Adenosine analogs as inhibitors of tyrosyl-tRNA synthetase: Design, synthesis and antibacterial evaluation

Article abstract of DOI:10.1016/j.bmc.2015.09.018

Herein we describe the synthesis and evaluation of a series of adenosine analogs for in vitro antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Out of these compounds, compound c6 has much stronger antibact

Full text of DOI:10.1016/j.bmc.2015.09.018

Accepted Manuscript
Adenosine analogues as inhibitors of tyrosyl-tRNA synthetase: design, synthe-
sis and antibacterial evaluation
Wei Wei, Wei-Kang Shi, Peng-Fei Wang, Xiao-Tong Zeng, Pan Li, Ji-Rong
Zhang, Qian Li, Zhi-Ping Tang, Jia Peng, Lang-Zhou Wu, Mei-Qun Xie, Chan
Liu, Xian-Hui Li, Yin-Chun Wang, Zhu-Ping Xiao, Hai-Liang Zhu
PII:
S0968-0896(15)30043-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.09.018
BMC 12565
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 August 2015
8 September 2015
9 September 2015
Please cite this article as: Wei, W., Shi, W-K., Wang, P-F., Zeng, X-T., Li, P., Zhang, J-R., Li, Q., Tang, Z-P., Peng,
J., Wu, L-Z., Xie, M-Q., Liu, C., Li, X-H., Wang, Y-C., Xiao, Z-P., Zhu, H-L., Adenosine analogues as inhibitors
of tyrosyl-tRNA synthetase: design, synthesis and antibacterial evaluation, Bioorganic & Medicinal Chemistry
(2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.09.018
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Adenosine analogues as inhibitors of tyrosyl-tRNA synthetase: design, synthesis and
antibacterial evaluation
Wei Wei,a Wei-Kang Shi,a Peng-Fei Wang,b Xiao-Tong Zeng,a Pan Li,a Ji-Rong Zhang,a Qian
Li,a Zhi-Ping Tang,a Jia Peng,a Lang-Zhou Wu,a Mei-Qun Xie,a Chan Liu,a Xian-Hui Li,b
Yin-Chun Wang, a Zhu-Ping Xiao,a,c, and Hai-Liang Zhua,c,*
aCollege of Chemistry and Chemical Engineering, Jishou University, Jishou 416000, P. R.
China
b School of Medicine, Jishou University, Jishou 416000, P. R. China
c State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, P. R. China
Abstract
Herein we describe the synthesis and evaluation of a series of adenosine analogues for in
vitro antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa. Out of these compounds, compound c6 has much stronger antibacterial potency
against Pseudomonas aeruginosa than ciprofloxacin, and was determined to target
tyrosyl-tRNA synthetase with IC50 of 0.8±0.07 μM. Structure-activtiy relationship analysises
suggested that introduction of a fluorine atom at the 3′-position of benzene ring of the
phenylacetyl moiety significantly increased affinities to the enzyme. In comparison with
isopropylidene analogs, 2
′
,3′
displayed higher inhibitory activity.
Molecular dockings provided an explanation for observations in biological assays.
 Corresponding author. E-mail address: xiaozhuping2005@163.com
* Corresponding author. E-mail address: zhuhl@nju.edu.cn
1

Keywords: adenosine analogues; tyrosyl-tRNA synthetase inhibitors; antibacterial;
molecular docking;
1. Introduction
Infectious disease is the second major cause of death worldwide and the third leading
cause of death in developed countries [1]. Since the introduction of the first sulfonamide and
penicillin in 1935 and 1940, many synthetic and natural antibiotics have been lauched and
have saved millions of lives. The ability of bacteria to evade any form of established therapy
has become apparent, causing a rapid emergence and worldwide diffussness of pathogens
resistant to one or more antibiotics. Despite the impressive therapeutic successes of
antibiotics throughout recent decades, acute infectious diseases account for 25% of deaths
worldwide, killing 13-17 million people per year [2]. Therefore, there is a pressing need for
antiinfective drugs with a new mechanism or/and a novle scaffold.
Aminoacyl-tRNA synthetases (aaRSs) are a kind of key enzymes which catalyze the
transfer of amino acids to their cognate tRNAs in the process of protein synthesis [3-5]. These
enzymes are essential for protein synthesis， and this process was forced to terminate when
these enzymes were restrained [6-7]. A selective inhibition of aaRSs in bacteria is feasible on
account of the different functions between prokaryotes aaRSs and eucaryon. This concept is
proven by the success of the broad-spectrum antibacterial drug mupirocin, which targets the
bacterial isoleucyl-tRNA synthetase [8-10]. Therefore, aaRSs inhibitors as a new type of
antibacterial agents have been receiving significant attention.
To our knowledge, previous studies have identified a few inhibitors against bacterial
TyrRS including the naturally compound SB-219383 (Scheme 1) and several synthetic
2

compounds. Although SB-219383 and its semi-synthetic analogues exhibit IC50s<1 nM
against Staphylococcus aureus tyrosyl-tRNA synthetase (TyrRS), they show very weak in
vitro activity against bacterial intact cells such as Staphylococci and Streptococci [11-13]. This
encourages us to search the new antiinfective drugs targeting TyrRS.
Scheme 1 structure of SB-219383
It is well known that the TyrRS acts as other tRNA synthetase enzymes by a two-step
mechanism. In the first stage, the enzyme recognises a tyrosine and activates it by reaction
with ATP to produce a tyrosyl adenylate (Scheme 2). In the second step, the enzyme catalyzes
the transfer of the tyrosine onto its cognate tRNA to form the desired product. Therefore, a
series of analogues of adenosine which was similar to the tyrosyl-adenylate intermediate have
been selected as a potential target in a structure-based drug design approach. In this context,
the side-chain of the tyrosyl-adenylate intermediate was modified to afford analogues b1-b14
and c1-c25. They were subsequently evaluated for biological activities against a
representative Gram-positive organism (Staphylococcus aureus ATCC 6538) and two
Gram-negative organisms (Escherichia coli ATCC 8739; Pseudomonas aeruginosa ATCC
9027). The results demonstrated some of the synthesized compounds show very good
antibacterial activities.
Scheme 2 structure of tyrosyl adenylate
3

2. Materials and methods
2.1. Chemistry
All chemicals (reagent grade) used were purchased from Aldrich (U.S.A) and
Sinopharm Chemical Reagent Co., Ltd (China). Separation of the compounds by column
chromatography was carried out with silica gel (200-300 mesh) purchased from Qingdao
Haiyang Chemical Co.,Ltd (China). The quantity of silica gel used was 30-60 times the
weight charged on the column. Then, the eluates were monitored using thin-layer
chromatography (TLC) using silica gel GF254 plates from Qingdao Haiyang Chemical Co.,
Ltd (China) with an UV lamp (254 nm). Melting points (uncorrected) were determined on a
XT4 MP apparatus (Taike Corp., Beijing, China). EI mass spectra were obtained on a Waters
GCT mass spectrometer, and NMR spectra were recorded on Bruker AV-400 and AV600
spectrometer at 25°C with TMS and solvent signals allotted as internal standards. Chemical
shifts were reported in ppm (δ). Elemental analyses were performed on a CHN-O-Rapid
instrument and were within ± 0.4 % of the theoretical values.
2.1.1. General procedure for preparation of compounds b
Triphenylphosphine (1.5 mmol), 2',3'-O-isopropylideneadenosine (1.0 mmol), and an
appropriately substituted benzeneacetic acids (1.0 mmol) were dissolved in anhydrous THF
(20 mL). After the solution was cooled in an ice bath, diisopropyl azodicarboxylate (DIAD
2.0 mmol) was added dropwise, and the resulted mixture was stirred at room temperature for
several hours (monitored by TLC). Evaporation to dryness and flash chromatography
(AcOEt/petroleum ether, from 5/1 to 3/1) afforded compounds b (Scheme 3).
4

