U.S. patent application number 17/404281 was filed with the patent office on 2021-12-09 for pyrazolo-heteroaryl derivative, preparation method and medical use thereof.
The applicant listed for this patent is Jiangsu Hengrui Medicine Co., Ltd., Shanghai Hengrui Pharmaceutical Co., Ltd.. Invention is credited to Feng HE, Qiyue HU, Chunfeng SHU, Weikang TAO, Guobao ZHANG.
Application Number | 20210380593 17/404281 |
Document ID | / |
Family ID | 1000005787154 |
Filed Date | 2021-12-09 |
United States Patent
Application |
20210380593 |
Kind Code |
A1 |
ZHANG; Guobao ; et
al. |
December 9, 2021 |
PYRAZOLO-HETEROARYL DERIVATIVE, PREPARATION METHOD AND MEDICAL USE
THEREOF
Abstract
Disclosed are a pyrazolo-heteroaryl derivative, a preparation
method and medical use thereof. In particular, this invention
relates to a new pyrazolo-heteroaryl derivative as shown in the
general formula (I), a preparation method thereof and a
pharmaceutical composition containing the derivative and the use
thereof as a therapeutic agent, in particular as a TLR7 agonist,
wherein each substituent in the general formula (I) is defined in
the description. ##STR00001##
Inventors: |
ZHANG; Guobao; (Minhang
District, CN) ; SHU; Chunfeng; (Minhang District,
CN) ; HU; Qiyue; (Minhang District, CN) ; HE;
Feng; (Minhang District, CN) ; TAO; Weikang;
(Minhang District, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Jiangsu Hengrui Medicine Co., Ltd.
Shanghai Hengrui Pharmaceutical Co., Ltd. |
Lianyungang
Minhang District |
|
CN
CN |
|
|
Family ID: |
1000005787154 |
Appl. No.: |
17/404281 |
Filed: |
August 17, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16464341 |
May 28, 2019 |
11117898 |
|
|
PCT/CN2017/113007 |
Nov 27, 2017 |
|
|
|
17404281 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 487/04
20130101 |
International
Class: |
C07D 487/04 20060101
C07D487/04 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 28, 2016 |
CN |
201611066071.7 |
Claims
1. A method for treating an infection caused by a virus, comprising
administering to a subject in need thereof an effective amount of a
pharmaceutical composition comprising one or more pharmaceutically
acceptable carriers, diluents or excipients, and a compound of
formula (I): ##STR00071## or a tautomer, racemate, enantiomer,
diastereomer thereof, or mixture thereof, or a pharmaceutically
acceptable salt thereof, wherein: ring A is selected from the group
consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl; G is
CH or N; X.sup.1 is alkylene or S(O).sub.m, wherein the alkylene is
optionally substituted by one or more substituents selected from
the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy,
hydroxyalkyl, cyano, amino, nitro, cycloalkyl and heterocyclyl;
L.sup.1 is selected from the group consisting of --NR.sup.4--,
--O--, --S--, --C(O)--, --S(O).sub.m--, --N(R.sup.4)C(O)--,
--C(O)N(R.sup.4)--, --N(R.sup.4)S(O).sub.2--,
--S(O).sub.2N(R.sup.4)-- and a covalent bond; R.sup.1 is selected
from the group consisting of alkyl, alkoxy, haloalkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the
alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and
heteroaryl are each independently optionally substituted by one or
more substituents selected from the group consisting of alkyl,
alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino,
nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, --OR.sup.5,
--C(O)R.sup.5, --S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7; each R.sup.2 is identical or different and
each is independently selected from the group consisting of
hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl,
cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl,
wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl
are each independently optionally substituted by one or more
substituents selected from the group consisting of alkyl, alkoxy,
halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro,
cycloalkyl, heterocyclyl, aryl, heteroaryl, --OR.sup.5,
--C(O)R.sup.5, --S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7; L.sup.2 is alkylene, wherein the alkylene is
optionally substituted by one or more substituents selected from
the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy,
hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl,
heteroaryl, -OR.sup.5, --C(O)R.sup.5, --S(O).sub.mR.sup.5,
