A class of novel conjugates of substituted purine and Gly-AA-OBzl: Synthesis and evaluation of orally analgesic activity

Article abstract of DOI:10.1016/j.bmcl.2009.05.077

Aimed at the chemotherapy of chronic pain two kinds of analgesic pharmacophores, substituted purine and Gly-AA-OBzl, were coupled via a five-step-reaction procedure and 19 novel conjugates N-[2-chloro-9- (tetrahydropyran-2-yl)-9H-purin-6-yl]-N-cyclopropylglycylamino acid benzylesters were provided. On mouse-tail flick model their in vivo analgesic activities were assayed. The results indicate that introducing Gly-OC₂H ₅ into the 6-position of the substituted purine leads to ambiguous increase of the analgesic activity, while introducing Gly-AA-OBzl into this position leads to significant increase of the analgesic activity.

Full text of DOI:10.1016/j.bmcl.2009.05.077

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6157–6160
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A class of novel conjugates of substituted purine and Gly-AA-OBzl: Synthesis
and evaluation of orally analgesic activity
b
b
c
c
c
a,b,
Guifeng Kang a, Ming Zhao b, , Xiaoyi Zhang , Li Peng , Chunbo Li , Wei Mao , Weidong Ye , Shiqi Peng
*
*
a College of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
b College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
c Zhejiang Medicine Co. Ltd, East Huancheng Rd., Xinchang, Zhejiang 312500, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 February 2009
Revised 3 May 2009
Accepted 18 May 2009
Available online 24 May 2009
Aimed at the chemotherapy of chronic pain two kinds of analgesic pharmacophores, substituted purine
and Gly-AA-OBzl, were coupled via a five-step-reaction procedure and 19 novel conjugates N-[2-chloro-
9-(tetrahydropyran-2-yl)-9H-purin-6-yl]-N-cyclopropylglycylamino acid benzylesters were provided. On
mouse-tail flick model their in vivo analgesic activities were assayed. The results indicate that introduc-
ing Gly-OC2H5 into the 6-position of the substituted purine leads to ambiguous increase of the analgesic
activity, while introducing Gly-AA-OBzl into this position leads to significant increase of the analgesic
activity.
Keywords:
Substituted purine
Gly-AA-OBzl
Conjugate
Ó 2010 Published by Elsevier Ltd.
Analgesia
3D QSAR
Clinically, chronic pain from various etiologies, such as inflam-
mation and neural destruction, is generally resistant to the treat-
ments of simple analgesics or traditional agents. Neuropathic
pain is accompanied by hypersensitivity to mechanical or thermal
stimuli.1 While inflammatory pain is accompanied by various pain-
ful responses of injury of peripheral tissue and/or inflammation
produced by trauma, infection, surgery, burns, or diseases with
an inflammatory component.2,3 Chronic pain, due to relative lack
of response to current analgesics, represents an unmet medical
need. In the development of analgesics for treating chronic pain
two receptor families, the adenosine receptors (ARs) and GlyRs,
have been concerned.
ARs belong to the superfamily of G-protein-coupled receptors.
Among four sub- classes (A1, A2A, A2B, and A3) of ARs that have been
identified to date A1 subtype (A1AR) is the best-characterized
member, and has been clearly identified to produce antinocicep-
tion in the spinal cord by using selective agonists and antago-
nists.4–7 With A1AR as the target numerous adenosine derivatives
were reported as selective agonists,8–13 based on which purine ring
was identified as a pharmacophre.8,14,15
In the processing of motor and sensory signals, neuronal develop-
ment, inflammatory pain sensitization, and in inherited neuro-log-
ical disorders such as hyperekplexia GlyRs play predominant
roles.16,17 With GlyRs as the target a lot of glycine derivatives were
reported as selective agonists,18,19 based on which glycine ester
was identified as a pharmacophre.20,21
In this context the present paper reported the synthesis and
in vivo analgesic evaluation of 19 novel conjugates consisted of the
mentioned two pharmacophores, substituted purine and Gly-AA-
OBzl. Using a five-step-reaction procedure and the corresponding
reaction conditions (Scheme 1) N-[2-chloro-9-(tetrahydropyran-2-
yl)-9H-purin-6-yl]-N-cyclopropylglycylamino acid benzylesters
(6a–s) were prepared with 3, 4 and 5 as the intermediates. The yields
of 3, 4, 5 and 6a–s were 78%, 97%, 99% and 30–93%, respectively. The
synthetic and chemical physical data of all compounds are given in
the file of Supplementary data. The data imply that using this five-
step-reaction procedure 6a–s can be smoothly obtained.
