Discovery of novel purine nucleoside derivatives as phosphodiesterase 2 (PDE2) inhibitors: Structure-based virtual screening, optimization and biological evaluation

Accepted Manuscript

Discovery of novel purine nucleoside derivatives as phosphodiesterase 2

(PDE2) inhibitors: structure-based virtual screening, optimization and biologi-

cal evaluation

Xiaoxia Qiu, Yiyou Huang, Deyan Wu, Fei Mao, Jin Zhu, Wenzhong Yan, Hai-

Bin Luo, Jian Li

PII:

S0968-0896(17)31941-7

https://doi.org/10.1016/j.bmc.2017.11.022

BMC 14074

DOI:

Reference:

Bioorganic & Medicinal Chemistry

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Revised Date:

Accepted Date:

2 October 2017

30 October 2017

12 November 2017

Please cite this article as: Qiu, X., Huang, Y., Wu, D., Mao, F., Zhu, J., Yan, W., Luo, H-B., Li, J., Discovery of

novel purine nucleoside derivatives as phosphodiesterase 2 (PDE2) inhibitors: structure-based virtual screening,

optimization and biological evaluation, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/

j.bmc.2017.11.022

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Graphical Abstract

Discovery of novel purine nucleoside derivatives as phosphodiesterase 2 (PDE2)

inhibitors: structure-based virtual screening, optimization and biological

evaluation

Xiaoxia Qiu^a,†, Yiyou Huang^b,†, Deyan Wu^b, Fei Mao^a, Jin Zhu^a, Wenzhong Yan^c,*, Hai-Bin Luo^b,*, Jian Li^a,*

^aShanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and

Technology, 130 Mei Long Road, Shanghai 200237, China

^bSchool of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, China

^ciHuman Institute, ShanghaiTech University, 99 Haike Road, Shanghai 201210, China

Bioorganic & Medicinal Chemistry

journal h omepage: www.els evier. com

Discovery of novel purine nucleoside derivatives as phosphodiesterase 2 (PDE2)

inhibitors: structure-based virtual screening, optimization and biological evaluation

Xiaoxia Qiu^a,†, Yiyou Huang^b,†, Deyan Wu^b, Fei Mao^a, Jin Zhu^a, Wenzhong Yan^c,*, Hai-Bin Luo^b,*, Jian

Li^a,*

^aShanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237,

China

^bSchool of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, China

^ciHuman Institute, ShanghaiTech University, 99 Haike Road, Shanghai 201210, China

^†These authors contributed equally to this work.

* To whom correspondence should be addressed. jianli@ecust.edu.cn, luohb77@mail.sysu.edu.cn, yanwzh@shanghaitech.edu.cn.

ARTICLE INFO

ABSTRACT

Article history:

Received

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the

central nervous system (CNS) disorders and pulmonary hypertension. Herein, we identified that

Received in revised form

Accepted

Available online

clofarabine (4), an FDA-approved drug, displayed potential PDE2 inhibitory activity (IC₅₀=

3.12 ± 0.67 μM) by structure-based virtual screening and bioassay. Considering the potential

therapeutic benefit of PDE2, a series of purine nucleoside derivatives based on the structure and

binding mode of 4 were designed, synthesized and evaluated, which led to the discovery of the

best compound 14e with a significant improvement of inhibitory potency (IC₅₀= 0.32 ± 0.04

μM). Further molecular docking and molecular dynamic (MD) simulations studies revealed that

5’-benzyl group of 14e could interact with the unique hydrophobic pocket of PDE2 by forming

extra van der Waals interactions with hydrophobic residues such as Leu770, Thr768, Thr805 and

Leu809, which might contribute to its enhancement of PDE2 inhibition. These potential

compounds reported in this article and the valuable structure-activity relationships (SARs) might

bring significant instruction for further development of potent PDE2 inhibitors.

Keywords:

clofarabine

PDE2

inhibitors

purine nucleoside derivatives

virtual screening

1. Introduction

treatment

of memory and

cognitive

disorders,^20-23

neurodegenerative disease.²⁴In addition, PDE2 is also expressed

in the cardiovascular system as well as in the lung, and it has

been regarded as a potential therapeutic target for treatment of

heart failure,²⁵pulmonary hypertension.²⁶Although several

PDE2 inhibitors have been reported in the recent two decades,

none of these PDE2 inhibitors have reached the market yet.

EHNA²⁷(compound 1, Figure 1) is one of the first generation of

PDE2 inhibitors with IC₅₀of 800 nM but low-selectivity over

other PDEs. BAY60-7550²⁸(compound 2, Figure 1), which was

first described in 2002 by Bayer, selectively inhibits PDE2 with

IC₅₀of 4.7 nM and shows excellent in vivo curative effects in rat

models.²⁴However, the clinical application of 2 was limited due

to the lack of good pharmacokinetic properties. Pfizer also

published a series of pyrazolopyrimidine PDE2 inhibitors, and

the best compound PF-05180999²⁹(compound 3, Figure 1) had

been advanced into phase I clinical trial for the treatment of

schizophrenia and migraine in 2012,^8,21but no recent

development had been reported since 2014. Therefore, the

development of novel PDE2 inhibitors is still extensively

prospective and significant.

The

second

messengers

(cAMP) and

cyclic

adenosine

guanosine

3’,5’-monophosphate

3’-5’-monophosphate (cGMP) regulate many physiological

processes, including contraction and relaxation of vascular

smooth muscle and cardiac myocytes, steroid hormone function,

insulin secretion, glycogen synthesis and glycogenolysis,

lipogenesis and lipolysis, inflammation, apoptosis and

memory.^1-5Cyclic nucleotide phosphodiesterases (PDEs) are

solo enzymes that can selectively catalyze the hydrolysis of 3’

cyclic phosphate bonds of cAMP and/or cGMP to change their

intracellular concentrations. PDEs are divided into 11 distinct

families (PDE1-PDE11) based on their structure and

pharmacological properties. Due to their critical roles in the

cellular processes, PDEs have been regarded as therapeutic

targets for many years,^6-12and many family-selective PDE

inhibitors have been approved for clinical use, such as PDE3,¹³

PDE4^14-15and PDE5^16-19inhibitors.

Phosphodiesterase 2 (PDE2) is a dual-specific enzyme that

could hydrolyze both cAMP and cGMP.^6-7PDE2 is mainly

distributed in the central nervous system (CNS) and involved in

the modulations of neuronal signaling such as learning and

memory, which made it an attracting therapeutic target for

In recent years, structure-based virtual screening and

rational optimization have become important methods in the

process of drug discovery. The X-ray crystal structure of PDE2

in complex with 2 was reported in 2013,³⁰which revealed that a

binding induced hydrophobic pocket (H-pocket) of PDE2 might

play an important role in the enhancement of binding affinity and

selectivity for PDE2 besides the conserved glutamine-switch

mechanism³¹for substrates-PDEs recognitions. Furthermore,

another case of inhibitor design to achieve high potency and

selectivity by exploring this uncommon pocket of PDE2 was

reported by Buijnsters et al. in 2014.³²These structural evidences

provide helpful guidelines in the structure-based design of highly

selective PDE2 inhibitors. Herein, an in-house library with ∼800

old drugs was investigated by the method of structure-based

virtual screening, to identify potential PDE2 inhibitors starting

with good pharmacokinetic properties. Ultimately, clofarabine³³

(compound 4, Figure 1), an FDA-approved drug for treating

relapsed or refractory acute lymphoblastic leukaemia (ALL) in

children, was found to be a moderate PDE2 inhibitor with an

IC₅₀value of 3.12 μM by subsequent bioassay. Further

optimization of 4 was carried out to improve the PDE2 inhibitory

activity, and 33 derivatives were designed, synthesized and

tested with biological assays. The most potent compound 14e

with a ~10 folds enhancement of activity was identified, and the

following molecular docking and molecular dynamic (MD)

simulations made it clear that 14e could form extra interactions

with the unique H-pocket of PDE2.

inhibitory activities against PDE2, and lead compound 4

displayed a most potency with IC₅₀of 3.21 µM (Table 1).

Figure 2. Binding mode of 4 (carbon in cyan, oxygen in red, nitrogen in blue,

chlorine in green, fluorine in pale cyan) within the active site pocket of

human PDE2 (PDB ID: 4C1I). Hydrogen bonds are represented by dashed

lines.

2.2. Design and synthesis

The lead compound 4 can be divided into two regions: the

2’-deoxy-2’-fluoro-β-D-arabinofuranosyl moiety and the purine

ring. First, we designed and synthesized derivatives 6-9 by

replaced the 2’-deoxy-2’-fluoro-β-D-arabinofuranosyl moiety

with different kinds of sugar groups to determine whether this

region was necessary for the PDE2 inhibitory activity. Then, we

removed the 2-chloro group (10) or introduced a substituent

group to the 6-amino group (11) on the purine ring to identify

whether these two groups could affect the PDE2 inhibitory

potency. The docked pose of 4 with PDE2 is represented in

Figure 2, and it is possible to optimize the hydroxyl group at

3’-position or 5’-position of the arabinofuranosyl moiety to

introduce interaction with the H-pocket. Thus, these two

positions were modified with benzyl or a series of substituted

benzyl groups (12a, 13a, 14a-k), cyanomethyl (14l), propargyl

(14m), 2-oxo-2-phenylethyl (14n), and 2-chlorophenyl (14o),

and 17 derivatives were designed. In addition, compounds 12b,

13b, and 14p were designed by removing the 2-chloro group of

compounds 12a, 13a, and 14a to identify the importance of the

2-chloro group. Due to a series of 5’-benzyl substituted

compounds with enhanced PDE2 activity were identified by

subsequent bioassay, 5’-benzyl was remained in the following

structure modification while the 2-chloro group was replaced

Figure 1. Structures of compounds 1-3 and lead compound 4.

2. Result and discussion

with

a series of phenyl (15a-e), furan-3-yl (15f), and

2.1. Discovery of lead compound

thiophene-3-yl (15g), another seven new derivatives were

obtained.

Molecular docking was performed to screen our in-house

library of old drugs for discovery of novel PDE2 inhibitors. Both

of the crystal structures of 4HTX and 4C1I were utilized

regarding that the side chain of invariant residue Gln859 in

active site has two conformations rotated by 180°. Firstly, the

crystallized 1 and 2 were re-docked back into the active site and

the average root mean square distance (RMSD) values between

the original X-ray pose and the top 10 re-docking poses were

less than 1.5 Å, which implied our docking parameters were

suitable for the PDE2 screening. The mean scores of re-docking

top ten poses were 11.13 for 4HTX and 6.33 for 4C1I,

respectively, and were used as the thresholds to filter our

in-house library in dock screening. Then, the docked binding

poses of molecules with higher scores than thresholds were

further visually inspected and those that had the conserved π−π

stacking with Phe862 and hydrogen bonds with Gln859 were

retained. Finally, 65 old drugs were selected to test their in vitro

The general synthetic routes for preparation of derivatives

6-9 were described in Scheme 1. Various acetyl or

benzoyl-substituted furanose or pyranose were reacted with

2-chloro-6-aminopurine

in

the

presence

of

N,O-bis(trimethylsilyl)trifluoroacetamide and TsOH under reflux

in acetonitrile to afford intermediates 16-19. Then, 16-19 were

hydrolyzed by sodium methoxide to yield derivatives 6-9.