--NR.sup.6R.sup.7 and --C(O)NR.sup.6R.sup.7; R.sup.3 is selected
from the group consisting of haloalkyl, hydroxy, hydroxyalkyl,
cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl,
--OR.sup.5, --C(O)R.sup.5, --S(O).sub.mR.sup.5, --NR.sup.6R.sup.7
and --C(O)NR.sup.6R.sup.7, wherein the cycloalkyl, heterocyclyl,
aryl and heteroaryl are each independently optionally substituted
by one or more substituents selected from the group consisting of
alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano,
amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl,
--C(O)R.sup.8, --S(O).sub.mR.sup.8, --NR.sup.9R.sup.10 and
--C(O)NR.sup.9R.sup.10; R.sup.4 is selected from the group
consisting of hydrogen, alkyl, haloalkyl, cycloalkyl, heterocyclyl,
aryl and heteroaryl; R.sup.5 is selected from the group consisting
of hydrogen, alkyl, haloalkyl, amino, hydroxy, cycloalkyl,
heterocyclyl, aryl and heteroaryl; R.sup.6 and R.sup.7 are
identical or different and are each independently selected from the
group consisting of hydrogen, alkyl, haloalkyl, cycloalkyl,
heterocyclyl, aryl, heteroaryl, --C(O)R.sup.8, --S(O).sub.mR.sup.8
and --C(O)NR.sup.9R.sup.10, wherein the alkyl, cycloalkyl,
heterocyclyl, aryl and heteroaryl are each independently optionally
substituted by one or more substituents selected from the group
consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy,
hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; or,
R.sup.6 and R.sup.7 together with the nitrogen atom to which they
are attached form a heterocyclyl, wherein the heterocyclyl
optionally contains one or two identical or different heteroatoms
selected from the group consisting of N, O and S in addition to the
nitrogen atom, and the heterocyclyl is optionally substituted by
one or more substituents selected from the group consisting of
alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl,
cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.8 is selected
from the group consisting of hydrogen, alkyl, haloalkyl, amino,
hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.9 and
R.sup.10 are identical or different and are each independently
selected from the group consisting of hydrogen, alkyl, haloalkyl,
amino, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; n is
0, 1, 2, 3 or 4; m is 0, 1 or 2; said cycloalkyl refers to a
saturated or partially unsaturated monocyclic or polycyclic
hydrocarbon group having 3 to 20 carbon atoms, polycyclic
cycloalkyl includes a cycloalkyl having a spiro ring, fused ring or
bridged ring; said heterocyclyl refers to a 3 to 20 membered
saturated or partially unsaturated monocyclic or polycyclic
hydrocarbon substituent group wherein 1 to 4 atoms are heteroatoms
selected from the group consisting of N, O, S(O) and S(O).sub.2,
polycyclic heterocyclyl includes a heterocyclyl having a spiro
ring, fused ring or bridged ring; said heteroaryl refers to a 5 to
14 membered heteroaromatic system having 1 to 4 heteroatoms
selected from the group consisting of O, S and N; and wherein the
virus is selected from the group consisting of dengue virus, yellow
fever virus, West Nile virus, Japanese encephalitis virus,
tick-borne encephalitis virus, Kunjin virus, Murray Valley
encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic
fever virus, bovine viral disarrhea virus, Zika virus, HIV, HBV,
HCV, HPV, RSV, SARS and influenza virus.
2. The method according to claim 1, wherein R.sup.3 is
heterocyclyl, and the heterocyclyl is optionally substituted by one
or more substituents selected from the group consisting of alkyl,
alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl,
cycloalkyl, heterocyclyl, aryl and heteroaryl.
3. The method according to claim 1, wherein R.sup.3 is
--NR.sup.6R.sup.7, and R.sup.6 and R.sup.7 together with the
nitrogen atom to which they are attached form a heterocyclyl,
wherein the heterocyclyl optionally contains one or two identical
or different heteroatoms selected from the group consisting of N, O
and S in addition to the nitrogen atom, and the heterocyclyl is
optionally substituted by one or more substituents selected from
the group consisting of alkyl, alkoxy, halogen, amino, cyano,
nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and
heteroaryl.
4. The method according to claim 1, wherein the ring A is
phenyl.