Scheme 3 Synthetic route of analogues of adenosine
2.1.2. General procedure for preparation of compounds c
A solution of dichloromethane containing compound b (0.40 mmol) in 1.5 mL of 80%
aqueous trifluoroacetic acid was stirred at room temperature until complete by TLC.
Saturated sodium bicarbonate (30 mL) was added to neutralize trifluoroacetic acid. The
solution was extracted twice with 200 mL of AcOEt. The organic layer was dried over
MgSO4 followed by removal of the solvent under reduced pressure. The residue was then
purified by column chromatography on silica gel to give compound c (Scheme 3) in yields of
65%-80%.
2.1.3.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,
3]dioxol-4-yl)methyl 2-(3-bromophenyl)acetate (b1)
White powder, 63.1%, mp 174-176°C; 1H NMR (DMSO-d6): 1.33 (s, 3H, CH3); 1.54 (s,
3H, CH3); 3.56-3.67 (m, 2H, CH2); 4.16-4.20 (m, 1H, CH); 4.26-4.30 (m, 1H, CH); 4.36-4.39
(m, 1H, CH); 5.01-5.04 (m, 1H, CH); 5.42-5.44 (m, 1H, CH); 6.19 (s, 1H, CH); 7.16 (d,
J=8.3Hz, 2H, ArH); 7.39 (s, 2H, NH2); 7.47 (d, J=8.2Hz, 2H, ArH); 8.17 (s, 1H, CH purine);
8.30 (s, 1H, CH purine); EIMS m/z 503 (M+). Anal. Calcd for C21H22BrN5O5: C, 50.01; H, 4.40;
Br, 15.84; N, 13.89; Found: C, 50.17; H, 4.42; Br, 15.78; N, 13.84.
5

2.1.4.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,
3]dioxol-4-yl)methyl 2-(3-chlorophenyl)acetate (b2)
White powder, 82.4%, mp 144-146°C; 1H NMR (DMSO-d6): 1.33 (s, 3H, CH3); 1.55 (s,
3H, CH3); 3.64-3.74 (m, 2H, CH2); 4.18-4.22 (m, 1H, CH); 4.28-4.32 (m, 1H, CH); 4.36-4.40
(m, 1H, CH); 5.03-5.05 (m, 1H, CH); 5.43-5.45 (m, 1H, CH); 6.19 (d, J=2.2Hz, 1H, CH);
7.20-7.22 (m, 1H, ArH); 7.37 (s, 2H, NH2); 7.52-7.55 (m, 3H, ArH); 8.17 (d, J=1.9Hz, 1H,
CH purine); 8.30 (s, 1H, CH purine); EIMS m/z 459 (M+). Anal. Calcd for C21H22ClN5O5: C,
54.85; H, 4.82; Cl, 7.71; N, 15.23; Found: C, 54.67; H, 4.82; Cl, 7.74; N, 15.29.
2.1.5.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl 2-(3-bromophenyl)acetate (c1)
White powder, 55.4%, mp 168-170°C; 1H NMR (DMSO-d6): 3.72 (s, 2H, CH2); 4.10 (s,
1H, CH); 4.26 (s, 2H, CH2); 4.36 (d, J=11.6Hz, 1H, CH); 4.66 (s, 1H, CH); 5.38 (s, 1H, OH);
5.57 (s, 1H, OH); 5.91 (d, 1H, CH); 7.24-7.46 (m, 6H, ArH and NH2); 8.14 (d, J=2.6Hz, 1H,
CH purine); 8.30 (d, J=3.2Hz, 1H, CH purine); EIMS m/z 463 (M+). Anal. Calcd for
C18H18BrN5O5: C, 46.57; H, 3.91; Br, 17.21; N, 15.08; Found: C, 46.63; H, 3.90; Br, 17.27; N,
15.04.
2.1.6.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl 2-(3-chlorophenyl)acetate (c2)
White powder, 72.8%, mp 167-168°C; 1H NMR (DMSO-d6): 3.69 (s, 2H, CH2); 4.11 (s,
1H, CH); 4.22-4.30 (m, 2H, CH2); 4.38 (d, J=11.8Hz, 1H, CH); 4.67 (t, J=4.9Hz, 1H, CH);
5.42 (d, J=5.4Hz, 1H, OH); 5.59-5.63 (m, 1H, OH); 5.93 (s, 1H, CH); 7.19-7.22 (m, 1H,
ArH); 7.31-7.33 (m, 5H, ArH and NH2); 8.16 (d, J=5.0Hz, 1H, CH purine); 8.32 (d, J=3.2Hz,
13
1H, CH purine); C NMR (DMSO-d6, 100 MHz): δ 40.02; 64.90; 70.70; 73.28; 81.89; 88.17;
119.63; 127.31; 128.69; 129.83; 130.55; 133.29; 137.11; 140.22; 149.83; 153.15; 156.58;
171.15; EIMS m/z 419 (M+). Anal. Calcd for C18H18ClN5O5: C, 51.50; H, 4.32; Cl, 8.44; N,
16.68; Found: C, 51.34; H, 4.33; Cl, 8.47; N, 16.72.
6

2.1.7.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d]
[1,3]dioxol-4-yl)methyl 2-(4-fluorophenyl)acetate (b3)
White powder, 80.5%, mp 154-156°C; 1H NMR (DMSO-d6): 1.33 (s, 3H, CH3); 1.54 (s,
3H, CH3); 3.57-3.67 (m, 2H, CH2); 4.16-4.20 (m, 1H, CH); 4.26-4.30 (m, 1H, CH); 4.37-4.40
(m, 1H, CH); 5.02-5.04 (m, 1H, CH); 5.42-5.44 (m, 1H, CH); 6.18 (d, J=2.2Hz, 1H, CH);
7.07-7.13 (m, 2H, ArH); 7.21-7.25 (m, 2H, ArH); 7.38 (s, 2H, NH2); 8.17 (s, 1H, CH purine);
8.30 (s, 1H, CH purine); EIMS m/z 443 (M+). Anal. Calcd for C21H22FN5O5: C, 56.88; H, 5.00;
F, 4.28; N, 15.79; Found: C, 56.97; H, 5.01; F, 4.27; N, 15.73.
2.1.8.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl 2-(4-fluorophenyl)acetate (c3)
White powder, 62.1%, mp 172-174°C; 1H NMR (DMSO-d6): 3.64 (s, 2H, CH2); 4.18 (d,
J=1.9Hz, 1H, CH); 4.27-4.34 (m, 2H, CH2); 4.41 (d, J=11.9Hz, 1H, CH); 4.65 (d, J=4.0Hz,
1H, CH); 5.26 (d, J=5.2Hz, 1H, OH); 5.54 (d, J=4.8Hz, 1H, OH); 5.97 (d, J=1.7Hz, 1H, CH);
6.99-7.03 (m, 2H, ArH); 7.06 (s, 2H, NH2); 7.24 (t, J=6.0Hz, 2H, ArH); 8.11 (s, 1H, CH purine);
8.18 (s, 1H, CH purine); 13C NMR (DMSO-d6, 100 MHz): δ 40.02; 64.45; 70.59; 73.64; 81.78;
88.68; 115.23; 115.42; 119.89; 130.13; 131.26; 131.33; 139.56; 149.64; 152.96; 156.43;
160.52; 171.04; EIMS m/z 403 (M+). Anal. Calcd for C18H18FN5O5: C, 53.60; H, 4.50; F, 4.71;
N, 17.36; Found: C, 53.42; H, 4.51; F, 4.72; N, 17.41.
2.1.9.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d]
[1,3]dioxol-4-yl)methyl 2-(4-bromophenyl)acetate (b4)
White powder, 50.5%, mp 136-138°C; 1H NMR (DMSO-d6): 1.33 (s, 3H, CH3); 1.54 (s,
3H, CH3); 3.56-3.67 (m, 2H, CH2); 4.16-4.20 (m, 1H, CH); 4.26-4.30 (m, 1H, CH); 4.36-4.39
(m, 1H, CH); 5.01-5.04 (m, 1H, CH); 5.42-5.44 (m, 1H, CH); 6.19 (s, 1H, CH); 7.16 (d,
J=8.3Hz, 2H, ArH); 7.39 (s, 2H, NH2); 7.47 (d, J=8.2Hz, 2H, ArH); 8.17 (s, 1H, CH purine);
8.30 (s, 1H, CH purine); EIMS m/z 503 (M+). Anal. Calcd for C21H22BrN5O5: C, 50.01; H, 4.40;
Br, 15.84; N, 13.89; Found: C, 50.18; H, 4.39; Br, 15.79; N, 13.85.
7

