Novel Pyrazolo[4,3- d]pyrimidine as Potent and Orally Active Inducible Nitric Oxide Synthase (iNOS) Dimerization Inhibitor with Efficacy in Rheumatoid Arthritis Mouse Model

Article abstract of DOI:10.1021/acs.jmedchem.9b00039

In order to discover novel anti-inflammatory agents for treatment of arthritis and based on preliminary structure-activity relationships, four series (A-D) of total 90 new pyrazolo[4,3-d]pyrimidine compounds were designed and synthesized. All the compounds have been tested for their anti-inflammatory activities by inhibiting of LPS-induced NO production. A clear structure-activity relationship has been concluded step by step, and finally 3,4,5-trimethoxystyryl-1H-pyrazolo[4,3-d]pyrimidine was found to be the most active scaffold. Among them, compound D27 was discovered as the most potent anti-inflammatory agent (IC₅₀ = 3.17 μM) with low toxicity and strong inhibitory of NO release (IR = 90.4% at 10 μM). This compound also showed potent inhibition of iNOS with IC₅₀ value of 1.12 μM. Preliminary mechanism studies indicated that it could interfere with the stability and formation of active dimeric iNOS. The anti-inflammatory effect of this compound was determined by adjuvant-induced arthritis in rat model. We believe these findings would further support the study of rational design of more efficient iNOS inhibitors in the future.

Full text of DOI:10.1021/acs.jmedchem.9b00039

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC
Article
Novel Pyrazolo[4,3-d]pyrimidine as Potent and Orally
Active Inducible Nitric Oxide Synthase (iNOS) Dimerization
Inhibitor with Efficacy in Rheumatoid Arthritis Mouse Model
Jing Bo Shi, Liu Zeng Chen, Bao Shi Wang, Xin Huang, Ming
Ming Jiao, Ming Ming Liu, Wen-Jian Tang, and Xin-Hua Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00039 • Publication Date (Web): 29 Mar 2019
Downloaded from http://pubs.acs.org on March 29, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Novel Pyrazolo[4,3-d]pyrimidine as Potent and Orally Active Inducible Nitric
Oxide Synthase (iNOS) Dimerization Inhibitor with Efficacy in Rheumatoid
Arthritis Mouse Model
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
§
,
§,
Jing Bo Shi, † Liu Zeng Chen, † Bao Shi Wang,† Xin Huang,† Ming Ming Jiao,†
,
Ming Ming Liu,† Wen Jian Tang† and Xin Hua Liu* †
† School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China
ABSTRACT: In order to discover novel anti-inflammatory agents for treatment of
arthritis and based on preliminary structure-activity relationships, four series (A~D)
of total 90 new pyrazolo[4,3-d]pyrimidine compounds were designed and synthesized.
All the compounds have been tested for their anti-inflammatory activities by
inhibiting of LPS-induced NO production. A clear structure-activity relationship has
been
concluded
step
by
step,
and
finally
3
,4,5-trimethoxystyryl-1H-pyrazolo[4,3-d]pyrimidine was found to be the most active
scaffold. Among them, compound D27 was discovered as the most potent
anti-inflammatory agent (IC =3.17 μM) with low toxicity and strong inhibitory of
5
0
NO release (IR=90.4% at 10 μM). This compound also showed potent inhibition of
iNOS with IC value of 1.12 μM. Preliminary mechanism studies indicated that it
5
0
could interfere the stability and formation of active dimeric iNOS. The
anti-inflammatory effect of this compound was determined by adjuvant-induced
arthritis in rat model. We believe these findings would further support study of
rational design of more efficient iNOS inhibitors in the future.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
INTRODUCTION
a
Nitric oxide synthases (NOS ) are dimeric enzymes that catalyze the formation of
1
-6
nitric oxide (NO) and citrulline from arginine and molecular oxygen. Inflammation
can be regulated by the recruitment of macrophages and production of
proinflammatory cytokines. Macrophages produce proinflammatory cytokines
including nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL-6) and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
, 8
interleukin-1β (IL-1β).
NO, being an important signaling molecule, is a unique
gasotransmitter in the process of vasodilation, non-specific host defense and chronic
or acute inflammation, which is biogenetically produced by NOS. The approach of
inhibiting NOS is to either disrupt the function of active NOS dimer or prevent NOS
dimerization. Given that inducible NOS (iNOS) is controlled at the transcriptional
9, 10
level, NOS dimerization inhibitors may be expected to work best with iNOS. Thus,
iNOS should be a potential target for inflammatory diseases.
In the past decades, several molecules capable of disrupting NOS dimer’s stability
or preventing NOS dimerization have been developed including clotrimazole,
1
, 9, 11-16
miconazole, PPA250, KLYP961, BBS-1 and BBS-4.
However, most of them
are imidazole-based molecules, which would also inhibit P450 resulting in drug-drug
interaction. For example, inhibition of endothelial NOS (eNOS) can always cause
serious side effects. Thus, selectively inhibiting NOS has always been a challenge as
However, except KLYP961, a non-imidazole-based inhibitor which can prevent
1
,15-18
dimerization of iNOS and neuronal NOS (nNOS)
, no other potent
non-imidazole-based NOS inhibitor with low toxicity profile has been reported until
2
ACS Paragon Plus Environment