5. The method according to claim 1, wherein X.sup.1 is
alkylene.
6. The method according to claim 1, wherein G is N.
7. The method according to claim 1, wherein the compound is a
compound of formula (III): ##STR00072## or a tautomer, racemate,
enantiomer, diastereomer thereof, or mixture thereof, or a
pharmaceutically acceptable salt thereof, wherein: s is 0, 1 or
2.
8. The method according to claim 1, wherein L.sup.1 is selected
from the group consisting of --O--, --NR.sup.4--, --C(O)-- and
--C(O)N(R.sup.4)--, and R.sup.4 is hydrogen or alkyl.
9. The method according to claim 1, wherein R.sup.1 is alkyl
optionally substituted by one or more alkoxy.
10. The method according to claim 1, wherein the compound is
selected from the group consisting of: ##STR00073## ##STR00074## or
a tautomer, racemate, enantiomer, diastereomer thereof, or a
mixture thereof, or a pharmaceutically acceptable salt thereof.
11. A method for treating or preventing melanoma, non-small cell
lung carcinoma, hepatocellular carcinoma, basal cell carcinoma,
renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD,
ulcerative colitis or hepatic fibrosis, comprising administering to
a subject in need thereof an effective amount of a pharmaceutical
composition comprising one or more pharmaceutically acceptable
carriers, diluents or excipients, and a compound of formula (I):
##STR00075## or a tautomer, racemate, enantiomer, diastereomer
thereof, or mixture thereof, or a pharmaceutically acceptable salt
thereof, wherein: ring A is selected from the group consisting of
cycloalkyl, heterocyclyl, aryl and heteroaryl; G is CH or N;
X.sup.1 is alkylene or S(O).sub.m, wherein the alkylene is
optionally substituted by one or more substituents selected from
the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy,
hydroxyalkyl, cyano, amino, nitro, cycloalkyl and heterocyclyl;
L.sup.1 is selected from the group consisting of --NR.sup.4--,
--O--, --S--, --C(O)--, --S(O).sub.m--, --N(R.sup.4)C(O)--,
--C(O)N(R.sup.4)--, --N(R.sup.4)S(O).sub.2--,
--S(O).sub.2N(R.sup.4)-- and a covalent bond; R.sup.1 is selected
from the group consisting of alkyl, alkoxy, haloalkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the
alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and
heteroaryl are each independently optionally substituted by one or
more substituents selected from the group consisting of alkyl,
alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino,
nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, --OR.sup.5,
--C(O)R.sup.5, --S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7; each R.sup.2 is identical or different and
each is independently selected from the group consisting of
hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl,
cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl,
wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl
are each independently optionally substituted by one or more
substituents selected from the group consisting of alkyl, alkoxy,
halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro,
cycloalkyl, heterocyclyl, aryl, heteroaryl, --OR.sup.5,
--C(O)R.sup.5, --S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7; L.sup.2 is alkylene, wherein the alkylene is
optionally substituted by one or more substituents selected from
the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy,
hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl,
heteroaryl, --OR.sup.5, --C(O)R.sup.5, --S(O).sub.mR.sup.5,
--NR.sup.6R.sup.7 and --C(O)NR.sup.6R.sup.7; R.sup.3 is selected
from the group consisting of haloalkyl, hydroxy, hydroxyalkyl,
cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl,
--OR.sup.5, --C(O)R.sup.5, --S(O).sub.mR.sup.5, --NR.sup.6R.sup.7
and --C(O)NR.sup.6R.sup.7, wherein the cycloalkyl, heterocyclyl,
aryl and heteroaryl are each independently optionally substituted
by one or more substituents selected from the group consisting of
alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano,
amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl,
--C(O)R.sup.8, --S(O).sub.mR.sup.8, --NR.sup.9R.sup.1.degree. and
--C(O)NR.sup.9R.sup.10; R.sup.4 is selected from the group
consisting of hydrogen, alkyl, haloalkyl, cycloalkyl, heterocyclyl,
aryl and heteroaryl; R.sup.5 is selected from the group consisting
of hydrogen, alkyl, haloalkyl, amino, hydroxy, cycloalkyl,
heterocyclyl, aryl and heteroaryl; R.sup.6 and R.sup.7 are
identical or different and are each independently selected from the
group consisting of hydrogen, alkyl, haloalkyl, cycloalkyl,