Scheme 2 depicts the synthesis of derivatives 10-11 and

12a-b. Compound 4 was hydrogenated by hydrogen under the

catalysis of Pd/C to afford compound 10. Then, 4 or 10 were

reacted with 3.2 equivalents of t-butyldimethylsilyl chloride

(TBDMSCl) in the presence of imidazole to afford 20a or 20b,

respectively. Next, 20a was reacted with 1 equivalent of (Boc)₂O

to give 21. After removing the O-protecting groups of 21,

compound 11 was prepared. Intermediates 20a and 20b were

reacted with 4 equivalents of (Boc)₂O to afford 22a and 22b

respectively, which were further treated with

tetrabutylammonium fluoride (TBAF) to afford 23a and 23b,

bromide to afford 24a or 24b via nucleophilic substitution, and

followed by the deprotection reaction to obtain 12a or 12b,

respectively.

respectively. 23a or 23b was coupled with excess benzyl

Scheme 1. Synthesis of compounds 6-9. Reagents and conditions: (a) (i) N,O-bis(trimethylsilyl)trifluoroacetamide, MeCN, reflux, 1 h; (ii) acylsugar, TsOH,

reflux, 1 h; (b) MeOH, MeONa, r.t., 7 h.

Scheme 2. Synthesis of compounds 10-11, and 12a-b. Reagents and conditions: (a) 10% Pd/C, H₂, MeOH, r.t., 2 days; (b) TBDMSCl, imidazole, DMF, r.t.,

overnight; (c) Boc₂O (1 eq.), DMAP, DMF, r.t., overnight; (d) TBAF, THF, 0°C to r.t., 2 h; (e) Boc₂O (4 eq.), DMAP, DMF, r.t., overnight; (f) n-Bu₄NOH, NaI,

NaOH, DCM/H₂O, BnBr, r.t., 4 h; (g) silica gel, toluene, 80°C, 3 h.

Derivatives 13a-b and 14a-p were synthesized through the

route outlined in Scheme 3. Intermediates 23a and 23b were

reacted with 1.3 equivalents of TBDMSCl to afford 25a and 25b,

respectively. Then, 25a and 25b were coupled with benzyl

bromide via nucleophilic substitution to afford 26a and 26b,

which was converted to 27a and 27b after treating with TBAF.

The two N-protecting groups of 27a and 27b were removed

utilizing silica gel in toluene at 80°C to afford 13a and 13b,

respectively. In parallel, 25a and 25b were coupled with

4-methoxybenzyl bromide to afford 28a and 28b, followed by

removing of O-protecting group to give 29a and 29b. Then, 29a

and 29b were reacted with various kinds of substituted benzyl

bromides,

bromoacetonitrile,

3-bromopropyne

or

2-bromoacetophenone to yield 30a-n and 30p, which were

converted to 31a-n and 31p by removing the 4-methoxybenzyl

utilizing DDQ. Ultimately, target compounds 14a-n and 14p

were achieved after removing two N-protecting groups of 31a-n.

Besides, 23a was coupled with 2-chlorophenol by Mitsunobu

reaction to yield 32, and was deprotected to afford compound

14o.

The synthesis of derivatives 15a-g is presented in Scheme 4.

Intermediates 33a-g were obtained from 31a through Suzuki

coupling reaction, which were deprotected to provide 15a-g.

Scheme 3. Synthesis of compounds 13a-b and 14a-p. Reagents and conditions: (a) TBDMSCl, imidazole, DMF, 0°C to r.t., 2 days; (b) n-Bu₄NOH, NaI, NaOH,

DCM/H₂O, BnBr, r.t., 2 h; (c) TBAF, THF, 0°C to r.t., 4 h; (d) silica gel, toluene, 80°C, 3 h; (e) n-Bu₄NOH, NaI, NaOH, DCM/H₂O, 4-methoxybenzyl bromide,

r.t., 2 h; (f) n-Bu₄NOH, NaI, NaOH, DCM/H₂O, R²Br, r.t., 4 h; (g) DDQ, DCM, r.t., 4 h; (h) Diisopropyl azodicarboxylate, PPh₃, anhydrous THF, r.t., overnight.

Scheme 4. Synthesis of compounds 15a-g. Reagents and conditions: (a) R³B(OH)₂, K₂CO₃, Pd(PPh₃)₄, toluene, H₂O, ethanol, 110°C, 12 h; (b) silica gel, toluene,

80°C, 3 h.

2.3. In vitro PDE2 inhibitory activities

IC₅₀>10μM) or xylopyranosyl (9, IC₅₀>10μM), the PDE2

inhibitory activities were decreased significantly (Table 1).

Furthermore, removing 2-chloro group on the purine (10,

IC₅₀>10μM) remarkably affected the PDE2 inhibitory activities.

In addition, a substituent group was introduced to the 6-amino

group (11, IC₅₀>10μM), which resulting in a dramatic loss of

activities, and it was consistent with the binding mode of 4 (Fig.

2) that the 6-amino nitrogen on the purine had the conserved

hydrogen bonds with Gln859. Subsequently, we retained

2’-fluoro-β-D-arabinofuranosyl and 6-amino group on the purine,

and modified the 3’-position and 5’-position hydroxyl group of

arabinofuranosyl and 2-chloro group.

The prepared derivatives were tested for in vitro PDE2

inhibitory activities. EHNA (1) purchased from SIGMA was

used as the reference compound. The results are summarized in

Table 1-2. Firstly, we varied the groups on the arabinofuranose

moiety to afford compounds 6 and 7, and a commercial

compound 5 (Cladribine, purchased from Aladdin) were also

tested in order to study the structure-activity relationships

(SARs). Once the 2’-fluoro group was removed (5, IC₅₀>10μM)

or replaced by a hydroxyl group (6, IC₅₀>10μM), the PDE2

inhibitory activities abated (Table 1). Similarly, when the

arabinofuranosyl was replaced by the glucopyranosyl (8,

Table 1. Chemical structures of 5-11 and their PDE2 inhibitory activities.

PDE2 inhibitory assay

Compd.

Chemical structure

Inhibition ratio

IC₅₀(μM)^a

(1 μM)

32.13%

3.12±0.67

4

5

7.91%

>10

1.83%

3.07%

0.68%

>10

6

7

8

1.59%

5.66%

1.22%

>10

9

10

11

^aResults are expressed as the mean of at least three experiments.

The data in Table 2 revealed that the introduction of a

benzyl at the 3’-position and/or 5’-position of the

arabinofuranosyl (12a, 13a, and 14a) was beneficial for

improving the potency, especially for 5’-benzyl substituent

compound 14a (IC₅₀= 0.41 ± 0.01 μM). Then, we replaced the

5’-benzyl with various substituted benzyl (14b-k), cyanomethyl

(14l), propargyl (14m), 2-oxo-2-phenylethyl (14n), and

2-chlorophenyl (14o). Among them, most of benzyl substituent

compounds (14b-i) displayed optimized PDE2 inhibitory

potency. In general, the substituted positions at the benzyl

affected the activity, and ortho-position of the benzyl was the

best substitution site (14b vs 14c and 14d, 14e vs 14f and 14g).

Additionally, compounds 12b, 13b and 14p exhibited worse

activities compared to 12a, 13a and 14a, respectively, which

strongly supporting the previous summary (4 vs 10, Table 1) that

2-position substitution of the purine was crucial for the

biological activity. Thus, the 2-position of the purine was

considered as a modification site, and the following 2-substituted

and 5’-benzyl substituted derivatives 15a-g was prepared.

Unfortunately, 15a-g displayed a lower potency than 14a, which

indicated that 2-chloro substitution of the purine was mostly

beneficial. Notably, the most potent compound 14e (IC₅₀= 0.32

± 0.04 μM), was ~10-fold more potent than lead compound 4

(IC₅₀= 3.12 ± 0.67 μM, Table 1).

Table 2. Chemical structures of 12a-b, 13, 14a-o and 15a-g, and their PDE2 inhibitory activities.

PDE2 inhibitory assay

Inhibition ratio

Compd.

R¹

R²

R³

IC₅₀(μM)^a

(1 μM)

Cl

H

54.54%

0.74±0.09

12a

12b

13a

13b

14a

14b

14c

14d

14e

26.98%

38.73%

5.46%

—

Cl

H

—

Cl

H

74.93%

72.19%

43.89%

42.21%

81.29%

0.41±0.01

0.47±0.07

—

0.32±0.04

Cl

H

53.50%

1.15±0.06

14f

14g

14h

14i

Cl

H

62.91%

77.91%

67.22%

29.63%

32.10%

0.67±0.05

0.37±0.04

0.62±0.06

—

14j

14k

—

Cl

H

28.85%

41.34%

—

14l

14m

Cl

H

3.52%

—

14n

14o

14p

Cl

H

22.35%

43.41%

—

H

24.77%

—

15a

15b

15c

15d

H

17.40%

26.85%

27.06%

—

15e

15f

H

34.83%

42.87%

—

15g

42.31%

37.45%

—

2.46 ± 0.19

EHNA

^aResults are expressed as the mean of at least three experiments; “—” indicates no test.

2.4. The binding mode of 14e with PDE2

driving force for inhibitors’ binding. Furthermore, the 5’-benzyl

group of 14e could extend to the H-pocket and form great van

der Waals interactions with the hydrophobic residues such as

Leu770, Thr768, Thr805 and Leu809. The structural

superposition of 14e and lead compound 4 bound to PDE2

(Figure 3B) showed that 14e shifted to the inner region and the

hydrogen bond with Gln812 was unstable due to the steric

hindrance compared with 4. Also, the arabinofuranosyl moiety

rotated some degree to fit the binding with H-pocket which

seemed to be very important for its inhibitory activity.

Molecular docking and molecular dynamic (MD)

simulations were used to explore the interactions of PDE2 and

14e. Compound 14e was firstly docked into the binding pocket

of PDE2 by using the same protocol as virtual screening

described above. Then, the binding mode was selected in

consideration of high docking score and conserved interaction

with Gln859/Phe862. Since docking only reflects a possibly

instantaneous and unstable binding, 8 ns MD simulation was

further applied by using the docked complex structure as initial

model and the RMSD curve was monitored to be stable

indicating that the system was well equilibrated. Finally, the

binding pose was generated by averaging the 100 snapshots from

Besides these direct observations, the binding mode could

be also analyzed from the energy point of view. As shown in

Table 3, the predicted binding free energies calculated by

molecular

mechanism/Poison-Boltzman

surface

area

the last 1 ns MD trajectories.

(MM-PBSA) method were -23.3 and -20.79 kcal/mol for the

complex of PDE2-14e and PDE2-4, respectively, which was

consistent with their inhibitory potencies (0.32 and 3.12 µM). As

for the detailed energy items, it seemed that the van der

Waals/hydrophobic interactions were the predominant force

facilitate the binding of 14e, which might compensate the

relatively weak electrostatic interactions in the unstable

hydrogen bond with Gln812.

