI-103BE (9)
was synthesized from ethyl 3-aminofuro[2,3-b]pyridine-2-carboxylate (1) and 3-methoxybenzoyl
chloride (2) in seven steps. In vitro, the bioisostere 9 acts as an antiproliferative activity against a
panel of cancer cell lines with reduced potency compared to PI-103. In vivo pharmacokinetics
studies in mice demonstrate that PI-103BE (9) affords significantly improved bioavailability over
PI-103, which may be translatable to efficacy in inhibiting the growth of xenograft tumors that is
not achieved by PI-103.
EXPERIMENTAL SECTION
General information. All reactions were carried out under air atmosphere, unless otherwise
mentioned. Commercially available materials were used as received without further purification.
Reactions were monitored by thin-layer chromatography (TLC) carried out on commercial silica
gel plates using UV light as a visualizing agent. Commercial silica gel was used for column
1
13
chromatography. H and C NMR spectra were recorded on 400 MHz or 600 MHz spectrometer.
1H NMR spectra were referenced to Chloroform-d (7.26 ppm) or DMSO-d6 (2.50 ppm), and
reported as follows; chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m
1
3
=
multiplet). Chemical shifts of the C NMR spectra were measured relative to Chloroform-d
(77.23 ppm) or DMSO-d6 (39.51 ppm). Mass spectral data were obtained from Bruker Daltonics
Data analysis 3.2 mass spectrometer and Thermo LTQ Orbitrap-XL mass spectrometer in positive
ion modes. Unless specified otherwise, all tested compounds were confirmed to be >95% pure by
HPLC.
Chemical synthesis of PI-103 bioisostere
Ethyl 3-(3-methoxybenzamido)furo[2,3-b]pyridine-2-carboxylate (3): To a mixture of ethyl 3-
aminofuro[2,3-b]pyridine-2-carboxylate (1) (2.06 g, 10 mmol) and Et N (2.6 mL, 20 mmol) in
3
dichloromethane (30 mL) was added dropwise 3-methoxybenzoyl chloride (2) at 0℃. After stirred
at room temperature for 2.5 h, the reaction mixture was diluted with dichloromethane and washed
with saturated NaHCO and brine. The organic layer was dried over Na SO and concentrated in
3
2
4

vacuo. The crude was recrystallized from ethyl acetate to give 3 (2.4 g, 77% yield) as a colorless
1
solid: H NMR (400 MHz, Chloroform-d) δ 10.55 (s, 1H), 9.08 (dd, J = 8.1, 1.7 Hz, 1H), 8.54 (dd,
J = 4.7, 1.8 Hz, 1H), 7.61 – 7.55 (m, 2H), 7.45 (t, J = 7.9 Hz, 1H), 7.36 (dd, J = 8.1, 4.7 Hz, 1H),
7
.15 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 4.51 (q, J = 7.1 Hz, 2H), 3.91 (s, 3H), 1.48 (t, J = 7.2 Hz, 3H).
3
-(3-Methoxybenzamido)furo[2,3-b]pyridine-2-carboxamide (4): To a solution of 3 (2.1 g, 6.7
mmol) in MeOH (150 mL) was added 28% aqueous NH (175 mL). After stirred at room
3
temperature for 12 h, the reaction mixture was concentrated to give the product 4 (1.81 g, 87%
1
yield): H NMR (400 MHz, DMSO-d ) δ 11.26 (s, 1H), 8.80 (dd, J = 8.0, 1.8 Hz, 1H), 8.53 (dd, J
6
=
4.7, 1.8 Hz, 1H), 8.46 (s, 1H), 8.09 (s, 1H), 7.59 – 7.48 (m, 4H), 7.26 (ddd, J = 7.7, 2.8, 1.5 Hz,
1
H), 3.86 (s, 3H).