heterocyclyl, aryl, heteroaryl, --C(O)R.sup.8, --S(O).sub.mR.sup.8
and --C(O)NR.sup.9R.sup.10, wherein the alkyl, cycloalkyl,
heterocyclyl, aryl and heteroaryl are each independently optionally
substituted by one or more substituents selected from the group
consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy,
hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; or,
R.sup.6 and R.sup.7 together with the nitrogen atom to which they
are attached form a heterocyclyl, wherein the heterocyclyl
optionally contains one or two identical or different heteroatoms
selected from the group consisting of N, O and S in addition to the
nitrogen atom, and the heterocyclyl is optionally substituted by
one or more substituents selected from the group consisting of
alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl,
cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.8 is selected
from the group consisting of hydrogen, alkyl, haloalkyl, amino,
hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.9 and
R.sup.10 are identical or different and are each independently
selected from the group consisting of hydrogen, alkyl, haloalkyl,
amino, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; n is
0, 1, 2, 3 or 4; m is 0, 1 or 2; said cycloalkyl refers to a
saturated or partially unsaturated monocyclic or polycyclic
hydrocarbon group having 3 to 20 carbon atoms, polycyclic
cycloalkyl includes a cycloalkyl having a spiro ring, fused ring or
bridged ring; said heterocyclyl refers to a 3 to 20 membered
saturated or partially unsaturated monocyclic or polycyclic
hydrocarbon substituent group wherein 1 to 4 atoms are heteroatoms
selected from the group consisting of N, O, S(O) and S(O).sub.2,
polycyclic heterocyclyl includes a heterocyclyl having a spiro
ring, fused ring or bridged ring; and said heteroaryl refers to a 5
to 14 membered heteroaromatic system having 1 to 4 heteroatoms
selected from the group consisting of O, S and N.
12. The method according to claim 11, wherein R.sup.3 is
heterocyclyl, and the heterocyclyl is optionally substituted by one
or more substituents selected from the group consisting of alkyl,
alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl,
cycloalkyl, heterocyclyl, aryl and heteroaryl.
13. The method according to claim 11, wherein R.sup.3 is
--NR.sup.6R.sup.7, and R.sup.6 and R.sup.7 together with the
nitrogen atom to which they are attached form a heterocyclyl,
wherein the heterocyclyl optionally contains one or two identical
or different heteroatoms selected from the group consisting of N, O
and S in addition to the nitrogen atom, and the heterocyclyl is
optionally substituted by one or more substituents selected from
the group consisting of alkyl, alkoxy, halogen, amino, cyano,
nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and
heteroaryl.
14. The method according to claim 11, wherein the ring A is
phenyl.
15. The method according to claim 11, wherein X.sup.1 is
alkylene.
16. The method according to claim 11, wherein G is N.
17. The method according to claim 11, wherein the compound is a
compound of formula (III): ##STR00076## or a tautomer, racemate,
enantiomer, diastereomer thereof, or mixture thereof, or a
pharmaceutically acceptable salt thereof, wherein: s is 0, 1 or
2.
18. The method according to claim 11, wherein L.sup.1 is selected
from the group consisting of --O--, --NR.sup.4--, --C(O)-- and
--C(O)N(R.sup.4)--, and R.sup.4 is hydrogen or alkyl.
19. The method according to claim 11, wherein R.sup.1 is alkyl
optionally substituted by one or more alkoxy.
20. The method according to claim 11, wherein the compound is
selected from the group consisting of: ##STR00077## ##STR00078## or
a tautomer, racemate, enantiomer, diastereomer thereof, or a
mixture thereof, or a pharmaceutically acceptable salt thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/464,341 filed May 28, 2019, which is a
Section 371 of International Application No. PCT/CN2017/113007,
filed Nov. 27, 2017, which was published in the Chinese language on
May 31, 2018, under International Publication No. WO 2018/095426
A1, which claims priority under 35 U.S.C. .sctn. 119(b) to Chinese
Patent Application No. 201611066071.7, filed on Nov. 28, 2016, the
disclosures of which are incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel pyrazolo-heteroaryl
derivative of formula (I), a method for preparing the same and a
pharmaceutical composition comprising the same, as well as the use
thereof as a therapeutic agent, particularly as a TLR7 agonist.