2.2.0.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d]
[1,3]dioxol-4-yl)methyl 2-(4-chlorophenyl)acetate (b5)
White powder, 66.3%, mp 160-162°C; 1H NMR (DMSO-d6): 1.33 (s, 3H, CH3); 1.54 (s,
3H, CH3); 3.58-3.68 (m, 2H, CH2); 4.16-4.21 (m, 1H, CH); 4.26-4.30 (m, 1H, CH); 4.36-4.40
(m, 1H, CH); 5.02-5.04 (m, 1H, CH); 5.42-5.44 (m, 1H, CH); 6.18 (d, J=2.2Hz, 1H, CH);
7.22 (d, J=8.4Hz, 2H, ArH); 7.34 (d, J=8.4Hz, 2H, ArH); 7.39 (s, 2H, NH2); 8.17 (s, 1H, CH
purine); 8.30 (s, 1H, CH purine); EIMS m/z 459 (M+). Anal. Calcd for C21H22ClN5O5: C, 54.85;
H, 4.82; Cl, 7.71; N, 15.23; Found: C, 54.67; H, 4.82; Cl, 7.74; N, 15.29.
2.2.1.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl 2-(4-bromophenyl)acetate (c4)
White powder, 72.7%, mp 113-115°C; 1H NMR (DMSO-d6): 3.70 (s, 2H, CH2); 4.12 (s,
1H, CH); 4.22-4.26 (m, 2H, CH2); 4.34-4.37 (m, 1H, CH); 4.63 (t, J=4.8Hz, 1H, CH); 5.46 (d,
J=5.5Hz, 1H, OH); 5.62 (d, J=5.7Hz, 1H, OH); 5.94 (d, J=4.6Hz, 1H, CH); 7.20 (d, J=8.2Hz,
2H, NH2); 7.48 (d, J=8.1Hz, 2H, ArH); 8.33 (m, 3H, ArH and CH purine); 8.48 (s, 1H, CH
purine); EIMS m/z 463 (M+). Anal. Calcd for C18H18BrN5O5: C, 46.57; H, 3.91; Br, 17.21; N,
15.08; Found: C, 46.41; H, 3.91; Br, 17.27; N, 15.13.
2.2.2.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 2-(4-chlorophenyl)acetate (c5)
White powder, 68.3%, mp 86-88°C; 1H NMR (DMSO-d6): 3.70 (d, J=2.1Hz, 2H, CH2);
4.07-4.11 (m, 1H, CH); 4.21-4.29 (m, 2H, CH2); 4.34-4.38 (m, 1H, CH); 4.65-4.68 (m, 1H,
CH); 5.37 (d, J=5.4Hz, 1H, OH); 5.57 (d, J=5.7Hz, 1H, OH); 5.91 (d, J=4.9Hz, 1H, CH);
7.25 (d, J=8.5Hz, 2H, NH2); 7.32-7.36 (m, 4H, ArH); 8.16 (m, 1H, CH purine); 8.31 (s, 1H, CH
13
purine); C NMR (DMSO-d6, 100 MHz): δ 39.62; 64.79; 70.69; 73.27; 81.89; 88.18; 119.63;
128.69; 128.69; 131.76; 131.76; 132.04; 133.71; 140.22; 149.82; 153.12; 156.55; 171.24;
EIMS m/z 419 (M+). Anal. Calcd for C18H18ClN5O5: C, 51.50; H, 4.32; Cl, 8.44; N, 16.68;
Found: C, 51.31; H, 4.33; Cl, 8.47; N, 16.73.
8

2.2.3.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,
3]dioxol-4-yl)methyl 2-(3-fluorophenyl)acetate (b6)
White powder, 79.8%, mp 177-179°C; 1H NMR (DMSO-d6): 1.33 (s, 3H, CH3); 1.54 (s,
3H, CH3); 3.64-3.74 (m, 2H, CH2); 4.22 (d, J=6.1Hz, 1H, CH); 4.31 (d, J=11.2Hz, 1H, CH);
4.39 (s, 1H, CH); 5.03 (s, 1H, CH); 5.39 (d, J=5.6Hz, 1H, CH); 6.20 (s, 1H, CH); 7.10-7.18
(m, 2H, ArH); 7.25-7.32 (m, 2H, ArH); 7.39 (s, 2H, NH2); 8.17-8.19 (m, 1H, CH purine); 8.29
(s, 1H, CH purine); EIMS m/z 443 (M+). Anal. Calcd for C21H22FN5O5: C, 56.88; H, 5.00; F,
4.28; N, 15.79; Found: C, 56.69; H, 5.01; F, 4.28; N, 15.85.
2.2.4.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,
3]dioxol-4-yl)methyl 2-(4-hydroxyphenyl)acetate (b7)
White powder, 61.1%, mp 168-170°C; 1H NMR (DMSO-d6): 1.33 (s, 3H, CH3); 1.54 (s,
3H, CH3); 3.51 (d, J=3.7Hz, 2H, CH2); 4.16-4.20 (m, 1H, CH); 4.26-4.30 (m, 1H, CH);
4.36-4.39 (m, 1H, CH); 5.00-5.03 (m, 1H, CH); 5.39-5.41 (m, 1H, CH); 6.18 (d, J=2.4Hz, 1H,
CH); 6.58-6.64 (m, 3H, ArH); 7.05 (t, J=8.2Hz, 1H, ArH); 7.36 (s, 2H, NH2); 8.17 (s, 1H, CH
purine); 8.28 (s, 1H, CH purine); 9.35 (s, 1H, OH); EIMS m/z 441 (M+). Anal. Calcd for
C21H23N5O6: C, 57.14; H, 5.25; N, 15.86; Found: C, 57.33; H, 5.21; N, 15.80.
2.2.5.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d]
[1,3]dioxol-4-yl)methyl 2-(2-hydroxyphenyl)acetate (b8)
White powder, 83.5%, mp 142-144°C; 1H NMR (DMSO-d6): 1.32 (s, 3H, CH3); 1.54 (s,
3H, CH3); 3.51 (s, 2H, CH2); 4.16-4.20 (m, 1H, CH); 4.23-4.27 (m, 1H, CH); 4.38-4.41 (m,
1H, CH); 4.95-4.97 (m, 1H, CH); 5.21-5.23 (m, 1H, CH); 6.15 (d, J=2.6Hz, 1H, CH); 6.67 (t,
J=7.4Hz, 1H, ArH); 6.78 (d, J=7.6Hz, 1H, ArH); 6.99 (d, J=7.4Hz, 1H, ArH); 7.03-7.07 (m,
1H, ArH); 7.35 (s, 2H, NH2); 8.17 (s, 1H, CH purine); 8.22 (s, 1H, CH purine); 9.51 (s, 1H, OH);
EIMS m/z 441 (M+). Anal. Calcd for C21H23N5O6: C, 57.14; H, 5.25; N, 15.86; Found: C,
57.33; H, 5.21; N, 15.83.
9