Page 3 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
now. Therefore, development of potent, low-toxicity non-imidazole iNOS inhibitors is
in urgent need.
The pyrazolo[4,3-d]pyrimidine skeleton, an isostere of adenine, is an important
drug-like scaffold and its derivatives have been demonstrated with a myriad of
therapeutic indications including anti-inflammation, anti-cancer and anti-infectious
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9-23
effects.
Also, pyrazolo-pyrimidine derivatives have been reported for treatment of
a variety of inflammatory diseases. Based on above findings, we hypothesized that
pyrazolo-pyrimidine derivatives could be potential NOS inhibitors.
In our previous study, many pyrazole-pyrimidine derivatives have been
25
synthesized
and in-house compounds library screening showed that most
compounds had no toxicity against six tumor cell lines. Further activity evaluation
showed that they were even less toxic to RAW 264.7 cells. We then preliminarily
evaluated their anti-inflammatory activities (compounds of Series A, Figure 1) by
evaluating their inhibitory effects against LPS-stimulated NO, IL-6 and TNF-α
productions in RAW 264.7 cells.
R3
O
N
N
N
HN
O
O
Continue
N
N
R2
N
N
O
A
Figure 1. Our previous work
The results revealed that some compounds exhibited moderate anti-inflammatory
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
effects through decreasing the levels of NO, IL-6 and TNF-α. Interestingly, it was
found that most compounds showed stronger inhibitory effects on NO production than
IL-6 or TNF-α production (Table 1). Thus, in order to find potent anti-inflammatory
agents and focusing on improving their inhibitory effect of NO production,
compounds of series B~D were designed and synthesized based on SARs step by step
in this study (Figure 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R3
N
R4
N
N
N
N
N
N
N
O
O
O
Series B
Series A
R6
N
R5
N
N
N
N
O
N
N
O
N
O
O
Series C
Series D
Figure 2. Four series compounds (A~D) were designed step by step
RESULTS AND DISCUSSION
2
5,26
Design. 3,4,5-trimethoxyphenyl moiety is crucial to activity in drug design,
and its derivatives have a wide range of therapeutic indications such as
2
2,27,28
29-31
anti-inflammatory
and
antinarcotic
effects.
Therefore,
some
3
,4,5-trimethoxyphenyl pyrazolo[4,3-d]pyrimidines (Series A) were designed and
synthesized at first.
4
ACS Paragon Plus Environment

Page 5 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
To further improve the activity of the compounds, stilbene scaffold was
introduced into the structure, which has been proved to be an active moiety as a basic
element of natural product. Representative chemicals with stilbene moiety including
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
resveratrol (I), pterostilbene (II) and combretastatin A-4 (III) are of significant
32,33
interest for drug research
(Figure 3). Inspired by this, we then introduced styryl
group at C-5 of pyrazolo[4,3-d]pyrimidine scaffold and compounds of Series B were
designed.
OH
H CO
OH
3
HO
O
H CO
3
OCH3
III
OH
OH
I
O
OCH3
II
Figure 3. Structures of representative stilbene
In view of the preliminary SARs results, influence of alternative substituents at
C-5 of pyrazolo[4,3-d]pyrimidine was found. Based on this finding, the styryl group
was changed to 2-(furan-2-yl)vinyl group since it has been successfully used as a
3
4,35
good styryl bioisostere (Series C).
Considering the contribution of
3
,4,5-trimethoxyphenyl moiety to the activity and the re-summarized SAR, we at last
introduced trimethoxystyryl group at C-5 of pyrazolo[4,3-d]pyrimidine scaffold
(Series D).
Chemistry.
According
to
our
previous
work,
was
4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide
(compound
1)
synthesized and used as the lead material. All the following compounds (Series A~D)
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
6, 37
were synthesized according to the protocol outlined in Scheme 1.
O
O
O
N
HN
H N
N
2
N
N
H N
2
a
N
c
O
b
N
R1
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H N
H
2
R1
1
2
3
2
A~D
3A~D
O
O
O
R3
Cl
N
O
OH
f
N
HN
N
HN
HN
N
e
d
N
N
N
N
R1
N
N
N
N
N
R1
R1
R
1
N
N
N
4
6
7
8
4A~D
A13, B18, D34
A14 D35
A15-17, D36-38
g
,
R2
O
O
N
N
A R1
=
B: R1=
N
:
R1
N
O
O
O
5
O
A1-12, B1-17,
C1-17, D1-33
1
D: R1=
C: R =
O
a
Scheme 1. Synthesis of Compounds Series A, B, C, D
a
Reaction conditions and reagents: (a): (1) substituted carboxylic acid, (COCl)2,
o
o
CH Cl , DMF (Cat), 25 C, 3 h; (2) Compound 1, CH Cl , TEA, 25 C, 12 h; (b):
2
2
2
2
NaOEt, EtOH, reflux, 12 h; (c): POCl , reflux, 3-8 h; (d): methyl 4-aminobenzoate,
3
IPA, reflux; (e): MeOH, 2N NaOH, rt.; (f): amine derivatives, EDCI, HOBt, TEA, rt.;
(g): amine derivatives, IPA, reflux.
At first, 3,4,5-trimethoxyphenyl-pyrazolo[4,3-d]pyrimidine derivatives (Series A)
were synthesized. Compound 2A (Scheme 1) was obtained by two steps: the
corresponding acyl chloride was prepared with oxalyl chloride at room temperature
and formed amide 2A. Compound 3A was afforded through intramolecular
6
ACS Paragon Plus Environment

Page 7 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
cyclization reaction. Synthon 4A was a key intermediate for title compounds, which
38
was achieved by chlorination reaction in the presence of POCl3. Nucleophilic
substitution reactions of compound 4A with various amines gave title compounds
A1-13 (Scheme 1). Based on compound A13, compounds A14-17 were prepared
through simple derivation. Compounds of Series B-D were synthesized with similar
route.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H NMR spectrum of alkene (CH=CH, compound D3) exhibited chemical shift at
7
.15 ppm in CDCl with coupling constant of 15.7 Hz. These results demonstrated
3
that the configuration of compound D3 is E geometrical isomer and the result was
further confirmed by X-ray. Crystal data of compound A2 was shown as following:
o
Colorless crystals, yield=55%; mp: 202-203 C; C H N O , Monoclinic, space
25
29
5
3
group P2 /c; a=7.2308(3), b=20.7026(8), c=15.7198(6) (Å); α=90, β=96.463(4),
1
3
3
γ=90(°), V=2338.24(16) nm T=293(2) K, Z=4, Dc=1.271 g/cm , F(000)=952.0,
,
Reflections collected/unique =8504/4341, Data / restraints / parameters =4341/0/304,
2
2
Goodness of fit on F =0.978, Fine, R =0.0572, wR(F )= 0.1281 Crystal data of
1
.
o
compound D3: Colorless crystals, yield=56%; mp: 109-110 C; C H N O ,
2
6
29
5
3
Monoclinic, space group P2 /c; a=10.1561(4), b=19.6033(10), c=14.3506(6) (Å);
1
3
3
α=90, β=103.929(5), γ=90(°), V=2773.1(2) nm , T=293(2) K, Z=4, Dc=1.211 g/cm ,
F(000)=1080.0, Reflections collected/unique=10620/5436, Data/restraints/ parameters
2
2
=
5436/36/367, Goodness of fit on F =1.039, Fine, R =0.0725, wR(F ) = 0.1373.
1
Their structures were shown in Figure 4.
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound A2
Compound D3
Figure 4. ORTEP drawing of compounds A2 and D3
Inhibition of NO Production in RAW 264.7 Cells. Pre-research results showed
that most compounds (Series A) had stronger inhibitory effects on NO production
than IL-6 or TNF-α production (Table 1). Therefore, based on the inhibitory effect of
NO production, anti-inflammatory activity of these compounds was evaluated in this
study (Table 1).
8
ACS Paragon Plus Environment