BACKGROUND OF THE INVENTION
[0003] Toll-like receptors (TLRs) are a class of important protein
molecules involved in innate immunity. TLRs are single,
membrane-spanning, non-catalytic receptors, usually expressed on
sentinel cells such as macrophages and dendritic cells, and can
recognize structurally conserved molecules produced by microbes.
Once these microbes have broken through physical barriers such as
the skin or intestinal tract mucosa, they are recognized by TLRs,
which activate immune cell responses (Mahla, RS. et al., Front
Immunol. 4: 248 (2013)). The ability of immune system to broadly
recognize pathogenic microorganisms is, in part, due to the
widespread presence of toll-like immunoreceptors (TLRs).
[0004] There are at least ten different TLRs in mammals. Ligands
and corresponding signaling cascades have been identified for some
of these receptors. TLR7 is a member of the subgroup of TLRs (TLRs
3, 7, 8, and 9), localised in the endosomal compartment of cells
which are specialized to detect non-self nucleic acids. TLR7
.mu.lays a key role in anti-viral defense via the recognition of
ssRNA (Diebold S. S. et al, Science, 2004: 303, 1529-1531; and Lund
J. M. et al, PNAS, 2004: 101, 5598-5603). TLR7 has a restricted
expression-profile in human, and is expressed predominantly by B
cells and plasmacytoid dendritic cells (pDC), and to a lesser
extent by monocytes. Plasmacytoid DCs are a unique population of
lymphoid-derived dendritic cells (0.2-0.8% of Peripheral Blood
Mononuclear Cells (PBMCs)), which are the primary type I
interferon-producing cells secreting high levels of
interferon-alpha (IFN.alpha.) and interferon-beta OE9) in response
to viral infections (Liu Y-J, Annu. Rev. Immunol., 2005: 23,
275-306).
[0005] A number of diseases and disorders are related to
abnormalities in TLRs, such as melanoma, non-small cell lung
carcinoma, hepatocellular carcinoma, basal cell carcinoma, renal
cell carcinoma, myeloma, allergic rhinitis, asthma, chronic
obstructive pulmonary disease (COPD), ulcerative colitis, hepatic
fibrosis, and viral infections such as HBV, Flaviviridae viruses,
HCV, HPV, RSV, SARS, HIV, or influenza. Therefore, the use of a TLR
agonist to treat related diseases is very promising.
[0006] Since TLR7 and TLR8 are highly homologous, the ligand of
TLR7 in most cases is also the ligand of TLR8. TLR8 stimulation
mainly induces the production of cytokines such as tumor necrosis
factor .alpha. (TNF-.alpha.) and chemokine. Interferon .alpha. is
one of the main drugs for treating chronic hepatitis B or hepatitis
C, while TNF-.alpha. is a pro-inflammatory cytokine, and its
over-secretion may cause severe side effects. Therefore, the
selectivity for TLR7 and TLR8 is critical for the development of
TLR7 agonists for treating virus infective diseases.
[0007] There are currently patent applications related to TLR7
agonists, such as WO2005025583, WO2007093901, WO2008011406,
WO2009091032, WO2010077613, WO2010133882, WO2011031965 and
WO2012080730. However, there is still a need to continue to develop
TLR7 agonists that are safer and more therapeutically
effective.
[0008] In view of the above technical problems, the present
invention provides a pharmaceutical compound having a lower onset
concentration, better selectivity (selective for TLR7, and no
activation effect on TLR8), more effective activation effect and at
the same time, due to a weak inhibitory effect on CYP, it is a
safer and more effective TLR7 agonist.