On mouse model the in vivo pain threshold was assayed. The
mice were orally administered 0.2 ml of CMC-Na (0.3%, vehicle
control), 25
pounds) and 25
l
mol/kg of 3–5 in 0.2 ml of CMC-Na (reference com-
mol/kg of 6a–s in 0.2 ml of CMC-Na (treating
GlyRs act as pentameric anion channels belonging to the ‘cys-
teine-loop’ superfamily of ionotropic neurotransmitter receptors.
l
groups). Thirty min later the mice received a180-min tail flick tests
at 30-min intervals. The value of the basic pain threshold of each
mouse was measured for three times. Analgesic potency was indi-
cated by the pain threshold variation and calculated according to
PTV = AAPT Ä BPT, wherein PTV is the pain threshold variation,
BPT is the basic pain threshold and AAPT is the difference of pain
* Corresponding authors. Tel./fax: +86 10 83911535 (M.Z.); tel./fax: +86 10 8391
1528 (S.P.).
E-mail addresses: mzhao@mail.bjum.edu.cn (M. Zhao), sqpeng@mail.bjum.
edu.cn (S. Peng).
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.05.077

6158
G. Kang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6157–6160
and constituted one sample. The data are listed in Tables 1 and
2, and the statistical analysis is carried out using one way ANOVA
test with p < 0.05 as significant cut-off.
Cl
N
Cl
N
NH
N
N
N
N
N
N
N
N
i
ii
N
H
Cl
Cl
Cl
N
The data in Table 1 explore that compounds 3–5 are modest
analgesics. The duration of the analgesic action for 3 and 5 is
120 min, and for 4 is 150 min. The statistical analyses of the data
of 3 and 4 indicate that introducing N-cyclopropylglycine ethylest-
er into 6-position of substituted purine 3 does not significantly
change the analgesic activity. On the other hand however when
the substituted purine 4 was converted to 5 the analgesic activity
was significantly decreased. This comparison implies the impor-
tance of an ester group for the activity. The data in Table 2 explore
that 6a–s are good analgesics and the activity order is
6a,b,e,g,q > 6c,d,f,l,m,n > 6h,I,j,k,o,p,r,s. The duration of the anal-
gesic action of 6a,d,g,h,j,m,n,p,r is 120 min, while 6e,f,i,k,l,o,q,s
is 180 min. The statistical analyses of the data of 6a–s indicate that
replacing ethoxy group of 4 with amino acid benzylester results in
significant increase of analgesic activity.
O
O
1
2
3
iii
O
O
O
N
AA OBzl
v
N
OH
O
N
OC H
2
5
N
N
N
N
N
N
iv
N
N
N
Cl
N
6a-s
Cl
N
Cl
N
5
4
O
O
Scheme 1. Synthetic route of amino acid substituted purin derivatives. (i) Pyridine
tosylate and 2,3-dihydropyran; (ii) triethylamine and cyclopropylamine; (iii) NaH,
BrCH2CO2C2H5; (iv) aqueous solution of NaOH (3 M) and KHSO4 (5%); (v) AA-OBz l/
DCC/NMM. In 6a AA = Ala; 6b AA = Arg; 6c AA = Asn; 6d AA = Asp(OBzl); 6e
AA = Gln; 6f AA = Glu(OBzl); 6g AA = Gly; 6h AA = His; 6i AA = Ile; 6j AA = Leu; 6k
AA = Lys(Boc); 6l AA = Met; 6m AA = Phe; 6n AA = Pro; 6o AA = Ser; 6p AA = Thr; 6q
AA = Trp; 6r, AA = Tyr; 6s AA = Val.