Page 9 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 1. Inhibitory Effects of Compounds A1-17 Against NO Production in RAW
a
2
64.7 Cells
R2
N
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
Series A
b
NO
NO
2
2
Compd
R
inhibition
Compd
R
inhibition
(%) ± SD
(
%) ± SD
F
F
N
O
A1
A2
A3
NH
-
A10
A11
A12
5.2±11.3
3.1±9.6
F
N
-
H C
NH
3
NH
N
22.2±6.2
22.2±3.2
7.6±10.3
O
O
NH
Cl
NH
NH
A4
A5
A6
A7
A13
A14
A15
A16
A17
17.3±3.2
O
NH
HO
20.2±3.2
10.2±5.3
21.6±3.3
44.9±3.2
NH
O
O
NH
-
-
O
N
Me
H
NH
N
O
N
Me
Me
N
A8
A9
N
21.2±2.4
51.9±1.3
27.6±4.4
59.2±3.7
NH
Indomethacin
“-” : No anti-inflammatory activity.
a
The cells were treated with compounds for 1 h, and then stimulated with LPS (1
µg/mL) for 24 h. The level of NO in the culture medium was measured by nitrite and
nitrate assay. All date shown were at least three independent experiments.
b
The inhibition of NO production in LPS-induced RAW 264.7 was tested at 10 μM.
c
Indomethacin as a positive control.
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
It could be observed that most of 3,4,5-trimethoxyphenyl-pyrazolo[4,3-d]
pyrimidine derivatives (Series A) showed weak inhibitory activity against
LPS-induced NO production at 10 μM (inhibitory rates (IR) were less than 22.2%).
Among them, compounds A1, A2, A15 and A16 had no activities. In contrast,
compound A9, an analog with isopropylamine at C-7 resulted in moderate inhibitory
activity (IR=51.9%) compared to that of positive control Indomethacin. Based on
above, it is not difficult to find that inhibitory activity of series A is closely related to
the chemical structure. Because stilbene scaffold is a privileged structure, styryl was
then introduced to C-5 of pyrazolo[4,3-d]pyrimidine scaffold (Series B). Their
activities against NO production were shown in Table 2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
ACS Paragon Plus Environment

Page 11 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Table 2. Inhibitory Effects of Compounds B1-18 Against NO Production
R2
N
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Series B
NO
NO
2
2
Compd
R
inhibition
(%) ± SD
Compd
R
inhibition
(%) ± SD
M
e
Me
F
F
B1
B2
B3
B4
NH
14.6±6.2
15.3±3.3
4.9±6.3
B10
B11
B12
B13
N
27.1±5.9
53.9±6.3
33.3±5.3
10.2±6.3
F
NH
NH
H C
NH
3
NH
NH
N
N
Cl
24.2±3.5
NH
Cl
O
N
B5
10.7±3.3
B14
10.2±3.3
Cl
N
B6
B7
14.3±7.6
48.6±3.3
B15
B16
6.3±8.3
7.7±5.2
NH
NH
N
F
O
N
NH
B8
B9
43.9±3.7
B17
B18
42.4±3.3
42.4±3.6
NH
NH
O
O
NH
O
48.0±5.3
59.2±3.7
Indomethacin
a
Described as Table 1.
In these styryl-pyrazolo[4,3-d]pyrimidine derivatives (Series B), compounds
B1-3, with substituted or unsubstituted N-phenyl group at C-7, showed very weak
potencies which were very similar to compounds A1-3. Compound B4 (IR=24.2%),
with N-Benzyl group at C-7 negligibly showed any significant effects. After
replacement of the N-phenyl with N-phenethyl moiety, inhibition activities were
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
found to be greatly enhanced (compounds B5-9) with IR increasing from 10.7% to
4
8.6%. Although only compound B11 (IR=53.9%) showed moderate inhibitory
activity, one-third of the series B compounds contained inhibitory activities more than
40%, indicating the merit of derivation strategy. Provided with the results of these
preliminary SARs, the influence of alternative substituents at C-5 of
pyrazolo[4,3-d]pyrimidine should be further revealed. Thus, compounds C1-17 were
designed and synthesized. Their activities were screened (Table 3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 3. Inhibitory Effects of Compounds C1-17 Against NO Production
R2
N
N
N
O
N
Series C
NO
NO
2
2
Compd
R
inhibition
Compd
R
inhibition
(%) ± SD
(
%) ± SD
O
F
F
C1
C2
NH
NH
8.9±7.6
C10
C11
NH
52.3 ±2.3
50.2±3.5
F
NH
8.9±3.5
F3C
H
3
C
N
NH
Cl
C3
Cl
56.4±2.5
C12
79.2±5.6
NH
Cl
C4
C5
C6
C7
C8
C9
54.8±5.2
40.6±4.1
50.7±3.3
43.8±3.7
35.6±3.3
38.4±3.3
C13
C14
70.8±3.7
36.4±2.2
73.0±3.3
30.6±6.3
37.7±3.3
59.2±3.7
Cl
NH
NH
NH
Br
F
NH
NH
F
C15
NH
Me
Me
O
C16
N
H CO
3
NH
NH
C17
OCH3
NH
Indomethacin
a
Described as Table 1.
1
2
ACS Paragon Plus Environment