As shown in Figure 3A, 14e bound to the catalytic pocket

mostly by interacting with the Q and H sub-pockets. In the Q

region, the purine ring was sandwiched by the hydrophobic

residues of Phe862 on one side and Phe830/Ile826 on the other

side, and the amino hydrogen atom formed a hydrogen bond

with amide oxygen of Gln859 side chain. These two interactions

were conserved in many PDE inhibitors and could be the main

Figure 3. (A) Binding mode of 14e (carbon in cyan, oxygen in red, nitrogen in blue, chlorine in green, fluorine in pale cyan) within the active site pocket of

human PDE2 (PDB ID: 4C1I); (B) Binding mode of 14e (carbon in cyan, oxygen in red, nitrogen in blue, chlorine in green, fluorine in pale cyan) after

superposition on the binding pose of 4 (carbon in yellow, oxygen in red, nitrogen in blue, chlorine in green, fluorine in pale cyan) within the active site pocket of

human PDE2 (PDB ID: 4C1I). Hydrogen bonds are represented by dashed lines.

Table 3. Predicted free energies (kcal/mol) for binding of 4 or 14e to PDE2 by MM-PBSA binding free energies method.

Energy terms (kcal/mol)

PDE2 + 4

PDE2 + 14e

a

-34.59 ± 4.31

-26.20 ± 3.86

ΔG_ele

b

-33.06 ± 2.46

-67.65 ± 3.65

-3.16 ± 0.07

50.01 ± 2.78

46.86 ± 2.79

-20.79 ± 2.41

-51.16 ± 2.77

-77.37 ± 4.80

-6.27 ± 0.10

60.31 ± 5.85

54.04 ± 5.85

-23.33 ± 6.73

ΔG_vdW

c

ΔG_MM

d

ΔG_nonpol,sol

e

ΔG_ele,sol

f

ΔG_sol

g

ΔG_bind

b

^aΔG_eleare electrostatic interactions calculated by the MM force field. ΔG_vdWare van der Waals contributions from MM. ^cΔG_MM= ΔG_ele+ ΔG_vdW. ^dΔG_nonpol,solare

e

f

g

the nonpolar contribution to solvation. ΔG_ele,solare the polar contribution to solvation. ΔG_sol= ΔG_ele,sol+ ΔG_nonpol,sol. ΔG_bindare binding free energies in the

absence of entropic contribution.

Binding free energies of the complex of PDE2-14e and

PDE2-4 could further decomposed into single-residue

contributions in a quantitative way by using the MM-PBSA

method (Figure 4). In general, a residue is considered to be

important for recognition of ligands if the decomposed energy is

more negative than −1 kcal/mol. Our results suggested that the

following residues were important for the binding of 14e and 4

to PDE2: Gln812(-3 and -3.52 kcal/mol), Gln859(-3.71 and -3.4

kcal/mol), Ile826(-2.17 and -2.77 kcal/mol), Phe862(-2.45 and

-2.62 kcal/mol), Tyr655(-1.85 and -1.81 kcal/mol), Leu809(-1.78

and -1.12 kcal/mol), Thr768(-1.74 and 0.01 kcal/mol),

Thr805(-1.39 and -0.02 kcal/mol) and Leu770(-1.06 and -0.08

kcal/mol). As expected, the hydrophobic clamp(Phe862 and

Ile826) and the hydrogen bond sites(Gln859 and Gln812) mostly

contributed to the binding free energies, in agreement with the

proposed binding patterns, and the relatively weak interactions

of the purine ring of 14e with these four residues caused by

induced shift were also well reflected. Moreover, it clearly

shown that the hydrophobic residues (Leu770, Thr768, Thr805

and Leu809) located in the H-pocket of PDE2 had great

interactions with 14e, which might contribute to the

enhancement of its inhibitory activity. The binding energy

calculation and decomposition results helped to validate the

binding patterns from our MD simulations and were

supplementary to the binding affinity results.

Figure 4. Decomposition of the key residue contributions to the binding free energies for the complexes of PDE2 with clofarabine (4) and 14e.

3. Conclusions

To discover novel PDE2 inhibitors, a structure based virtual

screening was utilized to identify potential leads from an

in-house library of ~800 old drugs. Sixty-five old drugs were

picked out for further in vitro evaluation, and compound 4 (IC₅₀

= 3.12 ± 0.67μM) exhibited the most potent PDE2 inhibitory

activity. Based on the structure and binding mode of 4, a series

of purine nucleoside derivatives were synthesized and tested

with PDE2 inhibitory assays, which led to the identification of

seven compounds (12a, 14a-b, 14e, 14g-i) with significant

improvement of inhibitory potency. Among them, compound 14e

with an IC₅₀value of 0.32 μM demonstrated PDE2 inhibitory

activity about 10 times higher than that of the lead compound 4.

Binding mode of 14e with PDE2 showed that the 5’-benzyl

group of 14e could extend to the unique H-pocket and form great

van der Waals interactions, which mainly contributed to its

enhancement on PDE2 inhibition. This compound in our study

provides a basis for the rational design of novel PDE2 inhibitors

with high affinity.

4. Experimental section

2H), 5.77 (d, J = 5.2 Hz, 1H), 4.60 (t, J = 5.1 Hz, 1H), 4.00–3.92

(m, 2H), 1.31 (d, J = 6.3 Hz, 3H). ESI-MS m/z 286.1 [M+H]⁺;

HRMS (ESI) m/z calcd C₁₀H₁₃ClN₅O₃[M+H]⁺286.0707, found

286.0709.

4.1. Chemistry

Solvents and reagents were purchased from Acros,

Adamas-beta, Alfa Aesar, Energy Chemical, J&K, Shanghai

Chemical Reagent Co., and TCI, and were used without further

purification. Analytical thin-layer chromatography (TLC) was

performed on HSGF 254 (150−200 μm thickness; Yantai Huiyou

Co., China). Nuclear magnetic resonance (NMR) spectra were

recorded with a Bruker AMX-400 NMR (IS as TMS). Chemical

shifts were reported in parts per million (ppm, δ) downfield from

tetramethylsilane. Proton coupling patterns were described as

singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and

broad (br). Low- and high-resolution mass spectra (LRMS and

HRMS) were obtained by electric ionization (EI) and

electrospray ionization (ESI) using a Finnigan MAT-95. HPLC

data analysis of compounds 6-11, 12a-b, 13, 14a-o, 15a-g were

performed on an Agilent 1100 with a quaternary pump and

diode-array detector (DAD). The peak purity was verified with

UV spectra. All analogs were confirmed to be ≥95% pure (Table

S1, Supporting information).

4.1.4. 6-Amino-2-chloro-9-(β-D-glucopyranosyl)-9H-purine (8)

8 was synthesized by the general procedure of 6,

β-D-ribofuranose 1-acetate 2,3,5-tribenzoate was replaced by

D-glucose pentaacetate, to give 8 as a white solid (69% yield).

¹H NMR (400 MHz, DMSO-d6) δ: 8.36 (s, 1H), 7.80 (s, 2H),

5.39–5.27 (m, 3H), 5.16 (d, J = 5.5 Hz, 1H), 4.60 (d, J = 5.6 Hz,

1H), 3.92 (td, J = 9.1, 5.8 Hz, 1H), 3.71 (dd, J = 10.4, 5.0 Hz,

1H), 3.48–3.39 (m, 3H), 3.25 (dt, J = 14.4, 7.2 Hz, 1H). ESI-MS

m/z 332.0 [M+H]⁺; HRMS (ESI) m/z calcd C₁₁H₁₅ClN₅O₅

[M+H]⁺332.0756, found 332.0756.

4.1.5. 6-Amino-2-chloro-9-(β-D-xylopyranosyl)-9H-purine (9)

9 was synthesized by the general procedure of 6,

β-D-ribofuranose 1-acetate 2,3,5-tribenzoate was replaced by

D-xylopyranose tetraacetate, to give 9 as a white solid (63%

1

yield). H NMR (400 MHz, DMSO-d6) δ: 8.35 (s, 1H), 7.84 (s,

2H), 5.48 (s, 2H), 5.26 (m, 2H), 3.93 (m, 1H), 3.84 (dd, J = 11.1,

5.2 Hz, 1H), 3.49 (m, 1H), 3.31 (m, 1H), 3.17 (d, J = 4.6 Hz, 1H).

EI-MS m/z 301.1 (M⁺); 169 (100%); HRMS (EI) m/z calcd

C₁₀H₁₂ClN₅O₄(M⁺) 301.0578, found 301.0574.

4.1.1.

6-Amino-2-chloro-9-(2’,3’,5’-tri-O-benzoyl-β-D-ribofuranosyl)-

9H-purine (16)

A

mixture of N,O-bis(trimethylsilyl)trifluoroacetamide

4.1.6.

(4.55 g, 17.7 mmol) and 2-chloro-6-aminopurine (1 g, 5.9 mmol)

in acetonitrile (12 mL) was refluxed for 1 h under a nitrogen

atmosphere. When the reaction solution was nearly clarified,

p-toluenesulfonic acid (0.175 g, 1.02 mmol) and

β-D-ribofuranose 1-acetate 2,3,5-tribenzoate (2.85 g, 5.65 mmol)

was slowly added to the reaction solution, and keep refluxing for

1 h. Then, the reaction solution was cooled to room temperature

and treated with methyl tert-butyl ether (10 mL), washed with

saturated sodium bicarbonate (15 mL). The organic layer was

evaporated under reduced pressure and purified by silica gel

column chromatography, eluting with EtOAc/petroleum ether

6-Amino-9-(2’-deoxy-2’-fluoro-β-D-arabinofuranosyl)-9H-purin

e (10)

To a solution of 4 (100 mg, 0.33 mmol) in methanol (15 mL)

was added palladium on carbon (containing 10% palladium, 20

mg, 0.19 mmol), and the mixture was stirred at room

temperature for 2 days under an atmosphere of H₂supplied via a

balloon. After adjusting the PH of the mixture to neutral with a

small amount of aqueous ammonia, the solvent was evaporated

under reduced pressure and the residue was purified by silica gel

column chromatography, eluting with MeOH/CH₂Cl₂(1/10, v/v)

1

(1/4, v/v) to obtain 16 as a white solid (2.01 g, 58% yield). H

to afford 10 as a white solid (51 mg, 57% yield). H NMR (400

NMR (400 MHz, CDCl₃) δ: 8.09 (d, J = 7.8 Hz, 2H), 7.98 (m,

5H), 7.65–7.51 (m, 3H), 7.51–7.33 (m, 6H), 6.46 (d, J = 3.3 Hz,

1H), 6.18 (m, 4H), 4.90 (d, J = 12.1 Hz, 1H), 4.83 (d, J = 2.7 Hz,

1H), 4.73 (dd, J = 12.1, 4.0 Hz, 1H).

MHz, DMSO-d6) δ: 8.40 (s, 1H), 7.87 (s, 2H), 5.81 (d, J = 5.9

Hz, 1H), 5.13 (s, 1H), 4.49 (t, J = 5.4 Hz, 1H), 4.15–4.09 (m,

1H), 3.93 (q, J = 3.7 Hz, 1H), 3.69–3.50 (m, 2H). ESI-MS m/z

302.0 [M+H]⁺; HRMS (ESI) m/z calcd C₁₀H₁₃ClN₅O₄[M+H]⁺

302.0656, found 302.0658.

4.1.2. 6-Amino-2-chloro-9-(β-D-ribofuranosyl)-9H-purine (6)

4.1.7.