Recently, the interaction of GlyRs with substrate was deduced
from the interaction of lactose permease or glycerol-3-phosphate
transporter with the substrate, and thought to occur in a hydro-
philic cavity that extended into the center of the lipid bilayer, as
well as the interaction functioned via salt bridges and hydrogen
bonds.22 This knowledge not only explains the importance of the
threshold after administration minus the basic pain threshold. All
values of pain threshold variation for each mouse were averaged
Table 1
Effect of 3, 4, 5 on the pain threshold of the treated mice
Compda
Pain threshold variation (x SD %)
ꢀ
30 min
60 min
90 min
120 min
150 min
180 min
Vehicle
À1.0 2.80
10.27 4.80c
9.92 4.60c
5.21 3.64b
À6.5 1.79
0.20 3.75
0.69 1.38
3.98 4.03
5.72 3.96
11.85 4.58e
3.12 3.22
À5.10 2.24
5.92 2.61
5.33 4.09
3.41 3.64
3
4
5
20.63 4.38c
22.64 5.89c
15.24 4.22b
11.93 3.54b
13.04 3.01d
8.22 3.67b
12.64 4.56c
14.91 4.50d
7.68 3.38b
a
The statistical analyses are carried out for the data of same time point, n = 10, vehicle = 0.3% CMC-Na; dose = 25
Compare to vehicle p < 0.01.
Compare to vehicle p < 0.01, and to 5 p < 0.05.
Compare to vehicle and 5 p < 0.01.
Compare to vehicle, 4 and 5 p < 0.01.
lmol/kg.
b
c
d
e
Table 2
Effect of 6a–s on the pain threshold of the treated mice
Compda
Pain threshold variation (x SD %)
ꢀ
30 min
60 min
90 min
120 min
150 min
180 min
Vehicle
6a
6b
6c
6d
6e
À1.0 2.80
À6.5 1.79
0.20 3.75
0.69 1.38
3.98 4.03
-5.10 2.24
5.96 4.72b
7.97 7.10b
15.45 8.05b
0.03 5.24b
7.71 4.02b
10.93 6.73b
À0.06 5.63b
4.38 7.30b
9.15 8.62b
0.04 2.64b
10.06 11.99b
12.89 7.50b
1.79 6.09b
6.63 6.97b
12.85 8.20b
2.34 3.76b
5.15 4.17b
12.02 3.35b
5.65 7.88b
55.91 19.39b
34.27 12.82b
34.53 12.90b
31.81 10.99b
46.96 17.23b
32.61 10.74b
55.72 20.52b
24.98 9.29b
25.56 10.06b
26.20 9.25b
18.33 8.89c
30.04 9.31b
39.20 13.70b
32.25 10.69b
27.77 8.14b
23.61 8.76b
43.53 17.13b
20.53 8.58b
21.83 9.18b
53.00 19.58b
52.35 18.90b
30.54 9.63c
38.15 13.91b
39.40 13.14b
34.74 13.51c
40.44 17.57b
36.43 13.94b
27.35 10.27d
34.19 9.65b
32.66 10.11c
40.97 15.64b
45.54 17.78b
36.73 13.68b
46.71 17.84b
28.36 10.48d
51.90 12.39b
35.82 10.98b
34.85 11.31b
32.06 12.53b
61.58 21.80b
34.95 14.14b
25.07 8.83b
37.82 9.61b
25.20 9.78b
25.94 9.30b
14.45 7.15d
22.57 9.30b
25.30 8.56b
6.01 3.85
38.16 16.12b
29.15 10.84b
7.82 8.16d
19.60 9.86c
21.71 8.02b
0.06 5.94
22.34 8.30c
20.71 7.88c
17.23 9.21d
10.16 6.67d
20.42 8.34d
12.79 8.08d
28.39 10.32b
28.66 10.61b
26.29 9.89b
17.49 9.85d
29.20 10.13b
10.91 6.25d
22.58 9.81c
17.44 5.49d
25.79 8.54b
10.72 3.72d
14.14 9.23d
7.26 5.38
5.31 4.99
16.43 9.73d
6.47 3.57
17.20 9.10d
17.47 9.79d
3.49 9.34
7.85 4.05
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
28.22 8.86b
32.85 10.22b
42.70 14.41b