Page 13 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
As described in Table 3, compounds C1-2, with substituted N-phenyl group at
C-7, had very weak potency. Interestingly, compounds C3 and B4, though both
having N-4-chlorobenzyl group at C-7, showed dramatic difference at inhibitory
activity (56.4% and 24.2%). To summarize, nearly half of the compounds (Series C)
exhibited moderate potencies, with IR ranging from 50.2% to 79.2%. Among the
N-phenethyl or N-alkyl substituted analogues C12-15, C16-17, most of compounds
exhibited moderate inhibitory activities. Compounds C10, C11 and C13, substituted
with phenethyl amine moiety at the C-7, could dramatically elevate inhibitory
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
activities (79.2% 70.8% and 73.0% respectively). These results indicated that
,
inhibitory activities of compounds C series were much higher than that of compounds
series A and B. Focusing on above SARs, in order to improve activity, we will carry
out further structural optimization. Compounds D1-39 were then synthesized.
Inhibitory effects against NO production were listed in Table 4.
a
Table 4. Inhibitory Effects of Compounds D1-39 Against NO Production
R2
N
N
N
O
N
O
Series D
O
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NO
NO
2
2
Compd
R
inhibition
(%) ± SD
Compd
R
inhibition
(%) ± SD
NH
F
F
D1
D2
NH
NH
38.2±4.2
25.8±3.7
48.1±3.3
25.3±6.2
33.0±3.2
54.4±3.3
63.2±1.3
82.6±3.7
37.8±2.3
31.0±6.3
38.5±1.3
16.4±3.3
67.2±1.3
90.0±1.3
53.9±2.3
69.7±3.6
D20
D21
D22
D23
D24
D25
D26
D27
D28
D29
D30
D31
D32
D33
D34
D35
F3C
54.6±3.3
80.0±3.3
46.9±2.4
86.1±4.2
85.0±3.7
15.0±6.3
82.1±3.3
90.4±1.2
75.0±3.3
28.3±3.3
81.0±3.2
45.3±3.2
15.5±3.7
16.3±4.3
10.3±6.4
15.3±6.9
F
Cl
NH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H C
3
Cl
NH
D3
NH
NH
Cl
NH
D4
F
Br
NH
D5
NH
NH
HO
D6
NH
Me
Me
O
NH
D7
N
F3C
F
NH
D8
NH
F3C
NH
NH
N
D9
Br
NH
N
D10
D11
D12
D13
D14
D15
D16
Br
NH
O
N
F3CO
NH
NH
HO
N
Cl
N
N
NH
Cl
NH
O
O
Br
F
NH
NH
NH
NH
O
HO
O
N
NH
O
73.1±2.4
70.9±3.6
35.9±4.3
D17
D36
H CO
3
O
NH
NH
D18
D19
D37
D38
10.6±6.3
15.4±5.6
NH
O
O
N
NH
62.1±2.5
59.2±3.7
N
NH
Indomethacin
a
Described as Table 1.
1
4
ACS Paragon Plus Environment

Page 15 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
In the series D, it could be observed that compounds D1-3, with substituted or
unsubstituted N-phenyl group at C-7, had moderate potency (IR ranging from 25.0%
to 48.1%), which were found to be slightly better compared to compounds A1-3, B1-3
and C1-2. This means that trimethoxystyryl group may contribute to the activity.
Based on this, we continued to synthesize compounds D4-12, with substituted or
unsubstituted N-phenyl group at C-7 and the results showed most of them exhibited
potent activities. Among them, compounds D6-8, were found to possessing
dramatically improved activity with inhibitory rates of 54.4%, 63.2% and 82.6%
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
respectively. Compounds D13-20, with substituted N-benzyl group at C-7 also
,
exhibited dramatically improved activity compared to compounds D1-12 and D21-25,
with IR ranging from 53.9% to 90.0%. These results indicated that introduction of
substituted N-benzyl group at C-7 was critical to activity. Particularly compounds
D26-28 showed dramatically improved activity with IR ranging from 75.0% to 90.4%,
compared to compounds A8-9. To summarize, among the compounds of series D,
eight compounds’ inhibition rates are more than 80%, and among them two
compounds’ IRs are more than 90%, surpassing that of commodity drug Indomethacin.
Therefore, after third rounds optimization, lead compound was initially discovered.
Inhibition Activity of iNOS. To explore the relationship between IC values of
5
0
NO and iNOS in the interest of further confirmation of the mechanism of
anti-inflammatory, selected compounds with high or no NO inhibitory activities were
chosen to evaluate their IC50s against iNOS. The results were summarized in Table 5.
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. IC Values of Selected Compounds Against iNOS
50
NO IC
cytotoxicity IC (μM)
50
5
a
0
c
Compd
iNOS IC50 (μM)
(μM)
without LPS
with LPS
b
d
A9
A11
10.78
7.26
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-
-
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
A14
15.82
-
10.23
-
B3
B11
12.20
22.11
9.26
6.04
3.17
13.72
45.57
8.46
5.31
4.25
2.60
1.12
5.23
24.89
B12
C3
C12
D27
D31
e
Indomethacin
“-” : No anti-inflammatory activity.
a
Inhibition(%)=[LPS (OD )-compounds(OD )]/[LPS(OD )-control (OD )]×100
5
40
570
540
540
b
First calculate the inhibition rates for different concentrations. The IC50s of various
compounds were calculated by regression analysis through SPSS.
c
Inhibition (%) = [LPS (AW , EW )–compounds (AW , EW )]/[LPS (AW ,
495
515
495
515
495
EW )- control (AW , EW )]×100.
5
15
495
515
d
First calculate the inhibition rates for different concentrations. The IC50s of various
compounds were calculated by regression analysis through SPSS.
e
Indomethacin as a positive control.
1
6
ACS Paragon Plus Environment