A solution of sodium methoxide (30% in methanol, 0.1 mL)

was added to a solution of 16 (2.5 g, 4.1 mmol) in methanol (15

mL), and the resulting solution was stirred at room temperature

for 7 h. After cooling the mixture to room temperature, glacial

acetic acid (0.025 mL) was added, and the mixture was stirred at

-10 °C for 1 h. The precipitate was filtered and washed with a

small amount of cold methanol followed by drying to afford 6 as

a white solid (823 mg, 67% yield). ¹H NMR (400 MHz,

DMSO-d6) δ: 8.40 (s, 1H), 7.87 (s, 2H), 5.81 (d, J = 5.9 Hz, 1H),

5.13 (s, 1H), 4.49 (t, J = 5.4 Hz, 1H), 4.15–4.09 (m, 1H), 3.93 (q,

J = 3.7 Hz, 1H), 3.69–3.50 (m, 2H). ESI-MS m/z 302.0 [M+H]⁺;

HRMS (ESI) m/z calcd C₁₀H₁₃ClN₅O₄[M+H]⁺302.0656, found

302.0658.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-3’,5’-di-O-tert-butyldim

ethylsilyl-β-D-arabinofuranosyl)-9H-purine (20a)

A solution of 4 (1.228 g, 4.04 mmol), imidazole (2.35 g,

34.52 mmol), and t-butyldimethylsilyl chloride (1.95 g, 12.94

mmol) in anhydrous DMF (12 mL) was stirred at room

temperature overnight. The mixture was poured into saturated

NH₄Cl (80 mL) and extracted with dichloromethane. The organic

layer was dried with anhydrous Na₂SO₄and evaporated under

reduced pressure. The residue was purified by silica gel column

chromatography, eluting with EtOAc/petroleum ether (1/2, v/v)

1

to afford 20a as white foam (2 g, 93% yield). H NMR (400

MHz, CDCl₃) δ: 8.18 (s, 1H), 6.45 (dd, J = 16.7, 3.8 Hz, 1H),

6.16 (s, 2H), 5.03 (d, J = 52.1 Hz, 1H), 4.61 (d, J = 17.8 Hz, 1H),

3.99–3.93 (m, 1H), 3.90–3.80 (m, 2H), 0.92 (m, 18H), 0.12 (m,

12H).

4.1.3.

6-Amino-2-chloro-9-(5’-deoxy-β-D-ribofuranosyl)-9H-purine (7)

7 was synthesized by the general procedure of 6,

β-D-ribofuranose 1-acetate 2,3,5-tribenzoate was replaced by

1,2,3-triacetyl-5-deoxy-D-ribose, to give 7 as a white solid (58%

4.1.8.

6-Amino-9-(2’-deoxy-2’-fluoro-3’,5’-di-O-tert-butyldimethylsilyl

-β-D-arabinofuranosyl)-9H-purine (20b)

1

yield). H NMR (400 MHz, DMSO-d6) δ: 8.36 (s, 1H), 7.84 (s,

20b was synthesized by the general procedure of 20a to

afford 20b as a white solid (91% yield). H NMR (400 MHz,

CDCl₃) δ 8.37 (s, 1H), 8.12 (d, J = 1.8 Hz, 1H), 6.50 (dd, J =

18.3, 3.5 Hz, 1H), 6.03 (s, 2H), 5.01 (d, J = 52.0 Hz, 1H), 4.62 (d,

J = 17.1 Hz, 1H), 3.99 (dd, J = 8.8, 4.5 Hz, 1H), 3.85 (d, J = 4.8

Hz, 2H), 0.93 (d, J = 5.0 Hz, 18H), 0.15 (s, 6H), 0.11 (s, 6H).

(6.5 mL) was added a solution of tetrabutylammonium fluoride

(1.76 g, 6.73mmol) in tetrahydrofuran (6.5 mL) at 0 °C, and then

the reaction was allowed to warm to room temperature and

stirred for 2 h. The solvent was evaporated under reduced

pressure, and the residue was purified by silica gel column

chromatography, eluting with EtOAc/petroleum ether (1/1, v/v)

1

to afford 23a as white foam (1.049 g, 93% yield). H NMR (400

4.1.9.

MHz, Acetone-d6) δ: 8.74 (s, 1H), 6.62 (dd, J = 13.8, 4.4 Hz,

1H), 5.49–5.32 (m, 1H), 5.28 (d, J = 4.9 Hz, 1H), 4.73 (ddd, J =

18.4, 9.3, 4.7 Hz, 1H), 4.39 (t, J = 5.7 Hz, 1H), 4.10 (dd, J = 9.1,

4.6 Hz, 1H), 3.89 (m, 2H), 1.46 (s, 18H).

2-Chloro-N⁶-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro-3’,5’-

di-O-tert-butyldimethylsilyl-β-D-arabinofuranosyl)-9H-purine

(21)

To a solution of 20a (0.6 g, 1.13 mmol) in anhydrous DMF

(5 mL) was added di-tert-butyl dicarbonate (0.26 mL, 1.13 mmol)

and 4-Dimethylaminopyridine (0.166 mg, 1.36 mmol), and the

mixture was stirred at room temperature overnight under a

nitrogen atmosphere. The mixture was poured into saturated

NH₄Cl (50 mL) and extracted with dichloromethane. The organic

layer was dried with anhydrous Na₂SO₄and evaporated under

reduced pressure. The residue was purified by silica gel column

chromatography, eluting with EtOAc/petroleum ether (1/5, v/v)

4.1.13.

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-3’,5’-di-O-benzyl-β-D-arabinofuranosyl)-9H-purine (24a)

To

a

solution of 23a (0.12 g, 0.24 mmol) in

dichloromethane (0.5 mL) was added a mixture consisting of 1M

aqueous sodium hydroxide solution (0.72 mL), sodium iodide

(4.8 mg, 0.03 mmol), and tetrabutylammonium hydroxide (0.226

mL, 0.87 mmol), followed by the addition of a solution of benzyl

bromide (0.244 g, 1.43 mmol) in dichloromethane (0.5 mL). The

mixture was stirred at room temperature in the dark for 4 h. The

mixture was poured into water (10 mL), and extracted with

dichloromethane. The organic layer was separated, dried with

anhydrous Na₂SO₄and evaporated under reduced pressure. The

residue was purified by silica gel column chromatography,

eluting with EtOAc/petroleum ether (1/4, v/v) to afford 24a as

white foam (121 mg, 74% yield). ¹H NMR (400 MHz, CDCl₃) δ:

8.32 (d, J = 1.8 Hz, 1H), 7.42–7.28 (m, 12H), 6.52 (dd, J = 18.2,

3.5 Hz, 1H), 5.20 (d, J = 51.5 Hz, 1H), 4.72–4.53 (m, 4H), 4.34

(d, J = 17.6 Hz, 1H), 4.27 (dd, J = 9.2, 4.7 Hz, 1H), 3.74–3.65

(m, 2H), 1.51–1.41 (m, 18H).

1

to afford 21 as white foam (599 mg, 84% yield). H NMR (400

MHz, CDCl₃) δ: 8.22 (d, J = 2.5 Hz, 1H), 8.09 (s, 1H), 6.49 (dd,

J = 17.5, 3.8 Hz, 1H), 5.11–4.93 (m, 1H), 4.62 (ddd, J = 17.7,

3.9, 2.6 Hz, 1H), 3.97 (q, J = 4.5 Hz, 1H), 3.85 (d, J = 4.5 Hz,

2H), 1.56 (s, 9H), 0.94 (s, 9H), 0.93 (s, 9H), 0.17–0.10 (m, 12H).

4.1.10.

2-Chloro-N⁶-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro-β-D-

arabinofuranosyl)-9H-purine (11)

To a solution of 21 (0.6 g, 0.95 mmol) in tetrahydrofuran (3

mL) was added a solution of tetrabutylammonium fluoride (0.75

g, 2.85mmol) in tetrahydrofuran (3 mL) at 0 °C, and then the

reaction was allowed to warm to room temperature and stirred

for 2 h. The solvent was evaporated under reduced pressure, and

the residue was purified by silica gel column chromatography,

eluting with MeOH/CH₂Cl₂(1/20, v/v) to afford 11 as white

4.1.14.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-3’,5’-di-O-benzyl-β-D-a

rabinofuranosyl)-9H-purine (12a)

1

foam (314 mg, 82% yield). H NMR (400 MHz, Acetone-d6) δ:

To a solution of 24a (35 mg, 0.05 mmol) in toluene (5 mL)

was added dry silica gel (100-200 mesh, 300 mg), and then the

mixture was stirred at 80 °C for 3 h. The solvent was evaporated

under reduced pressure, and the residue was purified by silica gel

column chromatography, eluting with EtOAc/petroleum ether

9.43 (s, 1H), 8.51 (s, 1H), 6.54 (dd, J = 14.7, 4.3 Hz, 1H), 5.34

(dt, J = 52.3, 3.8 Hz, 1H), 4.71 (d, J = 18.3 Hz, 1H), 4.40 (s, 1H),

4.08 (q, J = 4.5 Hz, 1H), 3.96–3.79 (m, 2H), 1.53 (s, 9H).

ESI-MS m/z 404.1 [M+H]⁺; HRMS (ESI) m/z calcd C₁₅H₂₀FN₅O₃

[M+H]⁺404.11137, found 404.11135.

1

(1/2, v/v) to afford 12a as a white solid (17 mg, 70% yield). H

NMR (400 MHz, CDCl₃) δ: 8.20 (s, 1H), 7.36 (m, 10H), 6.47 (dd,

J = 17.5, 3.7 Hz, 1H), 5.30–5.11 (m, 1H), 4.73–4.54 (m, 4H),

4.39–4.30 (m, 1H), 4.27 (dd, J = 9.3, 4.7 Hz, 1H), 3.70 (d, J =

4.7 Hz, 2H). EI-MS m/z 483.1 (M⁺); 198 (100%); HRMS (EI)

m/z calcd C₂₄H₂₃ClFN₅O₃(M⁺) 483.1473, found 483.1471.

4.1.11.

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-3’,5’-di-O-tert-butyldimethylsilyl-β-D-arabinofuranosyl)-9H-pu

rine (22a)

To a solution of 20a (1.2 g, 2.25 mmol) in anhydrous DMF

(6 mL) was added di-tert-butyl dicarbonate (2.06 mL, 8.97 mmol)

and 4-Dimethylaminopyridine (1.1 g, 9.00 mmol), and the

mixture was stirred at room temperature overnight under a

nitrogen atmosphere. The mixture was poured into saturated

NH₄Cl (80 mL) and extracted with dichloromethane. The organic

layer was dried with anhydrous Na₂SO₄and evaporated under

reduced pressure. The residue was purified by silica gel column

chromatography, eluting with EtOAc/petroleum ether (1/5, v/v)

4.1.15.