2.2.6.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl 2-(3-fluorophenyl)acetate (c6)
White powder, 45.7%, mp 158-159°C; 1H NMR (DMSO-d6): 3.74 (s, 2H, CH2); 4.11 (s,
1H, CH); 4.23-4.27 (m, 2H, CH2); 4.36-4.39 (m, 1H, CH); 4.66 (d, J=3.4Hz, 1H, CH); 5.39
(d, J=4.2Hz, 1H, OH); 5.58 (d, J=4.5Hz, 1H, OH); 5.92-5.93 (m, 1H, CH); 7.08 (t, J=8.2Hz,
3H, ArH); 7.32-7.36 (m, 3H, ArH and NH2); 8.16 (d, J=2.0Hz, 1H, CH purine); 8.30 (d,
J=1.1Hz, 1H, CH purine); EIMS m/z 403 (M+). Anal. Calcd for C18H18FN5O5: C, 53.60; H,
4.50; F, 4.71; N, 17.36; Found: C, 53.46; H, 4.51; F, 4.71; N, 17.41.
2.2.7.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d]
[1,3]dioxol-4-yl)methyl 2-(3-hydroxyphenyl)acetate (b9)
White powder, 81.3%, mp 172-174°C; 1H NMR (DMSO-d6): 1.33 (s, 3H, CH3); 1.54 (s,
3H, CH3); 3.51 (d, J=3.7Hz, 2H, CH2); 4.16-4.20 (m, 1H, CH); 4.26-4.30 (m, 1H, CH);
4.36-4.39 (m, 1H, CH); 5.00-5.03 (m, 1H, CH); 5.39-5.41 (m, 1H, CH); 6.18 (d, J=2.4Hz, 1H,
CH); 6.58-6.64 (m, 3H, ArH); 7.05 (t, J=8.2Hz, 1H, ArH); 7.36 (s, 2H, NH2); 8.17 (s, 1H, CH
purine); 8.28 (s, 1H, CH purine); 9.35 (s, 1H, OH); EIMS m/z 441 (M+). Anal. Calcd for
C21H23N5O6: C, 57.14; H, 5.25; N, 15.86; Found: C, 57.34; H, 5.21; N, 15.81.
2.2.8.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 2-(4-hydroxyphenyl)acetate (c7)
White powder, 39.8%, mp 137-139°C; 1H NMR (DMSO-d6): 3.58 (s, 2H, CH2); 4.12 (s,
1H, CH); 4.19-4.25 (m, 2H, CH2); 4.33-4.37 (m, 1H, CH); 4.64 (s, 1H, CH); 5.39 (m, 1H,
OH); 5.58 (s, 1H, OH); 5.92 (d, J=4.7Hz, 1H, CH); 6.61-6.65 (m, 3H, ArH); 7.07 (t, J=7.6Hz,
1H, ArH); 7.41 (s, 2H, NH2); 8.17 (s, 1H, CH purine); 8.33 (s, 1H, CH purine); 9.39 (s, 1H, OH);
EIMS m/z 401 (M+). Anal. Calcd for C18H19N5O6: C, 53.86; H, 4.77; N, 17.45; Found: C, C,
53.69; H, 4.78; N, 17.51.
2.2.9.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 2-(2-hydroxyphenyl)acetate (c8)
White powder, 40.3%, mp 125-127°C; 1H NMR (DMSO-d6): 3.55 (s, 2H, CH2); 4.10 (s,
10

1H, CH); 4.19-4.25 (m, 2H, CH2); 4.31-4.37 (m, 1H, CH); 4.65 (s, 1H, CH); 5.39 (m, 1H,
OH); 5.58 (s, 1H, OH); 5.91 (d, J=4.7Hz, 1H, CH); 6.62-6.65 (m, 3H, ArH); 7.06 (t, J=7.6Hz,
1H, ArH); 7.42 (s, 2H, NH2); 8.19 (s, 1H, CH purine); 8.33 (s, 1H, CH purine); 9.37 (s, 1H, OH);
EIMS m/z 401 (M+). Anal. Calcd for C18H19N5O6: C, 53.86; H, 4.77; N, 17.45; Found: C,
53.71; H, 4.78; N, 17.49.
2.3.0.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 2-(3-hydroxyphenyl)acetate (c9)
White powder, 57.3%, mp 112-114°C; 1H NMR (DMSO-d6): 3.57 (s, 2H, CH2); 4.10 (s,
1H, CH); 4.19-4.25 (m, 2H, CH2); 4.33-4.37 (m, 1H, CH); 4.64 (s, 1H, CH); 5.39 (m, 1H,
OH); 5.58 (s, 1H, OH); 5.91 (d, J=4.7Hz, 1H, CH); 6.62-6.65 (m, 3H, ArH); 7.06 (t, J=7.6Hz,
1H, ArH); 7.41 (s, 2H, NH2); 8.17 (s, 1H, CH purine); 8.32 (s, 1H, CH purine); 9.37 (s, 1H, OH);
EIMS m/z 401 (M+). Anal. Calcd for C18H19N5O6: C, 53.86; H, 4.77; N, 17.45; Found: C,
53.95; H, 4.76; N, 17.39.
2.3.1.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,
3]dioxol-4-yl)methyl 2-p-tolylacetate (b10)
1
White powder, 64.5%, mp 83-85°C; H NMR (DMSO-d6): 1.32 (s, 3H, CH3); 1.54 (s,
3H, CH3); 2.25 (s, 3H, CH3); 3.54 (d, J=3.1Hz, 2H, CH2); 4.16-4.20 (m, 1H, CH); 4.26-4.29
(m, 1H, CH); 4.38 (s, 1H, CH); 5.00 (d, J=2.9Hz, 1H, CH); 5.38 (d, J=4.8Hz, 1H, CH); 6.17
(d, J=2.4Hz, 1H, CH); 7.06 (s, 4H, ArH); 7.38 (s, 2H, NH2); 8.17 (d, J=5.7Hz, 1H, CH purine);
8.27 (d, J=3.2Hz, 1H, CH purine); EIMS m/z 439 (M+). Anal. Calcd for C22H25N5O5: C, 60.13;
H, 5.73; N, 15.94; Found: C, 60.32; H, 5.71; N, 15.89.
2.3.2.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,
3]dioxol-4-yl)methyl 2-(3,4-dichlorophenyl)acetate (b11)
1
White powder, 50.8%, mp 90-92°C; H NMR (DMSO-d6): 1.33 (s, 3H, CH3); 1.55 (s,
3H, CH3); 3.64-3.74 (m, 2H, CH2); 4.18-4.22 (m, 1H, CH); 4.28-4.32 (m, 1H, CH); 4.36-4.40
(m, 1H, CH); 5.03-5.05 (m, 1H, CH); 5.43-5.45 (m, 1H, CH); 6.19 (d, J=2.2Hz, 1H, CH);
7.20-7.22 (m, 1H, ArH); 7.37 (s, 2H, NH2); 7.52-7.55 (m, 2H, ArH); 8.17 (d, J=1.9Hz, 1H,
CH purine); 8.30 (s, 1H, CH purine); EIMS m/z 493 (M+). Anal. Calcd for C21H21Cl2N5O5: C,
11

51.02; H, 4.28; Cl, 14.34; N, 14.17; Found: C, 51.20; H, 4.27; Cl, 14.31; N, 14.13.
2.3.3.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 2-(3,4-dichlorophenyl)acetate (c11)
1
White powder, 70.7%, mp 172-174°C; H NMR (DMSO-d6): 3.71 (d, J=2.6Hz, 2H,
CH2); 4.08-4.12 (m, 1H, CH); 4.22-4.27 (m, 2H, CH2); 4.34-4.37 (m, 1H, CH); 4.63 (t,
J=5.0Hz, 1H, CH); 5.37 (d, J=5.4Hz, 1H, OH); 5.57 (d, J=5.7Hz, 1H, OH); 5.91 (d, J=4.9Hz,
1H, CH); 7.19-7.21 (m, 1H, ArH); 7.48-7.51 (m, 2H, ArH); 7.66 (s, 2H, NH2); 8.21 (m, 1H,
13
CH purine); 8.37 (s, 1H, CH purine); C NMR (DMSO-d6, 100 MHz): δ 39.12; 64.90; 70.64;
73.41; 81.98; 88.29; 119.53; 130.07; 130.41; 130.77; 131.22; 131.97; 135.77; 140.77; 149.59;
151.73; 155.40; 170.94; EIMS m/z 453 (M+). Anal. Calcd for C18H17Cl2N5O5: C, 47.59; H,
3.77; Cl, 15.61; N, 15.42; Found: C, 47.47; H, 3.78; Cl, 15.67; N, 15.45.
2.3.4.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 2-p-tolylacetate (c10)
1
White powder, 70.4%, mp 80-82°C; H NMR (DMSO-d6): 2.26 (s, 3H, CH3); 3.62 (s,
2H, CH2); 4.10 (s, 1H, CH); 4.20-4.27 (m, 2H, CH2); 4.34 (d, J=11.2Hz, 1H, CH); 4.65 (d,
J=4.2Hz, 1H, CH); 5.40 (s, 1H, OH); 5.60 (s, 1H, OH); 5.90-5.93 (m, 1H, CH); 7.09 (s, 4H,
ArH); 7.34 (s, 2H, NH2); 8.15-8.17 (m, 1H, CH purine); 8.28 (t, J=2.2Hz, 1H, CH purine); EIMS
m/z 399 (M+). Anal. Calcd for C19H21N5O5: C, 57.14; H, 5.30; N, 17.53; Found: C, 57.33; H,
5.29; N, 17.47.
2.3.5.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,
3]dioxol-4-yl)methyl 2-(3,4-dimethoxyphenyl)acetate (b12)
White powder, 80.5%, mp 132-134°C; 1H NMR (DMSO-d6): 1.33 (s, 6H, CH3); 1.55 (s,
6H, CH3); 3.64-3.74 (m, 2H, CH2); 4.18-4.22 (m, 1H, CH); 4.28-4.32 (m, 1H, CH); 4.36-4.40
(m, 1H, CH); 5.03-5.05 (m, 1H, CH); 5.43-5.45 (m, 1H, CH); 6.19 (d, J=2.2Hz, 1H, CH);
7.20-7.22 (m, 1H, ArH); 7.37 (s, 2H, NH2); 7.52-7.55 (m, 2H, ArH); 8.17 (d, J=1.9Hz, 1H,
CH purine); 8.30 (s, 1H, CH purine). Anal. Calcd for C23H27N5O7: C, 56.90; H, 5.61; N, 14.43;
Found: C, 56.83; H, 5.62; N, 14.47.
12