2
-(3-Methoxyphenyl)pyrido[3',2':4,5]furo[3,2-d]pyrimidin-4(3H)-one (5): To a mixture of 4
(1.8 g, 5.8 mmol) in i-PrOH (70 mL) was added 2 N NaOH (27 mL). After refluxed for 3 h, the
reaction mixture was neutralized with concentrated HCl and the resulting solid was collected to
1
give 5 (1.5 g, 88% yield) as a canary yellow solid: H NMR (400 MHz, DMSO-d ) δ 13.25 (s,
6
1
H), 8.67 (dd, J = 4.9, 1.8 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.75 (t, J = 2.1 Hz, 1H), 7.64 (dd, J
7.7, 4.8 Hz, 1H), 7.49 (t, J = 8.0 Hz, 2H), 7.18 (dd, J = 8.2, 2.6 Hz, 1H), 3.88 (s, 3H).
=
3
-(4-Oxo-3,4-dihydropyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl)phenyl acetate (6):
A
mixture of 5 (770 mg, 2.7 mmol), 48% HBr (50 mL), and AcOH (50 mL) was refluxed for 48 h.
The reaction mixture was concentrated in vacuo. Acetic anhydride (30 mL) and NaOAc (100 mg)
were added and the reaction mixture was refluxed for 0.5 h. After concentration, the solid obtained
1
was washed with methanol and Et O to give 6 (0.50 g, 65% yield) as a gray solid: H NMR (400
2
MHz, DMSO-d ) δ 13.29 (s, 1H), 8.68 – 8.62 (m, 2H), 8.10 – 8.05 (m, 1H), 7.97 (t, J = 2.1 Hz,
6
1
H), 7.66 – 7.60 (m, 2H), 7.40 (dd, J = 8.0, 2.3 Hz, 1H), 2.34 (s, 3H).
3
-(4-Morpholinopyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl)phenol (7): Compound 6 (1.0 g,
3
.6 mmol) in phosphorus oxychloride (10 mL) was refluxed for 3 h and concentrated in vacuo.
The residue was dissolved in THF (15 mL) and morpholine (15 mL) was added to it. After refluxed
for 1.5 h, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic
layer was washed with brine, dried over anhydrous MgSO4. After concentration, the solid obtained
1
was washed with CHCl /MeOH (96:4) to give 7 (0.40g, 32% yield) as a colorless solid: H NMR
3

(
400 MHz, DMSO-d ) δ 9.55 (s, 1H), 8.66 (dd, J = 11.9, 6.2 Hz, 2H), 7.93 – 7.85 (m, 2H), 7.62
6
(dd, J = 7.7, 4.8 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 6.88 (dd, J = 7.9, 2.5 Hz, 1H), 4.11 (t, J = 4.6
1
3
Hz, 4H), 3.85 (t, J = 4.7 Hz, 4H). C NMR (101 MHz, DMSO-d6): δ 162.7, 158.9, 158.0, 150.4,
1
48.9, 147.0, 139.5, 133.3, 132.2, 129.8, 121.3, 119.2, 117.6, 115.1, 115.0, 66.5, 45.8. HRMS (ESI)
+
calcd C H N O [M + H] 349.1300, found 349.1294.
1
9
17
4
3
3
-(4-Morpholinopyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl)phenyltrifluoromethane
sulfonate (8): To the phenol 7 (174mg, 0.5 mmol) in anhydrous CH Cl was added pyridine (0.1ml,
2
2
1
mmol) and the solution was cooled to 0 °C. Trifluoromethanesulfonic anhydride (0.13ml, 0.6
mmol) was added dropwise and the mixture was warmed to r.t. The reaction was complete within
0 min. The reaction mixture was diluted with dichloromethane and washed successively with
1
saturated NaHCO and brine. After drying over MgSO4, the solution was concentrated and the
3
residue was purified by column chromatography on silica gel with hexane-ethyl acetate (4:1 to
1
2
:1) as eluent to give the triflates 8 (0.20 g, 84% yield) as pale yellow: H NMR (400 MHz, DMSO-
d6) δ 8.72 – 8.67 (m, 2H), 8.55 (dt, J = 7.8, 1.4 Hz, 1H), 8.39 (t, J = 2.0 Hz, 1H), 7.74 (t, J = 8.0
Hz, 1H), 7.67 – 7.62 (m, 2H), 4.13 (t, J = 4.7 Hz, 4H), 3.85 (t, J = 4.7 Hz, 4H).