Page 17 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Ten compounds were exposed to investigate the inhibitory effects against NO
compared to iNOS. From the Table 5, it could be found that compounds A11 and B3
had no inhibitory activity against LPS-induced NO production at 10 μM. They also
didn’t show any activity against iNOS. In contrast, for compounds B12,C12 and D27,
there was a good correlation between IC50 of NO and the iNOS, indicating that
inhibition of the NO may result in inhibiting of iNOS. As anticipated, 5-substituted
styryl derivative (compound D27) exhibited the most potent inhibitory activity against
iNOS with the IC of 1.12 μM (Selectivity=22 times with Indomethacin), this was
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
consistent with the above SARs analysis. Thus, compound D27 was identified as the
title compound for further study.
Inhibition of iNOS Dimerization. From Tables 1~5, compound D27 showed
very good inhibitory effect against NO inhibition. NO plays an important role in
inflammation-related diseases, which is also importantly related to modulation
2
expression of iNOS. Thus, the inhibitory effect of compound D27 against expression
of iNOS in LPS-mediated RAW cells was analyzed by Western blot. From Figure 5,
we found that this compound could not affect the expression of iNOS. It was reported
1
that iNOS would dimerize to be in an active dimer form in LPS-mediated condition.
From previous result, our title compound indicated good NO inhibitory activity but
could not affect expression of iNOS. Thus, it would be very possible that inhibition of
NO production resulted from inhibition of iNOS dimerization. To confirm this, the
dimer of the iNOS in the presence of title compound was investigated using Native
Gel Sample Loading Buffer low-temperature SDS-PAGE, followed by
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
immunoblotting with antibody recognizing the protein of iNOS. Within a certain time,
dimeric iNOS was resolved on the basis of having distinct mobility (Figure 6). Title
compound D27 was found to inhibit iNOS from forming dimer. This result suggests
that compound D27 may be interfere the formation of stable dimer iNOS so as to
reflects anti-inflammatory activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Figure 5. Compound D27 inhibit expression of iNOS
a
iNOS expression was detected by Western blot analysis. β-actin was used as loading
#
##
control; p<0.001 compared with unstimulated cells, Data were obtained by at least
three independent experiments, and each was performed in duplicate.
a
Figure 6. Compound D27 destabilizes murine iNOS dimer in cells
a
RAW 264.7 cells were pretreated with the indicated concentrations of compound
D27 for 1 h and treated with LPS (1 μg/mL) for 24 h. Detect proteins were subjected
to 8% (iNOS) SDS-PAGE.
1
8
ACS Paragon Plus Environment

Page 19 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Anti-inflammatory Activity in Vivo. AIA as a rheumatoid arthritis model was
often used for testing anti-inflammatory activity. In this model, 80~90% of rats
developed arthritis within 14~21 days after adjuvant injection. Title compound was
administered intragastrically from day 14 to 28 after AA immunization. Intragastric
frequency was administered once a day. During the whole treatment period, doses (40
or 80 mg/kg) of compound D27 were administered intragastric administration to the
test groups. Aspirin was also administered by intragastric administration. Paw
swelling and arthritis index were measured every 3 days. As shown in Figure 7A, the
swelling peak of AA rats appeared on day 15. Administration of title compound D27
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(40 mg/kg) showed a significant inhibitory effect on hind paw swelling on day 30;
which was similar to the aspirin-treated positive control group. As displayed in
Figure 7B, body weight loss in group of rats treated with compound D27 was
significantly reduced.
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Effects of compound D27 on hind paw swelling and body weight loss in
a
rats with AA
a
###
Data are mean±s.d., n=5 per group. Aspirin was the positive control. p<0.01
compared with control; *** p<0.001, ** p<0.01, * p<0.05 vs AA group.
Typical photos of non-injected hind paw from sham and AIA rats were taken on
day 30 after AIA induction (Figure 8). There was a significant increase of secondary
hind paw swelling but title compound could treat secondary hind paw swelling.
,
a
Figure 8. Representative photos of hind paw on day 30 after arthritis induction
a
Representative photos of hind paw on day 30 after arthritis induction. (A) Normal;
(B) AA; (C) Aspirin 50 mg/kg; (D) Compound D27, 40 mg/kg; (E) Compound D27
80 mg/kg; Aspirin was the positive control.
To investigate whether compound D27 relieve the histological changes in knee
2
0
ACS Paragon Plus Environment

Page 21 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
joint of AA rats by photomicrographs of knee joints sections stained with H&E. No
inflammation was seen in normal rats (Figure 9A). AA rats exhibited extensive
inflammation, synovial hyperplaisia, bone or cartilage destruction and inflammatory
cells infiltration (Figure 9B).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
AA rats treated with compound D27 (40 mg/kg) exhibited moderate synovial
hyperplasia and inflammatory cells infiltration (Figure 9D). Compound D27 (80
mg/kg) and Aspirin (40 mg/kg) ameliorated inflammatory cells infiltration and
cartilage destruction, although slight synovial hyperplasia remained (Figures 9C and
9E).
Figure 9. Photomicrographs of sections stained with H&E illustrated the effects of
title compound D27 on joint histology for AA disease
(A) Normal; (B) AA; (C) Aspirin 50 mg/kg; (D) Compound D27 40 mg/kg; (E)
Compound D27 80 mg/kg, Aspirin was the positive control.
The serum levels of NO, TNF-α, IL-1β and IL-6 significantly elevated in AA
model group compared with control group (p<0.01). AA rats treatment with title
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound D27 (40, 80 mg/kg) showed a dose dependent decrease in serum
concentrations of NO, TNF-α, IL-1β and IL-6 compared with AA model group
(Figure 10). As a positive control, Aspirin showed similar inhibitory effects on IL-1β,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TNF-α, NO and IL-6 production in serum.
Figure 10. Effects of compound D27 on productions of NO, TNF-α, IL-1β and IL-6
a
by serum in rats with adjuvant arthritis (AA)
a
Data are mean±s.d., n=5 per group. Aspirin used as the positive control. ###p<0.01
compared with control group; *** p<0.001, ** p<0.01, * p<0.05 vs AA group.
CONCLUSIONS
In summary, based on the preliminary screening results of inhibitory effects
against NO production, guided by structure-activity relationships step by step, four
series of total 90 novel pyrazolo[4,3-d]pyrimidine derivatives have been designed、
2
2
ACS Paragon Plus Environment