2.3.6.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 2-(3,4-dimethoxyphenyl)acetate (c12)
White powder, 88.3%, mp 162-164°C; 1H NMR (DMSO-d6): 1.32 (s, 3H, CH3); 1.53 (s,
3H, CH3); 3.60-3.70 (m, 2H, CH2); 4.18-4.29 (m, 2H, CH2); 4.39-4.42 (m, 1H, CH);
4.96-4.98 (m, 1H, CH); 5.29-5.31 (m, 1H, CH); 5.46 (d, J=5.5Hz, 1H, OH); 5.62 (d, J=5.7Hz,
1H, OH); 6.15 (d, J=1.9Hz, 1H, CH); 7.23 (d, J=8.4Hz, 2H, NH2); 7.32-7.34 (m, 3H, ArH);
8.11 (s, 1H, CH purine); 8.25 (s, 1H, CH purine); EIMS m/z 445 (M+). Anal. Calcd for
C20H23N5O7: C, 53.93; H, 5.20; N, 15.72; Found: C, 53.75; H, 5.21; N, 15.66.
2.3.7.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,
3]dioxol-4-yl)methyl 2-(2-chlorophenyl)acetate (b13)
White powder, 54.8%, mp 155-156°C; 1H NMR (DMSO-d6): 1.32 (s, 3H, CH3); 1.54 (s,
3H, CH3); 3.54 (d, J=3.1Hz, 2H, CH2); 4.16-4.20 (m, 1H, CH); 4.26-4.29 (m, 1H, CH); 4.38
(s, 1H, CH); 5.00 (d, J=2.9Hz, 1H, CH); 5.38 (d, J=4.8Hz, 1H, CH); 6.17 (d, J=2.4Hz, 1H,
CH); 7.06 (s, 4H, ArH); 7.38 (s, 2H, NH2); 8.17 (d, J=5.7Hz, 1H, CH purine); 8.27 (d, J=3.2Hz,
1H, CH purine); EIMS m/z 459 (M+). Anal. Calcd for C21H22ClN5O5: C, 54.85; H, 4.82; Cl,
7.71; N, 15.22; Found: C, 54.97; H, 4.81; Cl, 7.69; N, 15.18.
2.3.8.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 2-(2-chlorophenyl)acetate (c13)
1
White powder, 77.1%, mp 185-187°C; H NMR (DMSO-d6): 3.83 (d, J=3.6Hz, 2H,
CH2); 4.09-4.12 (m, 1H, CH); 4.23-4.28 (m, 2H, CH2); 4.37-4.41 (m, 1H, CH); 4.60-4.64 (m,
1H, CH); 5.38 (d, J=5.4Hz, 1H, OH); 5.56 (d, J=5.9Hz, 1H, OH); 5.91 (d, J=5.2Hz, 1H, CH);
7.27-7.34 (m, 4H, ArH and NH2); 7.37-7.40 (m, 1H, ArH); 7.43-7.46 (m, 1H, ArH); 8.16 (m,
1H, CH purine); 8.26 (s, 1H, CH purine); 13C NMR (DMSO-d6, 100 MHz): δ 38.69; 64.95; 70.74;
73.34; 82.00; 88.02; 119.62; 127.68; 129.54; 129.57; 132.60; 132.96; 134.17; 140.06; 149.87;
153.16; 156.57; 170.45; EIMS m/z 419 (M+). Anal. Calcd for C18H18ClN5O5: C, 51.50; H,
4.32; Cl, 8.44; N, 16.68; Found: C, 51.67; H, 4.32; Cl, 8.42; N, 16.62.
13

2.3.9.((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,
3]dioxol-4-yl)methyl 2-(2-fluorophenyl)acetate (b14)
White powder, 78.9%, mp 104-106°C; 1H NMR (DMSO-d6): 1.33 (s, 3H, CH3); 1.54 (s,
3H, CH3); 3.64-3.74 (m, 2H, CH2); 4.22 (d, J=6.1Hz, 1H, CH); 4.31 (d, J=11.2Hz, 1H, CH);
4.39 (s, 1H, CH); 5.03 (s, 1H, CH); 5.39 (d, J=5.6Hz, 1H, CH); 6.20 (s, 1H, CH); 7.10-7.18
(m, 2H, ArH); 7.25-7.32 (m, 2H, ArH); 7.39 (s, 2H, NH2); 8.17-8.19 (m, 1H, CH purine); 8.29
(s, 1H, CH purine); EIMS m/z 443 (M+). Anal. Calcd for C21H22FN5O5: C, 56.88; H, 5.00; F,
4.28; N, 15.79; Found: C, 56.72; H, 5.00; F, 4.29; N, 15.84.
2.4.0.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 2-(2-fluorophenyl)acetate (c14)
1
White powder, 67.3%, mp 186-187°C; H NMR (DMSO-d6): 3.76 (s, 2H, CH2);
4.10-4.14 (m, 1H, CH); 4.25-4.29 (m, 2H, CH2); 4.38-4.42 (m, 1H, CH); 4.63-4.67 (m, 1H,
CH); 5.41 (d, J=5.4Hz, 1H, OH); 5.60 (d, J=5.8Hz, 1H, OH); 5.94 (d, J=5.1Hz, 1H, CH);
7.12-7.20 (m, 2H, ArH); 7.31-7.35 (m, 4H, ArH and NH2); 8.18 (m, 1H, CH purine); 8.29 (s,
13
1H, CH purine); C NMR (DMSO-d6, 100 MHz): δ 34.22 (J=2.61Hz); 64.98; 70.72; 73.35;
81.97; 88.08; 115.56 (J=21.37Hz); 119.62; 121.93 (J=16.00Hz); 124.80 (J=3.49Hz); 129.76
(J=8.18Hz); 132.44 (J=4.03Hz); 140.09; 149.86; 153.17; 156.58; 161.10 (J=243.37Hz);
170.62; EIMS m/z 403 (M+). Anal. Calcd for C18H18FN5O5: C, 53.60; H, 4.50; F, 4.71; N,
17.36; Found: C, 53.81; H, 4.49; F, 4.70; N, 17.32.
2.4.1.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl acetate (c15)
White powder, 65,8%, mp 198-201°C; 1H NMR (DMSO-d6): 0.84 (t, J=7.4Hz, 3H, CH3);
4.07-4.10 (m, 1H, CH); 4.17-4.22 (m, 1H, CH); 4.25-4.29 (m, 1H, CH); 4.33-4.37 (m, 1H,
CH); 4.66-4.70 (m, 1H, CH); 5.42 (d, J=4.8Hz, 1H, OH); 5.63 (d, J=5.7Hz, 1H, OH); 5.91 (d,
J=4.8Hz, 1H, CH); 7.35 (s, 2H, NH2); 8.16 (s, 1H, CH purine); 8.33 (s, 1H, CH purine); EIMS
m/z 309 (M+). Anal. Calcd for C12H15N5O5: C, 46.60; H, 4.89; N, 22.64; Found: C, 46.53; H,
4.89; N, 22.72.
14