SUMMARY OF THE INVENTION
[0009] The object of the present invention is to provide a compound
of formula (I):
##STR00002##
[0010] or a tautomer, mesomer, racemate, enantiomer, diastereomer
thereof, or mixture thereof, or a pharmaceutically acceptable salt
thereof,
[0011] wherein:
[0012] ring A is selected from the group consisting of cycloalkyl,
heterocyclyl, aryl and heteroaryl;
[0013] G is CH or N;
[0014] X.sup.1 is alkylene or S(O).sub.m, wherein the alkylene is
optionally substituted by one or more substituents selected from
the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy,
hydroxyalkyl, cyano, amino, nitro, cycloalkyl and heterocyclyl;
[0015] L.sup.1 is selected from the group consisting of
--NR.sup.4--, --O--, --S--, --C(O)--, --C(O)--OR.sup.4,
--S(O).sub.m--, --N(R.sup.4)C(O)--, --C(O)N(R.sup.4)--,
--N(R.sup.4)S(O).sub.2--, --S(O).sub.2N(R.sup.4)-- and a covalent
bond;
[0016] R.sup.1 is selected from the group consisting of alkyl,
alkoxy, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl
and heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl,
heterocyclyl, aryl and heteroaryl are each independently optionally
substituted by one or more substituents selected from the group
consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy,
hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl,
heteroaryl, --OR.sup.5, --C(O)R.sup.5, --S(O).sub.mR.sup.5,
--NR.sup.6R.sup.7 and --C(O)NR.sup.6R.sup.7;
[0017] each R.sup.2 is identical or different and each is
independently selected from the group consisting of hydrogen,
halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano,
amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl,
wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl
are each independently optionally substituted by one or more
substituents selected from the group consisting of alkyl, alkoxy,
halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro,
cycloalkyl, heterocyclyl, aryl, heteroaryl, --OR.sup.5,
--C(O)R.sup.5, --S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7;
[0018] L.sup.2 is alkylene or a covalent bond, wherein the alkylene
is optionally substituted by one or more substituents selected from
the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy,
hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl,
heteroaryl, --OR.sup.5, --C(O)R.sup.5, --S(O).sub.mR.sup.5,
--NR.sup.6R.sup.7 and --C(O)NR.sup.6R.sup.7;
[0019] R.sup.3 is selected from the group consisting of haloalkyl,
hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl,
heterocyclyl, aryl, heteroaryl, --OR.sup.5, --C(O)R.sup.5,
--S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and --C(O)NR.sup.6R.sup.7,
wherein the cycloalkyl, heterocyclyl, aryl and heteroaryl are each
independently optionally substituted by one or more substituents
selected from the group consisting of alkyl, alkoxy, halogen,
haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl,
heterocyclyl, aryl, heteroaryl, --OR.sup.8, --C(O)R.sup.8,
--S(O).sub.mR.sup.8, --NR.sup.9R.sup.1.degree. and
--C(O)NR.sup.9R.sup.1.degree. ;
[0020] R.sup.4 is selected from the group consisting of hydrogen,
alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl and
heteroaryl;
[0021] R.sup.5 is selected from the group consisting of hydrogen,
alkyl, haloalkyl, amino, hydroxy, cycloalkyl, heterocyclyl, aryl
and heteroaryl;
[0022] R.sup.6 and R.sup.7 are identical or different and are each
independently selected from the group consisting of hydrogen,
alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,
--C(O)R.sup.8, --S(O).sub.mR.sup.8 and --C(O)NR.sup.9R.sup.10,
wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl
are each independently optionally substituted by one or more
substituents selected from the group consisting of alkyl, alkoxy,
halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl,
heterocyclyl, aryl and heteroaryl;
[0023] or, R.sup.6 and R.sup.7 together with the nitrogen to which
they are attached form a heterocyclyl, wherein the heterocyclyl
optionally contains one or two identical or different heteroatoms
selected from the group consisting of N, O and S in addition to one
nitrogen atom, and the heterocyclyl is optionally substituted by
one or more substituents selected from the group consisting of
alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl,
cycloalkyl, heterocyclyl, aryl and heteroaryl;
[0024] R.sup.8 is selected from the group consisting of hydrogen,
alkyl, haloalkyl, amino, hydroxy, cycloalkyl, heterocyclyl, aryl
and heteroaryl;
[0025] R.sup.9 and R.sup.10 are identical or different and are each
independently selected from the group consisting of hydrogen,
alkyl, haloalkyl, amino, hydroxy, cycloalkyl, heterocyclyl, aryl
and heteroaryl;
[0026] n is 0, 1, 2, 3 or 4; and
[0027] m is 0, 1 or 2.