Page 23 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
synthesized and evaluated. After structural optimization through third rounds, target
compounds exhibited significant inhibitory activity against NO production in
LPS-induced RAW 264.7 cells. A clear structure-activity relationships indicated that
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
substituted
3,4,5-trimethoxystyryl
at
C-5
and
alkylamine
at
C-7
pyrazolo[4,3-d]pyrimidine scaffold were beneficial to its anti-inflammatory activity.
Particularly, compound D27 with a propan-2-amine side chain displayed potent
inhibition towards NO production with IC50 value of 3.17 μM. It also showed
inhibitory activity against iNOS with IC of 1.12 μM. Preliminary mechanism studies
5
0
indicated that title compound could prevent iNOS dimerization. Furthermore, in order
to confirm its anti-inflammatory activity in vivo, rats models of acute and chronic
inflammation were investigated with title compound. For acute inflammatory models
in AA rats, treatment of compound D27 showed significant inhibitory effect on hind
paw swelling and body weight loss, which was similar to the effect observed in
aspirin-treated group. These observations suggest that title compound D27 may be an
effective agent for treatment of inflammatory diseases through regulation of iNOS
dimerization.
EXPERIMENTAL SECTION
General Methods for Chemistry. Reactions were monitored by thin layer
chromatography (TLC) on pre-coated silica GF254 plates. Column chromatography
was performed using 300-400 mesh silica gel purchased from Qingdao Haiyang
Chemical Co., Ltd. Melting point was determined on a XT4MP apparatus (Taike
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
13
1
Corp., Beijing, China). H and C NMR spectra are obtained by Brucker AM-300 ( H,
13
1
13
3
00 MHz; C, 75 MHz) or Agilent DD2 600ꢀMHz ( H, 600 MHz; C, 151 MHz)
spectrometer with CDCl or DMSO-d as the solvents and TMS as the internal
3
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
standard. Chemical shifts are reported in δ (parts per million) values. Coupling
constants J are reported in Hz. High-resolution electron impact mass spectra (HR-MS)
are recorded under electron impact (70 eV) condition using a Micro Mass GCT CA
0
55 instrument. All chemicals or reagents were purchased from standard commercial
suppliers and treated with standard methods before use. Solvents were dried in a
routine way and redistilled. The purity of all the tested compounds is at least 95% as
determined by HPLC. Crystallographic studies (Supporting Information).
General
Procedure
for
Synthesis
of
Intermediates
4A~D.
3
,4,5-trimethoxybenzoic acid (1.06 g, 5 mmol), oxalyl chloride (0.846 mL, 10 mmol)
and catalytic DMF were dissolved in dry DCM (10 mL), and the mixture was stirred
o
at 25~30 C for 3 h. Then the mixture was concentrated under vacuum and directly
used in next without any further purification. The obtained corresponding acyl
chloride was dissolved in DCM (1 mL) and the solution was added to the mixture of
3
6, 37
compound 1
(0.819 g, 4.5 mmol) and TEA (1.34 mL, 10 mmol) in dry DCM (10
mL). The solution was allowed to stir at room temperature for overnight. Reaction
mixture was washed with saturated NaHCO and water. Organic solvent was dried
3
over anhydrous Na SO , filtrated, and then evaporated to give a solid. The crude
2
4
solid product was further recrystallized in ethanol to give compound 2A.
Compound 2A (0.376 g, 1 mmol) and sodium ethoxide (0.340 g, 5 mmol) were
2
4
ACS Paragon Plus Environment