2.4.2.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl butyrate (c16)
White powder, 57.3%, mp 125-127°C; 1H NMR (DMSO-d6): 0.84 (t, J=7.4Hz, 3H, CH3);
1.46-1.55 (m, 2H, CH2); 2.26-2.29 (m, 2H, CH2); 4.07-4.10 (m, 1H, CH); 4.17-4.22 (m, 1H,
CH); 4.25-4.29 (m, 1H, CH); 4.33-4.37 (m, 1H, CH); 4.66-4.70 (m, 1H, CH); 5.42 (d,
J=4.8Hz, 1H, OH); 5.63 (d, J=5.7Hz, 1H, OH); 5.91 (d, J=4.8Hz, 1H, CH); 7.35 (s, 2H, NH2);
8.16 (s, 1H, CH purine); 8.33 (s, 1H, CH purine); EIMS m/z 337 (M+). Anal. Calcd for
C14H19N5O5: C, 49.85; H, 5.68; N, 20.76; Found: C, 49.97; H, 5.66; N, 20.72.
2.4.3.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl hexanoate (c17)
White powder, 64.3%, mp 164-166°C; 1H NMR (DMSO-d6): 0.81 (t, J=6.9Hz, 3H, CH3);
1.15-1.28 (m, 4H, 2CH2); 1.44-1.52 (m, 2H, CH2); 2.26-2.30 (m, 2H, CH2); 4.06-4.09 (m, 1H,
CH); 4.17-4.21 (m, 1H, CH); 4.25-4.28 (m, 1H, CH); 4.32-4.36 (m, 1H, CH); 4.66-4.70 (m,
1H, CH); 5.40 (d, J=5.5Hz, 1H, OH); 5.61 (d, J=5.8Hz, 1H, OH); 5.91 (d, J=4.9Hz, 1H, CH);
7.34 (s, 2H, NH2); 8.15 (s, 1H, CH purine); 8.32 (s, 1H, CH purine); EIMS m/z 365 (M+). Anal.
Calcd for C16H23N5O5: C, 52.59; H, 6.34; N, 19.17; Found: C, 52.64; H, 6.32; N, 19.11.
2.4.4.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl octanoate (c18)
White powder, 70.3%, mp 101-103°C; 1H NMR (DMSO-d6): 0.83 (t, J=6.9Hz, 3H, CH3);
1.20 (s, 8H, 4CH2); 1.46-1.49 (m, 2H, CH2); 2.28 (t, J=7.1Hz, 2H, CH2); 4.06-4.09 (m, 1H,
CH); 4.17-4.22 (m, 1H, CH); 4.25 (d, J=3.6Hz, 1H, CH); 4.31-4.35 (m, 1H, CH); 4.67 (d,
J=4.1Hz, 1H, CH); 5.40 (d, J=3.8Hz, 1H, OH); 5.60 (d, J=4.4Hz, 1H, OH); 5.90 (d, J=4.9Hz,
1H, CH); 7.34 (s, 2H, NH2); 8.15 (s, 1H, CH purine); 8.32 (s, 1H, CH purine); EIMS m/z 393
(M+). Anal. Calcd for C18H27N5O5: C, 54.95; H, 6.92; N, 17.80; Found: C, 54.77; H, 6.94; N,
17.85.
15

2.4.5.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl decanoate (c19)
1
White powder, 61.6%, mp 94-96°C; H NMR (DMSO-d6): 0.84 (t, J=6.8Hz, 3H, CH3);
1.20 (s, 12H, 6CH2); 1.46-1.54 (m, 2H, CH2); 2.28 (t, J=14.3Hz, 2H, CH2); 4.06-4.09 (m, 1H,
CH); 4.17-4.21 (m, 1H, CH); 4.25 (s, 1H, CH); 4.31-4.35 (m, 1H, CH); 4.67 (d, J=3.1Hz, 1H,
CH); 5.40 (s, 1H, OH); 5.60 (d, J=3.5Hz, 1H, OH); 5.91 (d, J=4.9Hz, 1H, CH); 7.39 (s, 2H,
NH2); 8.16 (s, 1H, CH purine); 8.33 (s, 1H, CH purine); EIMS m/z 421 (M+). Anal. Calcd for
C20H31N5O5: C, 56.99; H, 7.41; N, 16.62; Found: C, 56.81; H, 7.39; N, 16.58.
2.4.6.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl benzoate (c20)
1
White powder, 80.3%, mp 140-142°C; H NMR (DMSO-d6): 4.23-4.26 (m, 1H, CH);
4.45-4.49 (m, 2H, CH2); 4.59-4.63 (m, 1H, CH); 4.79 (d, J=4.4Hz, 1H, CH); 5.51 (d,
J=4.4Hz, 1H, OH); 5.69 (d, J=4.9Hz, 1H, OH); 5.95 (d, J=4.7Hz, 1H, CH); 7.35 (s, 2H, NH2);
7.52 (t, J=7.7Hz, 2H, ArH); 7.67 (t, J=7.4Hz, 1H, ArH); 7.95 (d, J=7.2Hz, 2H, ArH); 8.12 (s,
1H, CH purine); 8.32 (s, 1H, CH purine); EIMS m/z 371 (M+). Anal. Calcd for C17H17N5O5: C,
54.98; H, 4.61; N, 18.86; Found: C, 54.77; H, 4.62; N, 18.91.
2.4.7.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 4-chlorobenzoate (c21)
1
White powder, 60.1%, mp 122-124°C; H NMR (DMSO-d6): 4.23-4.27 (m, 1H, CH);
4.46-4.51 (m, 2H, CH2); 4.61-4.65 (m, 1H, CH); 4.78-4.82 (m, 1H, CH); 5.50 (d, J=5.6Hz,
1H, OH); 5.67 (d, J=5.6Hz, 1H, OH); 5.96 (d, J=4.6Hz, 1H, CH); 7.37 (s, 2H, NH2); 7.59 (d,
J=8.6Hz, 2H, ArH); 7.94 (d, J=8.6Hz, 2H, ArH); 8.13 (s, 1H, CH purine); 8.33 (s, 1H, CH purine);
EIMS m/z 405 (M+). Anal. Calcd for C17H16ClN5O5: C, 50.32; H, 3.97; Cl, 8.74; N, 17.26;
Found: C, 50.15; H, 3.98; Cl, 8.76; N, 17.31.
2.4.8.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 3-chlorobenzoate (c22)
1
White powder, 80.1%, mp 120-121°C; H NMR (DMSO-d6): 4.21-4.25 (m, 1H, CH);
16