6-Amino-9-(2’-deoxy-2’-fluoro-3’,5’-di-O-benzyl-β-D-arabinofu

ranosyl)-9H-purine (12b)

12b was synthesized by the general procedure of 12a to

afford 12b as a white solid (64% yield). H NMR (400 MHz,

1

CDCl₃) δ: 8.35 (s, 1H), 8.08 (d, J = 2.5 Hz, 1H), 7.43 – 7.28 (m,

10H), 6.50 (dd, J = 19.4, 3.4 Hz, 1H), 5.91 (s, 2H), 5.18 (d, J =

52.7 Hz, 1H), 4.75 – 4.54 (m, 4H), 4.38–4.30 (m, 1H), 4.27 (dd,

J = 9.5, 4.9 Hz, 1H), 3.77–3.64 (m, 2H). EI-MS m/z 449.2 (M⁺);

358 (100%); HRMS (EI) m/z calcd C₂₄H₂₄FN₅O₃(M⁺) 449.1863,

found 449.1871.

1

to afford 22a as yellow foam (1.438 g, 87% yield). H NMR

(400 MHz, CDCl₃) δ: 8.26 (s, 1H), 6.41 (dd, J = 16.6, 3.8 Hz,

1H), 4.92 (d, J = 52.1 Hz, 1H), 4.52 (d, J = 17.8 Hz, 1H),

3.90–3.84 (m, 1H), 3.79–3.69 (m, 2H), 1.34 (s, 18H), 0.82 (m,

18H), 0.01 (m, 12H).

4.1.16.

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-5’-O-tert-butyldimethylsilyl-β-D-arabinofuranosyl)-9H-purine

(25a)

4.1.12.

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-β-D-arabinofuranosyl)-9H-purine (23a)

To a solution of 23a (0.9 g, 1.79 mmol) and imidazole

To a solution of 22a (1.64 g, 2.24 mmol) in tetrahydrofuran

(0.316 g, 4.64 mmol) in anhydrous DMF (4 mL) was added a

solution of t-butyldimethylchlorosilane (0.352 g, 2.34 mmol) in

anhydrous DMF (1 mL) at 0 °C, and then the reaction was

allowed to warm to room temperature slowly and stirred for 2

days. The mixture was poured into saturated NH₄Cl (50 mL) and

extracted with dichloromethane. The organic layer was dried

with anhydrous Na₂SO₄and concentrated under reduced pressure.

The residue was purified by silica gel column chromatography,

eluting with EtOAc/petroleum ether (1/5, v/v) to afford 25a as

white foam (961 mg, 87% yield). ¹H NMR (400 MHz, CDCl₃) δ:

8.38 (d, J = 1.6 Hz, 1H), 6.55 (dd, J = 16.9, 3.7 Hz, 1H),

5.24–5.04 (m, 1H), 4.74–4.60 (m, 1H), 4.06 (dd, J = 9.3, 4.6 Hz,

1H), 3.96–3.86 (m, 2H), 1.46 (s, 18H), 0.92 (s, 9H), 0.11 (s, 6H).

yl)-9H-purine (13b)

13b was synthesized by the general procedure of 13a to

afford 13b as a white solid (51% yield). H NMR (400 MHz,

1

DMSO-d6) δ: 8.25 (d, J = 2.0 Hz, 1H), 8.16 (s, 1H), 7.47–7.29

(m, 7H), 6.40 (dd, J = 16.7, 4.3 Hz, 1H), 5.52 (d, J = 51.7 Hz,

2H), 5.22 (s, 1H), 4.71 (dd, J = 30.6, 11.7 Hz, 2H), 4.45 (d, J =

19.0 Hz, 1H), 4.04 (d, J = 4.6 Hz, 1H), 3.67 (dd, J = 10.8, 5.6 Hz,

2H). EI-MS m/z 359.1 (M⁺); 91 (100%); HRMS (EI) m/z calcd

C₁₇H₁₈FN₅O₃(M⁺) 359.1394, found 359.1388.

4.1.21.

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-3’-O-(4-methoxybenzyl)-5’-O-tert-butyldimethylsilyl-β-D-arabi

nofuranosyl)-9H-purine (28a)

4.1.17.

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-3’-O-benzyl-5’-O-tert-butyldimethylsilyl-β-D-arabinofuranosyl)

-9H-purine (26a)

28a was synthesized by the general procedure of 26a to

afford 28a as white foam (65% yield). H NMR (400 MHz,

1

CDCl₃) δ: 8.34 (s, 1H), 7.29 (d, J = 8.3 Hz, 2H), 6.91 (t, J = 8.9

Hz, 2H), 6.50 (dd, J = 18.6, 3.4 Hz, 1H), 5.25–5.09 (m, 1H),

4.60 (q, J = 11.4 Hz, 2H), 4.37 (d, J = 19.9 Hz, 1H), 4.16–4.10

(m, 1H), 3.82 (d, J = 4.7 Hz, 5H), 1.45 (s, 18H), 0.92 (s, 9H),

0.08 (d, J = 4.0 Hz, 6H).

To

a

solution of 25a (0.74 g, 1.20 mmol) in

dichloromethane (2 mL) was added a mixture consisting of 1M

aqueous sodium hydroxide solution (1.88 mL), sodium iodide

(12 mg, 0.08 mmol), and tetrabutylammonium hydroxide (0.573

mL, 2.20 mmol), followed by the addition of a solution of benzyl

bromide (0.614 g, 3.59 mmol) in dichloromethane (1.25 mL).

The mixture was stirred at room temperature in the dark for 2 h.

The mixture was poured into water (10 mL), and extracted with

dichloromethane. The organic layer was separated, dried with

anhydrous Na₂SO₄and evaporated under reduced pressure. The

residue was purified by silica gel column chromatography,

eluting with EtOAc/petroleum ether (1/5, v/v) to afford 26a as

white foam (653 mg, 77% yield). ¹H NMR (400 MHz, CDCl₃) δ:

8.25 (d, J = 2.5 Hz, 1H), 7.34–7.22 (m, 5H), 6.43 (dd, J = 18.7,

3.5 Hz, 1H), 5.20–5.03 (m, 1H), 4.59 (q, J = 11.8 Hz, 2H),

4.34–4.25 (m, 1H), 4.10–4.03 (m, 1H), 3.80–3.69 (m, 2H), 1.37

(s, 18H).

4.1.22.

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-3’-O-(4-methoxybenzyl)-β-D-arabinofuranosyl)-9H-purine

(29a)

29a was synthesized by the general procedure of 27a to

1

afford 29a as white foam (93% yield). H NMR (400 MHz,

CDCl₃) δ: 8.30 (s, 1H), 7.29 (t, J = 6.3 Hz, 2H), 6.91 (t, J = 8.9

Hz, 2H), 6.48 (dd, J = 18.3, 3.3 Hz, 1H), 5.28–5.10 (m, 1H),

4.64 (dd, J = 26.2, 11.5 Hz, 2H), 4.40 (d, J = 18.0 Hz, 1H), 4.15

(dd, J = 9.0, 4.8 Hz, 1H), 3.96–3.77 (m, 6H), 1.46 (s, 18H).

4.1.23.

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-3’-O-(4-methoxybenzyl)-5’-O-benzyl-β-D-arabinofuranosyl)-9H

-purine (30a)

4.1.18.

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-3’-O-benzyl-β-D-arabinofuranosyl)-9H-purine (27a)

To

a

solution of 29a (0.68 g, 1.09 mmol) in

To a solution of 26a (0.65 g, 0.92 mmol) in tetrahydrofuran

(5 mL) was added a solution of tetrabutylammonium fluoride

(0.36 g, 1.38 mmol) in tetrahydrofuran (2.2 mL) at 0 °C, and

then the mixture was allowed to warm to room temperature for 4

h. The solvent was evaporated under reduced pressure, and the

residue was purified by silica gel column chromatography,

eluting with EtOAc/petroleum ether (1/1, v/v) to afford 27a as

white foam (545 mg, 91% yield).

dichloromethane (2 mL) was added a mixture consisting of 1M

aqueous sodium hydroxide solution (1.64 mL), sodium iodide

(10.9 mg, 0.07 mmol), and tetrabutylammonium hydroxide

(0.512 mL, 1.96 mmol), followed by the addition of a solution of

benzyl bromide (0.56 g, 3.27 mmol) in dichloromethane (1.09

mL). The mixture was stirred at room temperature in the dark for

4 h. The mixture was poured into water (10 mL), and extracted

with dichloromethane. The organic layer was separated, dried

with anhydrous Na₂SO₄and evaporated under reduced pressure.

The residue was purified by silica gel column chromatography,

eluting with EtOAc/petroleum ether (1/2, v/v) to afford 30a as

white foam (490 mg, 63% yield). ¹H NMR (400 MHz, CDCl₃) δ:

8.32 (s, 1H), 7.41 – 7.20 (m, 7H), 6.89 (d, J = 8.3 Hz, 2H), 6.51

(dd, J = 18.2, 3.4 Hz, 1H), 5.24–5.08 (m, 1H), 4.66–4.52 (m, 4H),

4.32 (d, J = 17.6 Hz, 1H), 4.24 (dd, J = 9.1, 4.6 Hz, 1H), 3.82 (d,

J = 6.2 Hz, 3H), 3.67 (t, J = 11.3 Hz, 2H), 1.48 (s, 18H).

4.1.19.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-3’-O-benzyl-β-D-arabin

ofuranosyl)-9H-purine (13a)

To a solution of 27a (0.5 g, 0.84 mmol) in toluene (10 mL)

was added dry silica gel (100-200 mesh, 900 mg), and then the

mixture was stirred at 80 °C for 3 h. The solvent was evaporated

under reduced pressure, and the residue was purified by silica gel

column chromatography, eluting with EtOAc/petroleum ether

1

4.1.24.

(1/1, v/v) to afford 13a as a white solid (209 mg, 63% yield). H

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-5’-O-benzyl-β-D-arabinofuranosyl)-9H-purine (31a)

NMR (400 MHz, DMSO-d6) δ: 8.28 (d, J = 2.3 Hz, 1H), 7.92 (s,

2H), 7.45–7.27 (m, 5H), 6.32 (dd, J = 15.9, 4.4 Hz, 1H),

5.63–5.45 (m, 1H), 5.16 (t, J = 5.8 Hz, 1H), 4.71 (dd, J = 31.5,

11.7 Hz, 2H), 4.44 (ddd, J = 18.5, 4.9, 3.1 Hz, 1H), 4.04 (q, J =

4.7 Hz, 1H), 3.74–3.60 (m, 2H). EI-MS m/z 393.1 (M⁺); 91

(100%); HRMS (EI) m/z calcd C₁₇H₁₇ClFN₅O₃(M⁺) 393.1004,

found 393.1008.

To

a

solution of 30a (0.42 g, 0.59 mmol) in

dichloromethane (4.8 mL) was added DDQ (174 mg, 0.77 mmol)

and water (0.248 mL). The mixture was stirred at room

temperature for 4 h. The mixture was poured into water (30 mL),

and extracted with dichloromethane. The organic layer was

separated, dried with anhydrous Na₂SO₄and evaporated under

reduced pressure. The residue was purified by silica gel column

4.1.20.