Page 25 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
dissolved in ethanol (15 mL) and refluxed for 8~12 h. The mixture was concentrated,
and the residue was acidified with 1N HCl to pH 7. The precipitate was filtered,
washed with water, and dried to give compound 3A.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 3A (3.58 g, 10 mmol) was dissolved in phosphorus oxychloride
o
(
POCl ) (30 mL) and stirred at 100 C for 8~12 h under N atmosphere. After
3
2
completion of the reaction, the mixture was concentrated in vacuum to give a dark oil
residue. The residue was washed with cold H O and extracted with DCM (30 mL×3).
2
The combined organic layer was washed with saturated NaHCO solution, saturated
3
brine solution, dried over anhydrous Na SO , and concentrated in vacuum to give the
2
4
key intermediate 4A. Compounds 4B~D were obtained according to the same
procedure as compound 4A.
1
-methyl-3-propyl-N-(3-(trifluoromethyl)phenyl)-5-(3,4,5-trimethoxyphenyl)-1
H-pyrazolo[4,3-d]pyrimidin-7-amine (A1). Compound 4A (200 mg, 0.531 mmol),
and 3-trifluoromethylaniline (85.6 mg, 0.531 mmol) were dissolved in i-propanol (15
mL) and refluxed for 6~8 h. After completion of the reaction, the solvent was
removed, and the residue was purified by column chromatography (gradient elution of
PE/EtOAc 85/15 v/v) to obtain compound A1. Compounds A2-13 were obtained
according to the same procedure.
o
1
Yellow solid (179.2 mg, yield: 67.3%), mp: 164-165 C. H NMR (300 MHz, CDCl ):
3
δ 8.10 (s, 1H, ArH), 7.92 (d, J = 8.0 Hz, 1H, ArH), 7.72 (s, 2H, ArH), 7.52 (t, J = 7.9
Hz, 1H, ArH), 7.43 (d, J = 7.7 Hz, 1H, ArH), 7.02 (s, 1H, NH), 4.36 (s, 3H, OCH3),
3
.95 (s, 6H, 2×OCH ), 3.91 (s, 3H, NCH ), 3.02 (t, J=7.6 Hz, 2H, CH ), 2.03~1.84 (m,
3
3
2
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2H,CH ), 1.06 (t, J = 7.4 Hz, 3H,CH ). HR-MS (ESI): calcd for C H F N O
2
3
25 26
3
5
3
+
[
M+H] , 502.20580; found 502.20583.
1
-methyl-3-propyl-N-(m-tolyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
pyrimidin-7-amine (A2). Compound was isolated as a yellow solid in 55.4% yieid,
o
1
mp: 202-203 C. (recryst. from ethanol). H NMR (600 MHz, DMSO-d ): δ 8.81 (s,
6
1H), 7.69 (d, J = 8.3 Hz, 1H), 7.67 (s, 2H), 7.58 (s, 1H), 7.31 (t, J = 7.8 Hz, 1H), 6.98
(d, J = 7.5 Hz, 1H), 4.31 (s, 3H), 3.84 (s, 6H), 3.71 (s, 3H), 2.91 (t, J = 7.4 Hz, 2H),
2
.35 (s, 3H), 1.85 (dd, J = 14.8, 7.4 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H). HR-MS (ESI):
+
calcd for C H N O [M+H] , 448.2039; found 448.2039.
2
5
30
5
3
1
-methyl-N-phenyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]
pyrimidin-7-amine (A3). Compound was isolated as a yellow solid in 82% yield. mp:
o
1
1
65-166 C. H NMR (300 MHz, CDCl ): δ 7.76 (d, 4H, ArH), 7.41 (t, J = 7.9 Hz, 2H,
3
ArH), 7.18 (t, J = 7.4 Hz, 1H, ArH), 6.89 (s, 1H, NH), 4.32 (s, 3H, OCH ), 3.97 (s, 6H,
3
2 ×OCH ), 3.90 (s, 3H, NCH ), 3.02 (t, J = 7.6 Hz, 2H, CH ), 1.93 (dd, J = 15.0, 7.5
3
3
2
Hz, 2H, CH ), 1.06 (t, J = 7.4 Hz, 3H, CH ). HR-MS (ESI): calcd for C H N O
3
2
3
24 28
5
+
[M+H] , 434.21496; found 434.21496.
N-(4-chlorophenethyl)-1-methyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-
pyrazolo[4,3-d]pyrimidin-7-amine (A4). Compound was isolated as a yellow solid
o
1
in 79% yield, mp: 145-147 C. (recryst. from ethanol). H NMR (600 MHz, CDCl ): δ
3
7
.80 (s, 2H, ArH), 7.31 (d, J = 8.2 Hz, 2H, ArH), 7.21 (d, J = 8.2 Hz, 2H, ArH), 5.12
(s, 1H, NH), 4.09 (s, 3H, OCH ), 3.99 (m, 8H, 2×OCH +CH ), 3.91 (s, 3H, NCH ),
3
3
2
3
3
.09 (t, J = 6.9 Hz, 2H, NCH ), 2.98 (t, J = 7.6 Hz, 2H, CH ), 1.97~1.85 (m, 2H, CH ),
2 2 2
2
6
ACS Paragon Plus Environment