4.30-4.50 (m, 2H, CH2); 4.60-4.64 (m, 1H, CH); 4.76-4.80 (m, 1H, CH); 5.46 (d, J=5.5Hz,
1H, OH); 5.62 (d, J=5.7Hz, 1H, OH); 5.93 (d, J=4.8Hz, 1H, CH); 7.33 (s, 2H, NH2); 7.56 (t,
J=7.9Hz, 1H, ArH); 7.75-7.77 (m, 1H, ArH); 7.88-7.92 (m, 2H, ArH); 8.08 (s, 1H, CH purine);
8.32 (s, 1H, CH purine); EIMS m/z 405 (M+). Anal. Calcd for C17H16ClN5O5: C, 50.32; H, 3.97;
Cl, 8.74; N, 17.26; Found: C, 50.52; H, 3.96; Cl, 8.71; N, 17.20.
2.4.9.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 3-fluorobenzoate (c23)
White powder, 85.6%, mp 139-141°C; 1H NMR (DMSO-d6): 4.24 (d, J=3.4Hz, 1H, CH);
4.47-4.51 (m, 2H, CH2); 4.61 (d, J=11.8Hz, 1H, CH); 4.79 (d, J=4.8Hz, 1H, CH); 5.48 (d,
J=4.8Hz, 1H, OH); 5.64 (d, J=4.9Hz, 1H, OH); 5.94 (d, J=4.1Hz, 1H, CH); 7.34 (s, 2H, NH2);
7.52-7.60 (m, 2H, ArH); 7.71 (d, J=9.2Hz, 1H, ArH); 7.78 (d, J=7.2Hz, 1H, ArH); 8.10 (s, 1H,
CH purine); 8.32 (s, 1H, CH purine); EIMS m/z 389 (M+). Anal. Calcd for C17H16FN5O5: C,
52.44; H, 4.14; F, 4.88; N, 17.99; Found: C, 52.56; H, 4.14; F, 4.87; N, 17.92.
2.5.0.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 4-fluorobenzoate (c24)
1
White powder, 76.4%, mp 98-100°C; H NMR (DMSO-d6): 4.23-4.26 (m, 1H, CH);
4.44-4.50 (m, 2H, CH2); 4.60-4.63 (m, 1H, CH); 4.81 (t, J=4.9Hz, 1H, CH); 5.48 (d, J=4.8Hz,
1H, OH); 5.64 (d, J=4.9Hz, 1H, OH); 5.96 (d, J=4.6Hz, 1H, CH); 7.33-7.37 (m, 4H, ArH and
NH2); 7.99-8.02 (m, 2H, ArH); 8.12 (s, 1H, CH purine); 8.32 (s, 1H, CH purine); EIMS m/z 389
(M+). Anal. Calcd for C17H16FN5O5: C, 52.44; H, 4.14; F, 4.88; N, 17.99; Found: C, 52.31; H,
4.15; F, 4.88; N, 18.05.
2.5.1.((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)met
hyl 4-bromobenzoate (c25)
1
White powder, 67.5%, mp 204-206°C; H NMR (DMSO-d6): 4.20-4.24 (m, 1H, CH);
4.43-4.48 (m, 2H, CH2); 4.59-4.63 (m, 1H, CH); 4.75-4.79 (m, 1H, CH); 5.46 (d, J=5.6Hz,
1H, OH); 5.63 (d, J=5.6Hz, 1H, OH); 5.93 (d, J=4.6Hz, 1H, CH); 7.35 (s, 2H, NH2); 7.73 (d,
J=8.4Hz, 2H, ArH); 7.85 (d, J=8.4Hz, 2H, ArH); 8.11 (s, 1H, CH purine); 8.31 (s, 1H, CH purine);
EIMS m/z 449 (M+). Anal. Calcd for C17H16BrN5O5: C, 45.35; H, 3.58; Br, 17.75; N, 15.55;
17

Found: C, 45.46; H, 3.57; Br, 17.70; N, 15.52.
2.2. Extraction of the TyrRS and enzyme assay
S. aureus TyrRS was over-expressed in E. coli and purified to near homogeneity (~98%
as judged by SDS-PAGE) using standard purification procedures. TyrRS activity was
measured by aminoacylation using modifications to previously described methods [17]. The
assays were performed at 37°C in a mixture containing (final concentrations) 100 mM Tris/Cl
pH 7.9, 50 mM KC1, 16 mM MgCl2, 5 mM ATP, 3 mM DTT, 4 mg/ml E. coli MRE600
tRNA (Roche) and 10 μM L-tyrosine (0.3 μM L-[ring-3,5-3H] tyrosine (PerkinElmer,
Specific activity: 1.48-2.22TBq/mmol), 10 μM carrier). TyrRS (0.2 nM) was preincubated
with a range of inhibitor concentrations for 10 min at room temperature followed by the
addition of pre-warmed mixture at 37°C. After specific intervals, the reaction was terminated
by adding aliquots of the reaction mix into ice-cold 7% trichloroacetic acid and harvesting
onto 0.45 mm hydrophilic Durapore filters (Millipore Multiscreen 96-well plates) and
counted by liquid scintillation. The rate of reaction in the experiments was linear with respect
to protein and time with less than 50% total tRNA acylation. IC50s correspond to the
concentration at which half of the enzyme activity is inhibited by the compound. The results
are presented in Table 1.
Table 1 In vitro inhibitory activity data of the synthesized compounds against S.aureus TyrRS
and
R
R
entry
IC50 (μM) entry
IC50 (μM)
3.7±0.4
23±0.1
b1
c7
18

6.3±0.1
7.1±0.4
44±1.2
9.3±0.6
b2
b3
c8
c9
5.6±0.2
8.4±0.2
15±0.7
97±0.5
b4
b5
c10
c11
1.1±0.3
>100
b6
c12
19±1.1
55±1.3
9.6±0.3
47±0.1
40±0.5
b7
b8
b9
c13
c14
c15
CH3
17±0.1
>100
65±0.7
77±2.2
b10
b11
c16
c17
>100
93±1.5
b12
c18
47±1.1
44±0.7
3.3±0.2
>100
b13
b14
c1
c19
c20
c21
39±0.2
43±0.3
6.0±0.1
7.7±0.5
5.2±0.4
28±0.1
11±0.1
51±0.4
c2
c3
c4
c22
c23
c24
19

8.1±0.7
62±0.2
c5
c6
c25
0.8±0.07
2.3. Antimicrobial activity
The antibacterial activities of the synthesized compounds were tested against
Gram-positive bacterial strain (S. aureus ATCC 6538, penicillin G sodium as positive control)
and two Gram-negative bacterial strains (E. coli ATCC 8739, ciprofloxacin as positive
control and P. aeruginosa ATCC 9027, ciprofloxacin as positive control) using LB medium.
The MTT proliferation assay was used to measure the MIC50s of the test compounds by
determining the absorbance of the cells in culture [14]. A stock solution of the synthesized
compound (1000 µg/ml) in DMSO was prepared with different concentrations using
sterilized liquid medium (50% (v/v) of DMSO in PBS). A specified quantity of the medium
containing the test compound was added into 96-well plates, which was replaced by the
sterilized liquid medium as blank control. Suspension of the microorganism was prepared to
contain approximate 105 cfu/mL and applied to 96-well plates with serially diluted
compounds (or blank control) to be tested and incubated at 37°C. In the case of fungi, plates
were incubated at 28°C. Fifty µL of PBS containing 3mg of MTT/mL was added to each well.
Incubation was continued at room temperature for 4-5 h. The content of each well was
removed, and 100 L of 10% sodium dodecyl sulfate containing 5% isopropanol and 10
mol/mL HCl was added to extract the dye. After 8-10 h of incubation at room temperature,
the control OD value (at 570 nm) should be controlled under 1.0-2.0 to achieve reliable
results. The observed MIC50s were presented in Table 2.
20

Table 2 Inhibitory activity (MIC50) of the synthetic compounds against microbes
Compound
MIC50 (μg/mL)
B
A
40.6
27.8
76.2
＞100
15.2
60.3
44.6
37.2
18.9
27
C
38.3
18.8
22
b1
b2
b3
b4
b5
b6
b7
b8
b9
b10
b11
b12
b13
b14
c1
44.9
＞100
＞100
7.2
24.8
21.2
30.6
12.9
17.4
23
＞100
＞100
＞100
＞100
＞100
40.2
25.9
14.1
19.3
30.1
28
34
47.3
29
＞100
＞100
＞100
＞100
49.2
25.7
38.1
20.2
＞100
88.9
7.7
25.5
27.4
35.1
26.1
19.3
19.9
0.08
25.9
26
c2
＞100
42.2
c3
c4
13.8
c5
1.3
9.2
c6
9.8
44.3
c7
15.9
37
37.8
c8
＞100
47.4
27.3
11.8
21.4
10.2
c9
16.4
19.4
16.5
c10
c11
c12
48.5
＞100
21