4
-Morpholino-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyrido[3',2':4,5]
furo[3,2-d]pyrimidine (PI-103BE, 9): A nitrogen-flushed 25 mL round-bottomed flask was
charged with PdCl (dppf) (8 mg, 0.01 mmol), KOAc (15 mg 0.15 mmol), bis(pinacolato)diboron
2
(34mg, 0.12 mmol), and the aryl triflate 8 (48mg, 0.1 mmol), then anhydrous dioxane (3 mL) was
added and the solution was stirred for 3 h at 80 °C. The reaction mixture was extracted with EtOAc,
the organic layer was washed with brine, and dried over anhydrous MgSO4. The organic solvent
was removed under reduced pressure and the crude was purified by column chromatography with
1
hexane-ethyl acetate (5:1 to 3:1) as eluent to afford the product 9 (36 mg, 76% yield): H NMR
(400 MHz, Chloroform-d) δ 8.89 (s, 1H), 8.62 (dd, J = 4.8, 1.5 Hz, 1H), 8.58 (d, J = 7.7 Hz, 1H),
7
1
.93 (d, J = 7.2 Hz, 1H), 7.56 – 7.45 (m, 3H), 4.26 (t, J = 4.7 Hz, 4H), 3.94 (t, J = 4.8 Hz, 4H),
1
.40 (s, 12H). H NMR (300 MHz, DMSO-d6): δ 8.76 (s, 1H), 8.73 (dd, J = 7.8, 1.8 Hz, 1H), 8.67
(dd, J = 5.1, 1.5 Hz, 1H), 8.53 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.61 (m, 1H), 7.53 (t,
1
3
J = 7.8 Hz, 1H), 4.11 (t, J = 4.7 Hz, 4H), 3.84 (t, J = 4.8 Hz, 4H), 1.34 (s, 12H). C NMR (75
MHz, DMSO-d6): 163.18, 159.34, 151.01, 149.54, 147.62, 138.16, 137.20, 134.65, 133.95, 133.05,

1
32.14, 129.17, 121.95, 115.47, 84.91, 67.08, 46.40, 25.80. HRMS (ESI) calcd C H BN O [the
19 18 4 4
+
free boronic acid of 9 + H] 377.1421, found 377.1416.
(3-(4-Morpholinopyrido[3’,2’:4,5]furo[3,2-d]pyrimidin-2-yl)phenyl)boronic acid (PI-103BA,
1
0): 9 (20 mg, 0.04 mmol) and sodium periodate (33 mg, 0.07 mmol) were dissolved in THF-H O
2
(5 mL, 4:1), to which 1N HCl (0,5 mL) was added. The mixture was stirred at room temperature
for 1h, the reaction was quenched with brine and ethyl acetate (15 mL, 1;1). The precipitate was
collected by filtration and washed with water and ethyl acetate 2-3 time, respectively to afford PI-
1
1
03BA (10) (13 mg, 82 % yield). The purity is > 98% based on HPLC. H NMR (300 MHz,
DMSO-d6): δ 8.85 (s, 1H), 8.72-8.66 (m, 2H), 8.47 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 7.2 Hz, 1H),
1
3
7
.63 (m, 1H), 7.48 (t, J = 7.5 Hz, 1H), 4.14 (t, J = 5.1 Hz, 4H), 3.85 (t, J = 4.8 Hz, 4H). C NMR
(75 MHz, DMSO-d6): 163.17, 159.90, 150.93, 149.49, 147.60, 137.73, 136.90, 134.67, 133.85,
+
1
3
32.88, 130.69, 128.51, 121.91, 115.53, 67.09, 46.35. HRMS (ESI) calcd C H BN O [M + H]
19 18 4 4
77.1421, found 377.1419.