Page 27 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
+
1.04 (t, J = 7.4 Hz, 3H, CH ). HR-MS (ESI): calcd for C H ClN O [M+H] ,
3
26 31
5
3
4
96.2110; found 496.2112.
-methyl-N-phenethyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]
pyrimidin-7-amine (A5). Compound was isolated as a yellow solid in 68% yield, mp:
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
1
1
35-136 C. (recryst. from ethanol). H NMR (600 MHz, CDCl ) δ 7.81 (s, 2H, ArH),
3
7.35 (t, J = 7.5 Hz, 2H, ArH), 7.28 (d, J = 7.3 Hz, 3H, ArH), 5.11 (t, J = 5.2 Hz, 1H,
NH), 4.06~4.01 (m, 5H, OCH +NCH ), 3.99 (s, 6H, 2×OCH ), 3.91 (s, 3H, NCH ),
3
2
3
3
3
.10 (t, J = 6.8 Hz, 2H, NCH ), 2.97 (t, J = 7.6 Hz, 2H, CH ), 1.94~1.86 (m, 2H, CH ),
2 2 2
+
1.04 (t, J = 7.4 Hz, 3H, CH ). HR-MS (ESI): calcd for C H N O [M+H] , 462.2500;
3
26 32
5
3
found 462.2502.
N-(4-methoxyphenethyl)-1-methyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-
pyrazolo[4,3-d]pyrimidin-7-amine (A6). Compound was isolated as a yellow solid
o
1
in 77% yield, mp:146-148 C. (recryst. from ethanol). H NMR (300 MHz, CDCl ): δ
3
7.81 (s, 2H ArH), 7.18 (d, J = 8.5 Hz, 2H, ArH), 6.87 (d, J = 8.6 Hz, 2H, ArH), 5.16 (t,
,
J = 5.3 Hz, 1H, NH), 4.04 (s, 3H, OCH ), 3.99 (s, 6H, 2×OCH ), 3.95 (d, J = 5.9 Hz,
3
3
2H, NCH ), 3.91 (s, 3H, NCH ), 3.79 (s, 3H, OCH ), 3.12~2.88 (m, 4H, 2×CH ), 1.90
2
3
3
2
(
m, 2H, CH ), 1.04 (t, J = 7.4 Hz, 3H, CH ). HR-MS (ESI): calcd for C H N O
2 3 27 34 5 4
+
[
M+H] , 492.25700; found 492.25700.
N,1-dimethyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin
-
7-amine (A7). Compound was isolated as a yellow solid in 65% yield, mp:184-185
o
1
C. (recryst. from ethanol). H NMR (600 MHz, CDCl ): δ 7.80 (s, 2H, ArH), 5.15 (d,
3
J = 4.2 Hz, 1H, NH), 4.22 (s, 3H, OCH ), 3.99 (s, 6H, 2×OCH ), 3.90 (s, 3H, NCH ),
3
3
3
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3.27 (d, J = 4.8 Hz, 3H NCH ), 2.98 (t, J = 7.6 Hz, 2H, CH ), 1.95~1.86 (m, 2H,
,
3
2
+
CH ), 1.04 (t, J = 7.4 Hz, 3H, CH ). HR-MS (ESI): calcd for C H N O [M+H] ,
2
3
19 26
5
3
3
72.20302; found 372.20122.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N,N,1-trimethyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]
pyrimidin-7-amine (A8). Compound was isolated as a yellow solid in 49% yield, mp:
o
1
122-123 C. (recryst. from ethanol). H NMR (600 MHz, CDCl ): δ 7.80 (s, 2H, ArH),
3
4
3
.13 (s, 3H, OCH ), 3.99 (s, 6H, 2×OCH ), 3.91 (s, 3H, NCH ), 3.20 (s, 6H, 2×CH ),
3 3 3 3
.02 (t, J = 7.6 Hz, 2H, CH ), 2.00~1.86 (m, 2H, CH ), 1.06 (t, J = 7.3 Hz, 3H, CH ).
2
2
3
+
HR-MS (ESI): calcd for C H N O [M+H] , 386.2187; found 386.2189.
20
28
5
3
N-isopropyl-1-methyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]
pyrimidin-7-amine (A9). Compound was isolated as a white solid yield 68%,
o
1
mp:166-167 C , H NMR (600 MHz, CDCl ): δ 7.79 (s, 2H, ArH), 4.87 (d, J = 6.5 Hz,
3
1
H, NH), 4.62 (dq, J = 13.0, 6.5 Hz, 1H, 2×CH ), 4.23 (s, 3H, OCH ), 3.99 (s, 6H,
3 3
2×OCH ), 3.90 (s, 3H, NCH ), 2.98 (t, J = 7.6 Hz, 2H, CH ), 1.95~1.85 (m, 2H, CH ),
3
3
2
2
1
.42 (d, J = 6.5 Hz, 6H, 2×CH ), 1.04 (t, J = 7.3 Hz, 3H, CH ). HR-MS (ESI): calcd
3 3
+
for C H N O [M+H] , 400.2343; found 400.2345.
2
1
30
5
3
4
-(1-methyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin
-7-yl)morpholine (A10). Compound was isolated as a yellow solid, yield 74%, mp:
o
1
145-146 C. (recryst. from ethanol). H NMR (600 MHz, CDCl ): δ 7.78 (s, 2H, ArH),
3
4
.12 (s, 3H, OCH ), 4.00 (s, 6H, 2×OCH ), 3.98~3.93 (m, 4H, 2×OCH ), 3.91 (s, 3H,
3 3 2
NCH ), 3.66~3.57 (m, 4H, 2×NCH ), 3.03 (t, J = 7.6 Hz, 2H, CH ), 1.93 (m, 2H,
3
2
2
13
CH ), 1.06 (t, J = 7.3 Hz, 3H, CH ). C NMR (151 MHz, CDCl ): δ 156.41, 153.86,
2
3
3
2
8
ACS Paragon Plus Environment

Page 29 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
153.25, 147.75, 145.67, 139.58, 134.18, 124.41, 105.30, 66.52, 61.08, 56.29, 50.11,
+
3
8.60, 28.02, 22.18, 14.29. HR-MS (ESI): calcd for C H N O [M+H] , 428.2292;
2
2
30
5
4
found 428.2292.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1-methyl-3-propyl-7-(pyrrolidin-1-yl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[4,3-d]pyrimidine (A11). Compound was isolated as a white solid in 59% yield, mp:
o
1
142-143 C. H NMR (600 MHz, CDCl ): δ 7.78 (s, 2H, ArH), 4.17 (s, 3H, OCH ),
3
3
3
.99 (s, 6H, 2×OCH ), 3.90 (s, 3H, NCH ), 3.87 (Brs, 4H, 2×NCH ), 3.01 (t, J = 7.6
3 3 2
Hz, 2H, CH ), 2.04 (Brs, 4H, 2×CH ), 1.91 (dt, J = 14.7, 7.4 Hz, 2H, CH ), 1.06 (t, J
2
2
2
1
3
= 7.3 Hz, 3H, CH ). C NMR (151 MHz, CDCl ): δ 155.99, 153.07, 151.09, 146.56,
3
3
1
45.09, 139.42, 134.78, 123.30, 105.27, 61.00, 56.21, 50.36, 41.47, 27.94, 25.71,
+
22.18, 14.28. HR-MS (ESI): calcd for C H N O [M+H] , 412.2343; found
2
2
30
5
3
4
12.2346.
-methyl-7-(piperidin-1-yl)-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo
[4,3-d]pyrimidine (A12). Compound was isolated as a white solid in 61% yield, mp:
1
o
1
1
29-130 C. H NMR (600 MHz,CDCl ): δ 7.80 (s, 2H, ArH), 4.11 (s, 3H, OCH ),
3 3
4.00 (s, 6H, 2×OCH ), 3.91 (s, 3H, NCH ), 3.55 (s, 4H, 2×NCH ), 3.02 (t, J = 7.6 Hz,
3
3
2
2
H, CH ), 1.97~1.88 (m, 2H, CH ), 1.82 (d, J = 4.9 Hz, 4H, 2×CH ), 1.75 (d, J = 4.6
2 2 2
13
Hz, 2H, CH ), 1.06 (t, J = 7.3 Hz, 3H, CH ). C NMR (151 MHz, CDCl ): δ 156.34,
2
3
3
154.55, 153.17, 147.38, 145.28, 139.56, 134.54, 124.62, 105.30, 61.03, 56.24, 50.66,
8.58, 28.02, 25.61, 24.59, 22.20, 14.27. HR-MS (ESI): calcd for C H N O
3
3
23
32
5
+
[M+H] , 426.2500; found 426.2503.
Methyl 4-((1-methyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]
2
9
ACS Paragon Plus Environment