7.4
23.6
77.6
＞100
＞100
＞100
＞100
33.7
28.1
11.3
5.3
＞100
51.4
26.7
35.8
58.2
87.3
＞100
＞100
＞100
21.9
19.3
8.7
c13
c14
＞100
＞100
＞100
＞100
＞100
67.4
c15
c16
c17
c18
c19
c20
＞100
9.4
c21
c22
20.4
3.5
c23
c24
24.8
10.1
0.67
/
＞100
＞100
/
11.2
22.7
0.1
c25
ciprofloxacin
penicillin G sodium
0.54
/
(A) E. coli ATCC 8739; (B) S. aureus ATCC 6538; (C) P. aeruginosa ATCC 9027
2.4. Protocol of docking study
The automated docking studies were carried out using AutoDock version 4.2. First,
AutoGrid component of the program pre-calculates a three-dimensional grid of interaction
energies based on the macromolecular target using the AMBER force field. A grid box of
65×75×75 Å size (x, y, z) with a spacing of 0.375 Å and grid maps were created representing
the catalytic active target site region where the native ligand was embedded. Then automated
docking studies were carried out to evaluate the binding free energy of the inhibitor within
the macromolecules. The genetic algorithm with local search (GA-LS) was chosen to search
for the best conformers. The parameters were set using the software ADT (AutoDockTools
package, version 1.5.4) on PC which is associated with AutoDock 4.2. Default settings were
22

used with an initial population of 50 randomly placed individuals, a maximum number of
7.5×106 energy evaluations, and a maximum number of 2.7×104 generations. A mutation rate
of 0.02 and a crossover rate of 0.8 were chosen. Results differing by less than 0.5 Ǻ in
positional root-mean-square deviation (RMSD) were clustered together and the results of the
most favorable free energy of binding were selected as the resultant complex structures.
3. Results and discussion
3.1. Chemistry
Thirty nine adenosine analogues were synthesized by the routes outlined in Scheme 3.
Compound b was prepared by esterification of the corresponding acid with
2′,3′-O-isopropylideneadenosine and Compound c was prepared by the deprotection of
isopropylidene group from compound b [15-16]. All of these target compounds were first
reported and fully characterized by elementary analyses, EIMS, 1H NMR and 13C NMR.
3.2. Inhibitory activities of the adenosine analogues against TyrRS from S. aureus
All synthesized compounds (b1-b14 and c1-c25) were tested for inhibitory activity
against TyrRS from S. aureus and IC50 values are presented in Table 1. In the initial phase of
our studies, we focused our efforts on introduction of varies substituents to the benzene ring
of the phenylacetyl moiety (b1-b14 and c1-c14). Meta-fluorine containing compounds (b6
and c6) showed IC50 values of 1.1 and 0.8 μM, being the most potent member in their own
series, and substitution with chloro, bromo or hydroxy groups led to a slight decrease in
activity. In comparison with substituents at the 3-position, introduction of chloro, fluoro,
brom or hydroxy substituents at the 2-position or 4-position led to a significant reduction in
23

potency. Modification at the 2-position or 4-position therefore seems to be detrimental to
enzyme inhibition. Introduction two substituents (b11, b12, c11 and c12) on the aryl ring
resulted in significant attenuation of the level of TyrRS inhibition, indicating that these
substituents may interfere the binding of ligand molecules to the enzyme. We then turned our
attention to exploring the structure-activity relationship between 2′,3′
(c1 to c14) and 2
′
,3′
(b1 to b14). The compounds (c1 to c14) with two
hydroxyl groups at the 2
′
-position and 3′-position of the ribose exerted the higher inhibitory
activity against S. aureus TyrRS than the corresponding parent compounds (b1 to b14). This
suggested that the inversion of hydrogen-bond donors and acceptors caused by the
isopropylidene group blocks the hydrogen-bonding interactions between ligand and receptor,
leading to a drease in binding energy. Therefore, the deprotected
suitable for further research.
seem to be more
Finally, replacement of the substituted phenylacetyl (c5, c2, c6, c3 and c4) with
corresponding substituted benzoyl (c21 to c25) resulted in some attenuation of potency.
Introduction of a fluorine atom at 3-position or 4-position in benzene ring led to a slight
increase in potency with respect to the corresponding chlorine or bromine analogues (c22 vs
c23 and c24 vs c25), which suggest that it is advisable to maintaining a fluorine atom at an
appropriate position .
Compounds (c15 to c19) with R1 being an aliphatic group showed weak activity against
TyrRS and the activities decrease as carbon chain increase. Out of tested compounds,
compound c6 was proved as the most potent, having IC50 of 0.8±0.07 M.
3.3. Antibacterial activity
24

All compounds were tested against a representative Gram-positive organism (S. aureus
ATCC 6538) and two Gram-negative organisms (E. coli ATCC 8739; P. aeruginosa ATCC
9027), and the results are presented in Table 2. In general, some of the evaluated compounds
exhibited good activities against Gram-negative bacteria, especially against P. aeruginosa
ATCC 9027, and all are inactive against Gram-positive bacteria. Compound c6 is the most
potent compound in all of the synthetic compounds, which is close to or slightly over that of
marketed antibiotics (ciprofloxacin). The compounds with strong inhibition against TyrRS
also exhibit good antibacterial activities, especially against P. aeruginosa ATCC 9027, which
shows that their inhibition of TyrRS may lead to the antibacterial activity.
3.4. Molecular docking
Docking study was undertaken using the AutoDock version 4.2 in order to gain insight
into the binding mode of compound c6. The binding mode of compound c6 within the
binding site of the TyrRS complex structure (1jij.pdb) [16] is shown in Fig. 1 and Fig. 2.
Fig. 1 Binding mode of compound c6 with TyrRS from S. aureus. For clarity, only
interacting residues were labeled. Hydrogen bonding interactions are shown in dash. This
figure was made using PyMol.
25

Fig. 2 Binding mode of compound c6 with TyrRS. The enzyme is shown as surface; while c6
docked structures are shown as sticks. This figure was made using PyMol.
In the binding model, adenosine moiety of c6 is in the vicinity of the entrance cavity,
which formed hydrogen-bonds with surrounding residues such as Gly193, Pro222 and
Val224. Oxygen atom of ribose-ring moiety is involved in hydrogen bond formation with
hydrogen of the NH2 group of Gly193 (distance = 2.399 Å), while nitrogen atom in adenine
interacts with hydrogen of the NH2 group of Gly193 at distances of 2.774 Å. The NH2 of the
adenine strongly contributes to complex stabilization by forming three hydrogen bonds with
hydrogen atoms offered by the NH2 group of Val224 (distance = 3.111 Å) and oxygen atom
…
of Pro222 residue, having H O bond lengths of 2.342 Å and 2.491 Å. Additionally, the
hydroxy group of the ribose ring makes a strong interaction with the backbone amino groups
of Gly38. Moreover, an H-bond (1.942 Å) formed between saturated oxygen and hydrogen of
NH2 group of Gly38 further stabilizes the complex. The part of benzene ring is located at the
bottom of the binding site. The fluorine atom of benzene ring interacted with Ala39 and
Tyr36 at a distance of 3.248 Å and 2.312 Å. The number of hydrogen bonds formed has the
most crucial role in complex stabilization, the nine hydrogen bonds assure stable complex
formation and explain the high inhibitory activity of this compound. Therefore, changing the
26

position of fluorine atom or replacing it with other substituent group significantly reduced the
enzyme inhibitory activity.
4. Conclusions
In this work, we synthesized a novel class of adenosine analogues, which have
distinctive structural difference from other antimicrobial agents. These novel compounds are
modified from the tyrosyl-adenylate intermediate. Their biological activities were tested
against TyrRS from S. aureus and microorganism. Summarizing the results of tables 1-2, we
can conclude that compound c6 have much stronger antibacterial potency against P.
aeruginosa ATCC 9027 than ciprofloxacin and good inhibitory activity with IC50 of 0.8±0.07
μM against TyrRS. Additionally, we also performed docking of c6 into S. aureus TyrRS
binding pocket. The docking model indicated that introduction of a fluorine atom at the
3
′-position of benzene ring of the phenylacetyl moiety significantly increased affinities to the
enzyme that may explain the excellent inhibitory activity of compound c6. Thus compound
c6 can be considered for developing into a potent antibacterial agent.
Acknowledgements
The work was financed by grants from National Natural Science Foundation (grant No.
21262013) of China, by a Project supported by Hunan Provincial Natural Science Foundation
of China (grant No. 2015JJ2116), by a research project of Jishou University (14JDY053) and
by the Specialty Plant Resources Development and Utilization of Wuling Mountain in
Innovation Base for the Graduate Student Training of Hunan Provincial (2014KFXM04).
27

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