6-Amino-9-(2’-deoxy-2’-fluoro-3’-O-benzyl-β-D-arabinofuranos

1

chromatography, eluting with EtOAc/petroleum ether (1/2, v/v)

to afford 31a as white foam (266 mg, 76% yield). ¹H NMR (400

MHz, DMSO-d6) δ: 8.75 (s, 1H), 7.38–7.25 (m, 5H), 6.55 (dd, J

= 11.7, 5.0 Hz, 1H), 6.24 (d, J = 4.5 Hz, 1H), 5.48 – 5.30 (m,

1H), 4.57 (s, 2H), 4.50 (ddd, J = 19.4, 10.6, 5.2 Hz, 1H), 4.07 (dt,

J = 15.2, 7.5 Hz, 1H), 3.79 (d, J = 4.7 Hz, 2H), 1.38 (d, J = 10.9

Hz, 18H).

afford 14e as a white solid (63% yield). H NMR (400 MHz,

DMSO-d6) δ: 8.17 (s, 1H), 7.90 (s, 2H), 7.58–7.41 (m, 2H), 7.34

(m, 2H), 6.35 (dd, J = 13.9, 4.6 Hz, 1H), 6.07 (d, J = 5.1 Hz, 1H),

5.37–5.16 (m, 1H), 4.65 (s, 2H), 4.48 (dd, J = 19.2, 4.3 Hz, 1H),

4.04 (d, J = 4.4 Hz, 1H), 3.82 (d, J = 4.2 Hz, 2H). ESI-MS m/z

428.0 [M+H]⁺; HRMS (ESI) m/z calcd C₁₇H₁₇Cl₂FN₅O₃[M+H]⁺

428.0687, found 428.0684.

4.1.25.

4.1.30.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-benzyl-β-D-arabin

ofuranosyl)-9H-purine (14a)

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(3-chlorobenzyl)-β

-D-arabinofuranosyl)-9H-purine (14f)

To a solution of 31a (70 mg, 0.12 mmol) in toluene (10 mL)

was added dry silica gel (100-200 mesh, 600 mg), and then the

mixture was stirred at 80 °C for 3 h. The solvent was evaporated

under reduced pressure, and the residue was purified by silica gel

column chromatography, eluting with EtOAc/petroleum ether

14f was synthesized by the general procedure of 14a to

afford 14f as a white solid (62% yield). H NMR (400 MHz,

1

DMSO-d6) δ: 8.17 (s, 1H), 7.90 (s, 2H), 7.45–7.25 (m, 4H), 6.35

(dd, J = 13.8, 4.6 Hz, 1H), 6.07 (d, J = 5.1 Hz, 1H), 5.26 (dt, J =

52.7, 4.4 Hz, 1H), 4.58 (s, 2H), 4.46 (dd, J = 19.4, 4.4 Hz, 1H),

4.07–3.97 (m, 1H), 3.76 (d, J = 4.7 Hz, 2H). ESI-MS m/z 428.0

[M+H]⁺; HRMS (ESI) m/z calcd C₁₇H₁₇Cl₂FN₅O₃[M+H]⁺

428.0687, found 428.0682.

1

(1/2, v/v) to afford 14a as a white solid (32 mg, 70% yield). H

NMR (400 MHz, DMSO-d6) δ: 8.17 (s, 1H), 7.93 (s, 2H),

7.40–7.26 (m, 5H), 6.34 (dd, J = 13.3, 4.3 Hz, 1H), 6.08 (d, J =

5.1 Hz, 1H), 5.26 (d, J = 52.5 Hz, 2H), 4.56 (s, 2H), 4.46 (dd, J =

19.2, 4.8 Hz, 1H), 4.01 (d, J = 4.5 Hz, 1H), 3.75 (s, 2H). EI-MS

m/z 393.1 (M⁺); 91 (100%); HRMS (EI) m/z calcd

C₁₇H₁₇ClFN₅O₃(M⁺) 393.1004, found 393.1008.

4.1.31.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(4-chlorobenzyl)-β

-D-arabinofuranosyl)-9H-purine (14g)

14g was synthesized by the general procedure of 14a to

afford 14g as a white solid (67% yield). H NMR (400 MHz,

4.1.26.

1

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(2-fluorobenzyl)-β

-D-arabinofuranosyl)-9H-purine (14b)

DMSO-d6) δ: 8.17 (s, 1H), 7.90 (s, 2H), 7.39 (dd, J = 18.8, 8.5

Hz, 4H), 6.34 (dd, J = 13.5, 4.7 Hz, 1H), 6.07 (d, J = 5.2 Hz, 1H),

5.26 (dt, J = 52.6, 4.3 Hz, 1H), 4.56 (s, 2H), 4.52–4.38 (m, 1H),

4.05–3.96 (m, 1H), 3.75 (d, J = 4.7 Hz, 2H). ESI-MS m/z 428.0

[M+H]⁺; HRMS (ESI) m/z calcd C₁₇H₁₇Cl₂FN₅O₃[M+H]⁺

428.0687, found 428.0684.

14b was synthesized by the general procedure of 14a to

afford 14b as a white solid (51% yield). H NMR (400 MHz,

1

DMSO-d6) δ: 8.16 (s, 1H), 7.92 (s, 2H), 7.46 (t, J = 7.5 Hz, 1H),

7.38 (dd, J = 13.7, 6.7 Hz, 1H), 7.21 (m, 2H), 6.34 (dd, J = 13.6,

4.7 Hz, 1H), 6.07 (d, J = 5.2 Hz, 1H), 5.25 (dt, J = 52.7, 4.3 Hz,

1H), 4.62 (s, 2H), 4.50–4.39 (m, 1H), 4.05–3.97 (m, 1H), 3.77 (d,

J = 4.9 Hz, 2H). ESI-MS m/z 412.1 [M+H]⁺; HRMS (ESI) m/z

calcd C₁₇H₁₇ClF₂N₅O₃[M+H]⁺412.0983, found 412.0982.

4.1.32.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(2-bromobenzyl)-β

-D-arabinofuranosyl)-9H-purine (14h)

14h was synthesized by the general procedure of 14a to

afford 14h as a white solid (72% yield). H NMR (400 MHz,

4.1.27.

1

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(3-fluorobenzyl)-β

DMSO-d6) δ: 8.18 (d, J = 2.1 Hz, 1H), 7.91 (s, 2H), 7.63 (dd, J

= 7.9, 1.0 Hz, 1H), 7.51 (dd, J = 7.6, 1.5 Hz, 1H), 7.39 (td, J =

7.5, 1.1 Hz, 1H), 7.26 (td, J = 7.8, 1.7 Hz, 1H), 6.36 (dd, J = 13.9,

4.7 Hz, 1H), 6.09 (d, J = 5.2 Hz, 1H), 5.36–5.18 (m, 1H), 4.61 (s,

2H), 4.54–4.44 (m, 1H), 4.08–4.01 (m, 1H), 3.86–3.78 (m, 2H).

EI-MS m/z 471.1 (M⁺); 198 (100%); HRMS (EI) m/z calcd

C₁₇H₁₆BrClFN₅O₃(M⁺) 471.0109, found 471.0114.

-D-arabinofuranosyl)-9H-purine (14c)

14c was synthesized by the general procedure of 14a to

afford 14c as a white solid (58% yield). H NMR (400 MHz,

1

DMSO-d6) δ: 8.17 (s, 1H), 7.92 (s, 2H), 7.40 (dd, J = 14.2, 8.0

Hz, 1H), 7.24–7.05 (m, 3H), 6.35 (dd, J = 13.6, 4.7 Hz, 1H),

6.08 (d, J = 5.1 Hz, 1H), 5.26 (dt, J = 52.6, 4.4 Hz, 1H), 4.59 (s,

2H), 4.47 (dd, J = 19.3, 4.8 Hz, 1H), 4.02 (dd, J = 10.1, 5.1 Hz,

1H), 3.83–3.67 (m, 2H). ESI-MS m/z 412.1 [M+H]⁺; HRMS

(ESI) m/z calcd C₁₇H₁₇ClF₂N₅O₃[M+H]⁺412.0983, found

412.0980.

4.1.33.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(2-cyanobenzyl)-β

-D-arabinofuranosyl)-9H-purine (14i)

14i was synthesized by the general procedure of 14a to

afford 14i as a white solid (61% yield). H NMR (400 MHz,

4.1.28.

1

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(4-fluorobenzyl)-β

-D-arabinofuranosyl)-9H-purine (14d)

DMSO-d6) δ: 8.17 (d, J = 2.0 Hz, 1H), 8.01–7.81 (m, 3H), 7.71

(t, J = 7.2 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.53 (t, J = 7.5 Hz,

1H), 6.35 (dd, J = 14.3, 4.6 Hz, 1H), 6.08 (d, J = 5.1 Hz, 1H),

5.25 (dt, J = 52.5, 4.2 Hz, 1H), 4.74 (s, 2H), 4.55–4.42 (m, 1H),

4.04 (dd, J = 10.1, 5.2 Hz, 1H), 3.88–3.77 (m, 2H). EI-MS m/z

418.1 (M⁺); 116 (100%); HRMS (EI) m/z calcd C₁₈H₁₆ClFN₆O₃

(M⁺) 418.0956, found 418.0956.

14d was synthesized by the general procedure of 14a to

afford 14d as a white solid (53% yield). H NMR (400 MHz,

1

DMSO-d6) δ: 8.17 (s, 1H), 7.92 (s, 2H), 7.39 (dd, J = 8.2, 5.8 Hz,

2H), 7.18 (t, J = 8.8 Hz, 2H), 6.34 (dd, J = 13.4, 4.7 Hz, 1H),

6.07 (d, J = 5.1 Hz, 1H), 5.26 (dt, J = 52.8, 4.4 Hz, 1H), 4.54 (s,

2H), 4.51–4.38 (m, 1H), 4.05–3.96 (m, 1H), 3.73 (d, J = 4.9 Hz,

2H). ESI-MS m/z 412.1 [M+H]⁺; HRMS (ESI) m/z calcd

C₁₇H₁₇ClF₂N₅O₃[M+H]⁺412.0983, found 412.0984.

4.1.34.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(2-(trifluoromethyl

)benzyl)-β-D-arabinofuranosyl)-9H-purine (14j)

4.1.29.

14j was synthesized by the general procedure of 14a to

afford 14j as a white solid (74% yield). H NMR (400 MHz,

DMSO-d6) δ: 8.16 (d, J = 2.1 Hz, 1H), 7.91 (s, 2H), 7.76–7.64

(m, 3H), 7.53 (t, J = 7.4 Hz, 1H), 6.35 (dd, J = 14.0, 4.7 Hz, 1H),

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(2-chlorobenzyl)-β

-D-arabinofuranosyl)-9H-purine (14e)

1

14e was synthesized by the general procedure of 14a to

4.1.40.

6.08 (d, J = 5.2 Hz, 1H), 5.36–5.18 (m, 1H), 4.74 (s, 2H),

4.54–4.41 (m, 1H), 4.04 (dd, J = 10.3, 5.1 Hz, 1H), 3.82 (d, J =

4.8 Hz, 2H). EI-MS m/z 461.1 (M⁺); 302 (100%); HRMS (EI)

m/z calcd C₁₈H₁₆ClF₄N₅O₃(M⁺) 461.0878, found 461.0883.