[0028] In a preferred embodiment of the present invention, in the
compound of formula (I), R.sup.3 is heterocyclyl, and the
heterocyclyl is optionally substituted by one or more substituents
selected from the group consisting of alkyl, alkoxy, halogen,
amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl,
heterocyclyl, aryl and heteroaryl.
[0029] In a preferred embodiment of the present invention, in the
compound of formula (I), R.sup.3 is --NR.sup.6R.sup.7, and R.sup.6
and R.sup.7 together with the nitrogen to which they are attached
form a heterocyclyl, wherein the heterocyclyl optionally contains
one or two identical or different heteroatoms selected from the
group consisting of N, O and S in addition to one nitrogen atom,
and the heterocyclyl is optionally substituted by one or more
substituents selected from the group consisting of alkyl, alkoxy,
halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl,
heterocyclyl, aryl and heteroaryl.
[0030] In a preferred embodiment of the present invention, in the
compound of formula (I), the ring A is selected from the group
consisting of phenyl and pyridyl.
[0031] In a preferred embodiment of the present invention, in the
compound of formula (I), the pyridyl is selected from the group
consisting of
##STR00003##
[0032] In a preferred embodiment of the present invention, in the
compound of formula (I), X.sup.1 is alkylene.
[0033] In a preferred embodiment of the present invention, the
compound of formula (I) is a compound of formula (II):
##STR00004##
[0034] or a tautomer, mesomer, racemate, enantiomer, diastereomer
thereof, or mixture thereof, or a pharmaceutically acceptable salt
thereof,
[0035] wherein G, L.sup.1.about.L.sup.2, R.sup.1.about.R.sup.2,
R.sup.6.about.R.sup.7 and n are as defined in formula (I).
[0036] In a preferred embodiment of the present invention, in the
compound of formula (I), G is N.
[0037] In a preferred embodiment of the present invention, in the
compound of formula (I), L.sup.2 is alkylene.
[0038] In a preferred embodiment of the present invention, the
compound of formula (I) is a compound of formula (III):
##STR00005##
[0039] or a tautomer, mesomer, racemate, enantiomer, diastereomer
thereof, or mixture thereof, or a pharmaceutically acceptable salt
thereof,
[0040] wherein:
[0041] s is 0, 1 or 2;
[0042] L.sup.1, R.sup.1.about.R.sup.2 and n are as defined in
formula (I).
[0043] In a preferred embodiment of the present invention, in the
compound of formula (I), L.sup.1 is selected from the group
consisting of --O--, --NR.sup.4--, --C(O)-- and --C(O)N(R.sup.4)--,
and R.sup.4 is hydrogen or alkyl.
[0044] In a preferred embodiment of the present invention, in the
compound of formula (I), R.sup.1 is alkyl optionally substituted by
one or more alkoxy.
[0045] In a preferred embodiment of the present invention, in the
compound of formula (I), each R.sup.2 is identical or different and
each is independently hydrogen or halogen.
[0046] Typical compounds of the present invention include, but are
not limited to:
TABLE-US-00001 Example No. Structure and name of the compound 1
##STR00006##
6-Butoxy-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-
4-amine 2 ##STR00007##
1-(4-(Azetidin-1-ylmethyl)benzyl)-6-butoxy-1H-pyrazolo[3,4-d]pyrimidin-4-
amine 3 ##STR00008##
6-Butoxy-1-(4-(piperidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-
4-amine 4 ##STR00009##
6-Butoxy-1-(3-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-
4-amine 5 ##STR00010##
1-(3-(Azetidin-1-ylmethyl)benzyl)-6-butoxy-1H-pyrazolo[3,4-d]
pyrimidin-4-amine 6 ##STR00011##
6-Butoxy-1-(3-(piperidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-
4-amine 7 ##STR00012##
6-(2-Methoxyethoxy)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d-
] pyrimidin-4-amine 8 ##STR00013##
6-((1-Methoxypropan-2-yl)oxy)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine 9 ##STR00014##
6-Butoxy-1-(3-fluoro-4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]
pyrimidin-4-amine 10 ##STR00015##
N.sup.6-Butyl-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]
pyrimidine-4,6-diamine 11 ##STR00016##
4-Amino-N-propyl-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]
pyrimidine-6-carboxamide 12 ##STR00017##
1-(4-Amino-1-(4-pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]
pyrimidin-6-yl)pentan-1-one
[0047] or a tautomer, mesomer, racemate, enantiomer, diastereomer
thereof, or mixture thereof,
[0048] or a pharmaceutically acceptable salt thereof.