37.73 13.75b
23.31 9.41b
32.17 10.37b
23.38 12.52c
38.93 9.63b
36.45 11.45b
30.14 10.86b
18.32 9.51d
6.53 5.50
11.45 5.56d
8.71 6.65
21.08 9.73c
6s
a
The statistical analyses are carried out for the data of same time point, n = 10, vehicle = 0.3% CMC-Na; dose = 25
l
mol/kg.
b
Compare to vehicle and 4 p < 0.01.
Compare to vehicle p < 0.01, and to 4 p < 0.05.
c
d
Compare to vehicle p < 0.01.

G. Kang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6157–6160
6159
hydrophobicity and the hydrogen bond for the interaction of GlyRs
with the substrate but also helps us to understand the distinct
activity. As depicted in Figure 1 the hydrophobicity of the com-
pounds is in the order of Bzl of 6 > C2H5 of 4 > H of 5, and the nucle-
ophilicity of the compounds is in the order of two carbonyls of
6 > ester carbonyl of 4 > acid carbonyl of 5. Therefore the interac-
tions of GlyRs with the compounds are in the order of 6 > 4 > 5.
To know the effect of the dose on the activity the most active
compounds (6a,b,g) were observed for dose-dependently analgesic
response. The mice were orally administered 30.0, 3.0 and
metabolites, 1, 2, 3, Gly and Gly-Ala-OBzl were used as reference
compound. The mice were orally administered 30.0 mol/kg of
l
them and the data are listed in Table 4. It is clear that the pain
threshold of the mice receiving reference compound and their mix-
ture is significantly lower than that of the mice receiving 6a. Thus,
we hypothesize 6a–s exert their in vivo analgesic activities not
through metabolism conversion.
In conclusion, though no any datum of synergistic or additive
analgesic action of AR and GlyR agonists is available in the litera-
ture, the observation that N-[2-chloro-9-(tetra-hydropyran-2-yl)-
9H-purin-6-yl]-N-cyclopropylglycylamino acid benzylesters exhi-
bit higher activities than substituted purine and glycine explores
that the conjugation of AR agonist with GlyR agonist may result
in synergistic or additive analgesic action. In the literature1,3 aden-
osine receptor agonist 2-amino-3-(4-chlorobenzoyl)-5,6,7,8-tetra-
hydro-benzothiophene (T62)1 and glycine receptor agonist H-
0.3 lmol/kg of 6a,b,g and the data are listed in Table 3. It is clear
that with the increase of the dose the pain threshold of the mice
significantly increases. This dose-based difference may last
120 min.
To gain insight into the effect of metabolism on analgesic activ-
ity 6a was selected as model compound at random and its possible
Proton acceptor
O
Proton acceptor
O
Proton acceptors
O
O
N
N
OH
N
O C H
2 5
N
CH
R
C
O Bzl
H
N
N
N
N
N
N
N
N
N
hydrophilic group
hydrophilic group
Cl
N
Cl
N
Cl
N
6
5
4
O
O
O
Figure 1. Comparison of hydrophobicity and the ability of forming hydrogen bonds of 4, 5 and 6 in their interactions with receptor.