2-Chloro-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-deoxy-2’-fluoro

-5’-O-(2-chlorophenyl)-β-D-arabinofuranosyl)-9H-purine (32)

To

a

mixture of 23a (0.132 g, 0.26 mmol),

4.1.35.

triphenylphosphine (0.094 g, 0.36 mmol), and 2-chlorophenol

(0.033 g, 0.26 mmol) in anhydrous tetrahydrofuran (4 mL) was

added a solution of diisopropyl azodicarboxylate (0.072 g, 0.36

mmol) in anhydrous tetrahydrofuran (1 mL) dropwise under

nitrogen atmosphere. The mixture was stirred at room

temperature overnight. The solvent was evaporated under

reduced pressure and the residue was purified by silica gel

column chromatography, eluting with EtOAc/petroleum ether

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(2-nitrobenzyl)-β-

D-arabinofuranosyl)-9H-purine (14k)

14k was synthesized by the general procedure of 14a to

afford 14k as a white solid (73% yield). H NMR (400 MHz,

1

DMSO-d6) δ: 8.17 (d, J = 2.2 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H),

7.91 (s, 2H), 7.78–7.71 (m, 2H), 7.61–7.54 (m, 1H), 6.35 (dd, J

= 14.5, 4.6 Hz, 1H), 6.08 (d, J = 5.1 Hz, 1H), 5.35–5.17 (m, 1H),

4.91 (s, 2H), 4.52–4.40 (m, 1H), 4.04 (dd, J = 10.2, 5.1 Hz, 1H),

3.81 (d, J = 4.9 Hz, 2H). ESI-MS m/z 439.1 [M+H]⁺; HRMS

(ESI) m/z calcd C₁₇H₁₇ClFN₆O₅[M+H]⁺439.0933, found

439.0931.

1

(1/5, v/v) to afford 32 as white foam (76 mg, 47% yield). H

NMR (400 MHz, CDCl₃) δ: 8.47 (d, J = 1.4 Hz, 1H), 7.38 (d, J =

7.1 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 6.97 (dd, J = 16.8, 8.3 Hz,

2H), 6.63 (dd, J = 16.2, 3.9 Hz, 1H), 5.29–5.13 (m, 1H), 4.91 (dd,

J = 18.0, 3.4 Hz, 1H), 4.40 (dd, J = 8.6, 4.2 Hz, 1H), 4.38 – 4.30

(m, 2H), 1.44 (s, 18H).

4.1.36.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-cyanomethyl-β-D-

arabinofuranosyl)-9H-purine (14l)

4.1.41.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(2-chlorophenyl)-

β-D-arabinofuranosyl)-9H-purine (14o)

14l was synthesized by the general procedure of 14a to

afford 14l as a white solid (61% yield). H NMR (400 MHz,

1

To a solution of 32 (60 mg, 0.1 mmol) in toluene (8 mL)

was added dry silica gel (100-200 mesh, 500 mg), and then the

mixture was stirred at 80°C for 3 h. The solvent was evaporated

under reduced pressure, and the residue was purified by silica gel

column chromatography, eluting with EtOAc/petroleum ether

DMSO-d6) δ: 8.20 (d, J = 2.1 Hz, 1H), 7.92 (s, 2H), 6.35 (dd, J

= 14.7, 4.6 Hz, 1H), 6.12 (d, J = 5.1 Hz, 1H), 5.25 (dt, J = 52.5,

4.2 Hz, 1H), 4.56 (s, 2H), 4.44 (ddd, J = 18.9, 9.4, 5.1 Hz, 1H),

4.00 (dd, J = 9.5, 5.7 Hz, 1H), 3.90–3.76 (m, 2H). ESI-MS m/z

343.1 [M+H]⁺; HRMS (ESI) m/z calcd C₁₂H₁₃ClFN₆O₃[M+H]⁺

343.0716, found 343.0715.

1

(1/2, v/v) to afford 14o as a white solid (27 mg, 67% yield). H

NMR (400 MHz, DMSO-d6) δ: 8.27 (d, J = 1.3 Hz, 1H), 7.93 (s,

2H), 7.46 (d, J = 7.9 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.23 (d, J

= 8.1 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 6.43 (dd, J = 11.8, 5.1 Hz,

1H), 6.20 (d, J = 5.1 Hz, 1H), 5.36 (dt, J = 52.9, 4.8 Hz, 1H),

4.64 (ddd, J = 19.5, 10.6, 5.1 Hz, 1H), 4.48–4.31 (m, 2H), 4.21

(m, 1H). ESI-MS m/z 414.0 [M+H]⁺; HRMS (ESI) m/z calcd

C₁₆H₁₅Cl₂FN₅O₃[M+H]⁺414.0530, found 414.0523.

4.1.37.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-propargyl-β-D-ara

binofuranosyl)-9H-purine (14m)

14m was synthesized by the general procedure of 14a to

1

afford 14m as a white solid (64% yield). H NMR (400 MHz,

DMSO-d6) δ: 8.20 (d, J = 2.0 Hz, 1H), 7.91 (s, 2H), 6.33 (dd, J

= 14.0, 4.6 Hz, 1H), 6.08 (d, J = 5.2 Hz, 1H), 5.34–5.16 (m, 1H),

4.42 (ddd, J = 18.8, 9.6, 5.1 Hz, 1H), 4.23 (s, 2H), 3.98 (dd, J =

9.6, 5.7 Hz, 1H), 3.80–3.69 (m, 2H), 3.50 (t, J = 2.3 Hz, 1H).

ESI-MS m/z 342.1 [M+H]⁺; HRMS (ESI) m/z calcd

C₁₃H₁₄ClFN₅O₃[M+H]⁺342.0764, found 342.0763.

4.1.42.

2-(3,4-Dimethoxyphenyl)-N⁶,N⁶-di-(tert-butyloxycarbonyl)-9-(2’-

deoxy-2’-fluoro-5’-O-benzyl-β-D-arabinofuranosyl)-9H-purine

(33a)

To a solution of 31a (100 mg, 0.17mmol) in toluene (5 mL),

water (1 mL), and ethanol (1 mL) was added

3,4-dimethoxyphenylboronic acid (232 mg, 1.28 mmol) and

potassium carbonate (93 mg, 0.67 mmol), degassed the mixture

for about 10 min and Pd(PPh₃)₄(78 mg, 0.07 mmol) was added

then. The mixture was allowed to warm to 110°C and stirred for

12 h under the nitrogen atmosphere. The solvent was evaporated

under reduced pressure, and the residue was taken up in ethyl

acetate and washed with water. The organic layer was separated,

dried with anhydrous Na₂SO₄and evaporated under reduced

pressure. The residue was purified by silica gel column

chromatography, eluting with EtOAc/petroleum ether (1/4, v/v)

to afford 33a as a white solid (95 mg, 81% yield). ¹H NMR (400

MHz, CDCl₃) δ: 8.27 (d, J = 2.7 Hz, 1H), 8.13 (dd, J = 8.5, 1.9

Hz, 1H), 8.04 (d, J = 1.9 Hz, 1H), 7.43–7.31 (m, 5H), 6.95 (d, J

= 8.6 Hz, 1H), 6.72 (dd, J = 18.6, 3.6 Hz, 1H), 5.26–5.10 (m,

1H), 4.73–4.60 (m, 3H), 4.21 (dd, J = 9.6, 5.1 Hz, 1H), 4.00 (s,

3H), 3.95 (s, 3H), 3.83–3.73 (m, 2H), 1.45 (s, 18H).

4.1.38.

6-Amino-2-chloro-9-(2’-deoxy-2’-fluoro-5’-O-(2-oxo-2-phenylet

hyl)-β-D-arabinofuranosyl)-9H-purine (14n)

14n was synthesized by the general procedure of 14a to

1

afford 14n as a white solid (29% yield). H NMR (400 MHz,

DMSO-d6) δ: 8.34 (s, 1H), 7.92 (m, 4H), 7.65 (t, J = 7.3 Hz, 1H),

7.53 (t, J = 7.6 Hz, 2H), 6.36 (dd, J = 13.4, 4.6 Hz, 1H), 6.07 (d,

J = 5.0 Hz, 1H), 5.27 (d, J = 52.6 Hz, 2H), 4.99 (s, 2H),

4.56–4.46 (m, 1H), 4.04 (d, J = 3.9 Hz, 1H), 3.90–3.77 (m, 2H).

ESI-MS m/z 422.1 [M+H]⁺; HRMS (ESI) m/z calcd

C₁₈H₁₈ClFN₅O₄[M+H]⁺422.1026, found 422.1022.

4.1.39.

6-Amino-9-(2’-deoxy-2’-fluoro-5’-O-benzyl-β-D-arabinofuranos

yl)-9H-purine (14p)

14p was synthesized by the general procedure of 14a to

1

afford 14p as a white solid (67% yield). H NMR (400 MHz,

4.1.43.

DMSO-d6) δ: 8.16 (s, 1H), 8.15 (s, 1H), 7.33 (dd, J = 19.9, 5.2

Hz, 7H), 6.42 (dd, J = 14.3, 4.5 Hz, 1H), 6.07 (d, J = 5.0 Hz, 1H),

5.23 (dt, J = 52.6, 4.1 Hz, 1H), 4.57 (s, 2H), 4.48 (dd, J = 19.0,

4.6 Hz, 1H), 4.02 (d, J = 4.7 Hz, 1H), 3.72 (d, J = 17.2 Hz, 2H).

EI-MS m/z 359.1 (M⁺); 91 (100%); HRMS (EI) m/z calcd

C₁₇H₁₈FN₅O₃(M⁺) 359.1394, found 359.1401.

6-Amino-2-(3,4-dimethoxyphenyl)-9-(2’-deoxy-2’-fluoro-5’-O-be

nzyl-β-D-arabinofuranosyl)-9H-purine (15a)

To a solution of 33a (90 mg, 0.13 mmol) in toluene (8 mL)

was added dry silica gel (100-200 mesh, 600 mg), and then the

mixture was stirred at 80°C for 3 h. The solvent was evaporated

under reduced pressure, and the residue was purified by silica gel

column chromatography, eluting with EtOAc/petroleum ether

(1/2, v/v) to afford 15a as a white solid (46 mg, yield 71%). H

NMR (400 MHz, DMSO-d6) δ: 8.13 (d, J = 2.2 Hz, 1H),

8.01–7.92 (m, 2H), 7.42–7.23 (m, 7H), 7.04 (d, J = 8.6 Hz, 1H),

6.53 (dd, J = 13.7, 4.9 Hz, 1H), 6.11 (d, J = 5.1 Hz, 1H),

5.41–5.22 (m, 1H), 4.68–4.52 (m, 3H), 4.04 (dd, J = 10.2, 5.5 Hz,

1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.80–3.74 (m, 2H). EI-MS m/z

495.1 (M⁺); 300 (100%); HRMS (EI) m/z calcd C₂₅H₂₆FN₅O₅

(M⁺) 495.1918, found 495.1921.

(dd, J = 14.0, 4.8 Hz, 1H), 6.09 (d, J = 5.0 Hz, 1H), 5.29 (dt, J =

52.6, 4.4 Hz, 1H), 4.68–4.50 (m, 3H), 4.03 (dd, J = 10.0, 5.4 Hz,

1H), 3.83–3.68 (m, 2H). EI-MS m/z 425.1 (M⁺); 230 (100%);

HRMS (EI) m/z calcd C₂₁H₂₀FN₅O₄(M⁺) 425.1499, found

425.1498.

1

4.1.49.

6-Amino-2-(thiophene-3-yl)-9-(2’-deoxy-2’-fluoro-5’-O-benzyl-β

-D-arabinofuranosyl)-9H-purine (15g)

15g was synthesized by the general procedure of 15a to

1

afford 15g as a white solid (72% yield). H NMR (400 MHz,

4.1.44.