In vitro antiproliferative assay of PI-103 bioisostere toward cancer cell lines
For growth assay in the presence of PI-103 bioisosteres 9, the cancer cells were plated in plates at
a density of 50,000 each well in 5% FBS DMEM medium. The cells were then treated with PI-
1
03BE (9) separately at 6 different doses ranging from for 4 days, while equal treatment volume
of DMSO and PI-103 (7) were used as vehicle control and positive control, respectively. Cell
numbers were counted with a Coulter instrument (Beckman-Coulter). The ratio of drug-treated
cell numbers to vehicle-treated cell numbers was defined as survival ratio. IC values were
5
0
obtained from dose-response curves for each bioisostere. Experiments were conducted in triplicate
and data represented as mean ± SD.
Pharmacokinetics study of PI-103BE (9) in mice
Sample collection of plasma: Four to six weeks old female ovariectomized Nu/Nu mice were
purchased from Charles River Laboratories (Wilmington, MA). The above mice were used in the
pharmacokinetic study of PI-103BE (9) and PI-103 (7). In oral pharmacokinetic study, mice (n=5)

were given a single dose of 10 mg/kg of PI-103BE (9) or PI-103 (7) dissolved in the oral gavage
containing 5% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 400, 55% saline. In the
pharmacokinetic study by i.p. injection, mice were given a single dose of 10 mg/kg PI-103BE (9)
or PI-103 (7) solution in ethanol by i.p. injection. After drug administration, blood samples were
collected from the orbital sinus of the mice at 0.5, 1, 3, 8, and 24 h time points with each group of
mice subjected to only one sampling. Blood samples were collected with a sterile capillary into
1
.5 mL microcentrifuge tubes containing 0.1 mL of 10 % EDTA anticoagulant. Plasma was then
separated from cell pellets by centrifugation 3000 rpm, 5 minutes in a refrigerated centrifuge at
°C and transferred to a separate tube. Plasma samples were frozen at -80 °C until analysis.
4
Analysis of metabolites on TSQ Mass spectrometer: Plasma samples were processed following
3
2
a similar protocol. These samples were injected on the Hypersil GOLD C18 column (1.8µm,
.1mmx50mm) with UHPLC ultimate 3000 from Dionex coupled with a TSQ Vantage mass
2
spectrometer coupled with a UHPLC ultimate 3000 from Dionex to quantitate the concentration
of the main metabolites of PI-103BE (9) and PI-103 (7). The 10 µL samples were run with the
gradient starting at 0.6 mL/min from 10% mobile phase B (Acetonitrile with 0.05%
formic acid) and 90% mobile phase A (water with 0.05% formic acid) until 0.5min, up to 100%
B at 3 min, and until 6 min, then came back to 10% B until equilibration. The TSQ Vantage was
set at spray vantage at 3200V, vaporizer temperature at 365ºC, Sheath gas pressure at 42 PSI, Aug
Gas pressure at 12 psi, Capillary temperature at 350 ºC.
All procedures involving the animals were conducted in compliance with State and Federal laws,
standards of the U.S. Department of Health and Human Services, and guidelines established by
Xavier University Animal Care and Use Committee. The facilities and laboratory animals program
of Xavier University are accredited by the Association for the Assessment and Accreditation of
Laboratory Animal Care.
Declaration of Competing Interest: The authors declare that they have no known competing
financial interests or personal relationships that could have appeared to influence the work reported
in this paper.

Acknowledgment: This work was funded by Sichuan University-Lu Zhou Strategic Cooperation
Projects (2017 CDLZ-S34) (L. He) and supported by NIMHD at Xavier University of Louisiana
through Grant (U54MD007595) (G. Wang).
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