Table 3
Dose-dependent effect of 6a,b,g on the pain threshold of the treated mice
Compda
Pain threshold variation (x SD %)
ꢀ
30 min
60 min
90 min
120 min
150 min
180 min
Vehicle
6a
0.13 0.14
0.15 0.16
0.17 0.14
0.18 0.15
0.16 0.13
11.20 4.35
12.00 4.50
10.00 4.45
24.69 9.00
15.00 6.06
10.03 5.01
12.33 6.15
10.04 5.16
9.45 4.55
0.15 0.12
11.00 5.00
10.00 4.98
8.09 4.70
14.01 6.17
10.34 5.10
7.56 3.19
10.16 5.02
9.20 4.33
9.00 4.20
H
M
L
H
M
L
H
M
L
60.11 12.19b
43.25 11.07c
25.60 9.04
39.41 10.24b
20.55 8.32d
12.15 5.28
60.84 12.50b
45.27 11.07c
18.00 7.70
58.18 11.52b
39.81 10.11c
21.56 8.11
57.51 11.73b
41.07 10.29c
25.11 9.33
45.52 11.60b
29.07 9.62c
16.24 6.59
37.16 10.03b
25.34 9.17c
11.43 6.10
66.71 12.81b
50.14 11.08c
32.88 9.20
31.04 9.22b
20.25 6.20d
11.20 5.22
30.44 9.12b
18.03 8.66d
11.03 6.62
43.30 11.21b
29.09 9.18c
12.14 6.22
22.31 8.15b
14.53 5.10d
10.02 4.21
6b
6g
a
The statistical analyses are carried out for the data of same time point, n = 10, vehicle = 0.3% CMC-Na; dose of 6a,b,g: H = 30 lmol/kg, M = 3 lmol/kg and L = 0.3 lmol/kg.
b
Compare to 3 lmol/kg p < 0.01.
c
Compare to 0.3
lmol/kg p < 0.01.
d
Compare to 0.3
lmol/kg p < 0.05.
Table 4
Effect of building block of 6a on the pain threshold of the treated mice
Compda
Pain threshold variation (x SD%)
ꢀ
30 min
60 min
90 min
120 min
150 min
180 min
Vehicle
1
2
3
Gly
Gly-Ala-OBzl
3 + Gly
3 + Gly-Ala-OBzl
6a
0.13 0.14
0.15 0.16
0.17 0.14
0.18 0.15
0.16 0.13
0.15 0.12
12.22 3.89c
14.30 4.37c
15.20 4.42c
10.13 2.95c
12.25 2.99c
15.39 4.51c
15.53 4.48c
60.11 12.19b
21.62 4.86c
23.99 4.81c
25.61 4.77c
11.21 3.72c
13.32 3.69c
26.08 4.85c
25.30 4.69c
58.18 11.52b
14.02 3.61c
15.01 3.81c
16.96 3.99c
11.31 3.88c
13.45 3.57c
18.11 4.26c
17.05 3.89c
37.16 10.03b
12.97 3.46c
14.13 4.00c
15.66 4.11c
12.03 3.39c
13.57 3.42c
16.04 4.13c
15.49 4.23c
30.44 9.12b
8.77 3.21c
9.89 3.50c
10.78 3.88c
11.54 3.37c
13.00 3.42c
11.12 3.79c
13.29 3.90c
11.20 3.35c
7.99 2.57c
8.03 2.71c
9.95 2.98c
10.96 2.62c
12.99 2.71c
10.21 2.96c
11.01 2.89c
11.00 3.00c
a
The statistical analyses are carried out for the data of same time point, n = 10, vehicle = 0.3% CMC-Na; dose of 1, 2, 3, Gly, Gly-Ala-OBzl and 6a: 30 lmol/kg.
Compare to the others p < 0.01.
Compare to vehicle p < 0.01.
b
c

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G. Kang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6157–6160
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