DMSO-d6) δ: 8.21–8.09 (m, 2H), 7.78 (d, J = 4.9 Hz, 1H), 7.58

(dd, J = 4.9, 3.1 Hz, 1H), 7.43 – 7.24 (m, 7H), 6.49 (dd, J = 14.1,

4.8 Hz, 1H), 6.10 (d, J = 5.0 Hz, 1H), 5.30 (dt, J = 52.7, 4.3 Hz,

1H), 4.66 – 4.51 (m, 3H), 4.04 (dd, J = 9.8, 5.2 Hz, 1H), 3.84 –

3.71 (m, 2H). EI-MS m/z 441.1 (M⁺); 246 (100%); HRMS (EI)

m/z calcd C₂₁H₂₀FN₅O₃S (M⁺) 441.1271, found 441.1275.

6-Amino-2-(2-methoxyphenyl)-9-(2’-deoxy-2’-fluoro-5’-O-benzyl

-β-D-arabinofuranosyl)-9H-purine (15b)

15b was synthesized by the general procedure of 15a to

1

afford 15b as a white solid (69% yield). H NMR (400 MHz,

DMSO-d6) δ: 8.16 (d, J = 2.0 Hz, 1H), 7.51–7.24 (m, 9H), 7.08

(d, J = 8.4 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 6.42 (dd, J = 14.4,

4.7 Hz, 1H), 6.03 (d, J = 5.1 Hz, 1H), 5.33–5.15 (m, 1H),

4.60–4.44 (m, 3H), 4.00 (dd, J = 10.2, 5.1 Hz, 1H), 3.81–3.66 (m,

5H). EI-MS m/z 465.2 (M⁺); 374 (100%); HRMS (EI) m/z calcd

C₂₄H₂₄FN₅O₄(M⁺) 465.1812, found 465.1809.

4.2. Bioassay

4.2.1. Protein expression and purification

The subcloning and protein expressing of human PDE2A

(catalytic domain, residues 580-919) were reported

previously^34-36and are briefly described here. The recombined

vector pET15b-PDE2A (580-919) was transformed into E. coli

competent cell BL21(Codonplus) for over expression. The E.

coli cells carrying these plasmids were grown in 2XYT medium

(containing 100 μg/mL ampicillin and 20 μg/mL

chloramphenicol) at 37^oC until OD₆₀₀= 0.6-0.8, and then 0.1

mM isopropyl-β-D-thiogalactopyranoside was added for induced

growth at 16 C for 20-40 h. These recombinant proteins were

purified by Ni-NTA column (Qiagen). A typical batch of

purification yielded 10-20 mg of PDE2A from a 1.0 L cell

culture and the purity of these recombinant proteins was greater

than 90% as confirmed by SDS–PAGE.

4.1.45.

6-Amino-2-(3-methoxyphenyl)-9-(2’-deoxy-2’-fluoro-5’-O-benzyl

-β-D-arabinofuranosyl)-9H-purine (15c)

15c was synthesized by the general procedure of 15a to

1

afford 15c as a white solid (73% yield). H NMR (400 MHz,

DMSO-d6) δ: 8.16 (d, J = 2.2 Hz, 1H), 7.99–7.89 (m, 2H),

7.48–7.25 (m, 8H), 7.03 (dd, J = 8.2, 1.9 Hz, 1H), 6.54 (dd, J =

14.1, 4.8 Hz, 1H), 6.11 (d, J = 5.1 Hz, 1H), 5.41–5.22 (m, 1H),

4.65–4.51 (m, 3H), 4.09–4.01 (m, 1H), 3.81 (s, 3H), 3.80–3.72

(m, 2H). EI-MS m/z 465.1 (M⁺); 270 (100%); HRMS (EI) m/z

calcd C₂₄H₂₄FN₅O₄(M⁺) 465.1812, found 465.1811.

4.1.46.

4.2.2. Enzymatic assays

6-Amino-2-(4-methoxyphenyl)-9-(2’-deoxy-2’-fluoro-5’-O-benzyl

-β-D-arabinofuranosyl)-9H-purine (15d)

The enzymatic activities of the catalytic domains of PDE2A

were measured by using cGMP as substrates and EHNA

(compound 1, purchased from SIGMA) as the reference

compound. The assay buffer contains 20 mM Tris-HCl (pH 8.0),

15d was synthesized by the general procedure of 15a to

1

afford 15d as a white solid (70% yield). H NMR (400 MHz,

DMSO-d6) δ: 8.31 (d, J = 8.8 Hz, 2H), 8.12 (d, J = 2.1 Hz, 1H),

7.46–7.23 (m, 7H), 7.01 (d, J = 8.9 Hz, 2H), 6.53 (dd, J = 14.7,

4.6 Hz, 1H), 6.10 (d, J = 5.1 Hz, 1H), 5.39–5.21 (m, 1H),

4.62–4.49 (m, 3H), 4.09–4.01 (m, 1H), 3.82 (s, 3H), 3.80–3.71

(m, 2H). EI-MS m/z 465.2 (M⁺); 241 (100%); HRMS (EI) m/z

calcd C₂₄H₂₄FN₅O₄(M⁺) 465.1812, found 465.1817.

3

10 mM MgCl₂, 1 mM dithiothreitol and 10-30 nM H-cGMP

(20,000–30,000 cpm/assay, GE Healthcare). The reaction was

carried out at room temperature for 15 min and then terminated

3

by addition of 0.2 M ZnSO₄. The reaction product H-GMP was

precipitated by 0.2 N Ba(OH)₂, while unreacted ³H-cGMP

remained in the supernatant. Radioactivity of the supernatant

was measured in 2.5 mL Ultima Gold liquid scintillation

cocktails (Fisher Scientific) by a PerkinElmer 2910 liquid

scintillation counter. For the measurement of IC₅₀, at least eight

concentrations of inhibitors were used in the presence of suitable

4.1.47.

6-Amino-2-phenyl-9-(2’-deoxy-2’-fluoro-5’-O-benzyl-β-D-arabin

ofuranosyl)-9H-purine (15e)

15e was synthesized by the general procedure of 15a to

3

1

concentrations of H-cGMP and the enzymes that hydrolyze up

afford 15e as a white solid (67% yield). H NMR (400 MHz,

to 70% of the substrates. Each measurement was repeated at

least three times. The IC₅₀values were calculated by nonlinear

regression.

DMSO-d6) δ: 8.47–8.30 (m, 2H), 8.17 (s, 1H), 7.70–7.19 (m,

10H), 6.55 (dd, J = 14.6, 4.5 Hz, 1H), 6.12 (s, 1H), 5.43–5.21 (m,

1H), 4.67–4.45 (m, 3H), 4.12–4.00 (m, 1H), 3.87–3.69 (m, 2H).

EI-MS m/z 435.2 (M⁺); 211 (100%); HRMS (EI) m/z calcd

C₂₃H₂₂FN₅O₃(M⁺) 435.1707, found 435.1702.

4.3. Modeling simulations

4.3.1. Docking

4.1.48.

Surflex-dock³⁷method embedded in TriposSybyl 2.0 was

used to screen our internal collection of FDA drugs and to

explore the interactions between PDE2 and the synthesized

compounds. Herein, two crystal structures (4HTX and 4C1I)

were utilized as PDE2 receptor and the active site were defined

by using the co-crystallized ligands as reference. All ionizable

residues in the systems were set to their protonation states at a

6-Amino-2-(furan-3-yl)-9-(2’-deoxy-2’-fluoro-5’-O-benzyl-β-D-a

rabinofuranosyl)-9H-purine (15f)

15f was synthesized by the general procedure of 15a to

1

afford 15f as a white solid (74% yield). H NMR (400 MHz,

DMSO-d6) δ: 8.19 (s, 1H), 8.12 (d, J = 2.1 Hz, 1H), 7.75 (t, J =

1.6 Hz, 1H), 7.42–7.24 (m, 7H), 6.97 (d, J = 1.1 Hz, 1H), 6.45

neutral pH. The zinc and magnesium ions were retained and

assigned with a charge of 2⁺and the coordinated water around

them were also checked after which hydrogen atoms were

complemented. The co-crystallized EHNA (1) and BAY60-7550

(2) were firstly re-docked into the active site to check the

reliability of docking method and to determine the suitable

thresholds for filtering the docking results of the compound

collection. Besides the docking score, it’s remarkable that there

is a common scheme of inhibitor binding to PDEs in which the

hydrophobic interactions formed by the conserved hydrophobic

residues sandwich ligands in the catalytic site and

hydrogen-bonding interactions occur with the invariant

glutamine. On the basis of these given scheme, the filtered

ligands could be selected and the initial binding modes with

PDE2 were also determined, which could be subjected to the

MD simulations.

area (SASA) and is calculated by the following equations:

ΔG_solv= ΔG_PB+ ΔG_np

(4)

ΔG_np=γSASA + b (5)

The calculations of the entropy contribution for the

PDE2-ligand complexes were omitted, since it is extremely time

consuming for large protein−ligand systems.

Acknowledgements

Financial support for this research provided by the

National Key R&D Program of China (Grant 2017YFB0202600),

the National Natural Science Foundation of China (Grant

21672064), the “Shu Guang” project supported by the Shanghai

Municipal Education Commission and Shanghai Education

Development Foundation (Grant 14SG28), and the Fundamental

Research Funds for the Central Universities are gratefully

acknowledged.

4.3.2. Molecular dynamic and binding free energy calculations

MD simulations were further performed by Amber14³⁸to

predict the more precise and stable binding patterns of selected

ligands with PDE2. The parameters for MD and binding free

energy calculations were similar as previously reported.^{35, 39-41}

The docking poses of molecules in Data set3 were first

calculated for the partial atomic charges by using the

Hartree−Fock method at the 6-31G* level with Gaussian 03.

Then Antechamber was applied to fit the restricted electrostatic

potential (RESP) and assign the general amber force field

(GAFF) parameters. The amber03 force field was utilized for

this protein. The force field parameters for Zn2+ and Mg2+ were

assigned with the “nonbond model” method. This simple model

reproduced the structural and energetic properties of the solvated

ions in MD simulations. Å TIP3P water box in the form of a

truncated octahedron with Na+ (for 4HTX) or Cl− (for 4C1I)

ions was added for neutralizing. 8 ns MD simulations were

carried out in the NPT ensemble with a constant pressure of 1

atm and a constant temperature of 300 K. The periodic boundary

conditions were adopted, along with a 8 Å cutoff for long range

electrostatic interactions with the partial mesh Ewald (PME)

method. The SHAKE algorithm was utilized for restriction of all

bonds involving hydrogen atoms, and thereby the time step was

set to 2 fs. The subsequent routine included the extraction of 100

snapshots of the last 1 ns trajectories and MM-PBSA binding

free energy calculations with default parameters assigned.

Supplementary date

Supplementary data associated with this article can be

found in the online version at http://dx.doi.org/.

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Article Doi

Discovery of novel purine nucleoside derivatives as phosphodiesterase 2 (PDE2) inhibitors: Structure-based virtual screening, optimization and biological evaluation

DOI: 10.1016/j.bmc.2017.11.022

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Full text of DOI:10.1016/j.bmc.2017.11.022

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