[0049] In another aspect, the present invention relates to a
compound of formula (I-C):
##STR00018##
[0050] or a tautomer, mesomer, racemate, enantiomer, diastereomer
thereof, or mixture thereof, or a pharmaceutically acceptable salt
thereof,
[0051] wherein:
[0052] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0053] X is halogen, preferably chlorine;
[0054] ring A, G, X.sup.1, L.sup.2, R.sup.2.about.R.sup.3 and n are
as defined in formula (I).
[0055] The compounds of formula (I-C) include, but are not limited
to:
TABLE-US-00002 Example No. Structure and name of the compound 1e
##STR00019##
6-Chloro-N-(4-methoxybenzyl)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-
1H-pyrazolo[3,4-d]pyrimidin-4-amine 1e 2f ##STR00020##
1-(4-(Azetidin-1-ylmethyl)benzyl)-6-chloro-N-(4-methoxybenzyl)-
1H-pyrazolo[3,4-d]pyrimidin-4-amine 2f 3c ##STR00021##
6-Chloro-N-(4-methoxybenzyl)-1-(4-(piperidin-1-ylmethyl)benzyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine 3c 4b ##STR00022##
6-Chloro-N-(3-methoxybenzyl)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-
1H-pyrazolo[3,4-d]pyrimidin-4-amine 4b 5e ##STR00023##
1-(3-(Azetidin-1-ylmethyl)benzyl)-6-chloro-N-(4-methoxybenzyl)-
1H-pyrazolo[3,4-d]pyrimidin-4-amine 5e 6c ##STR00024##
6-Chloro-N-(3-methoxybenzyl)-1-(4-(piperidin-1-ylmethyl)benzyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine 6c 9f ##STR00025##
6-Chloro-1-(3-fluoro-4-(pyrrolidin-1-ylmethyl)benzyl)-N-(4-methoxy-
benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 9f
[0056] In another aspect, the present invention relates to a
compound of formula (I-E):
##STR00026##
[0057] or a tautomer, mesomer, racemate, enantiomer, diastereomer
thereof, or mixture thereof, or a pharmaceutically acceptable salt
thereof,
[0058] wherein:
[0059] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0060] ring A, G, X.sup.1, L.sup.1.about.L.sup.2,
R.sup.1.about.R.sup.3 and n are as defined in formula (I).
[0061] The compounds of formula (I-E) include, but are not limited
to:
TABLE-US-00003 Example No. Structure and name of the compound 1f
##STR00027##
6-Butoxy-N-(4-methoxybenzyl)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-
1H-pyrazolo[3,4-d]pyrimidin-4-amine 1f 2g ##STR00028##
1-(4-(Azetidin-1-ylmethyl)benzyl)-6-butoxy-N-(4-methoxybenzyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine 2g 3d ##STR00029##
6-Butoxy-N-(4-methoxybenzyl)-1-(4-(piperidin-1-ylmethyl)benzyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine 3d 4c ##STR00030##
6-Butoxy-N-(3-methoxybenzyl)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-
1H-pyrazolo[3,4-d]pyrimidin-4-amine 4c 5f ##STR00031##
1-(3-(Azetidin-1-ylmethyl)benzyl)-6-butoxy-N-(4-methoxybenzyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine 5f 6d ##STR00032##
6-Butoxy-N-(3-methoxybenzyl)-1-(4-(piperidin-1-ylmethyl)benzyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine 6d 7a ##STR00033##
N-(4-Methoxybenzyl)-6-(2-methoxyethoxy)-1-(4-(pyrrolidin-1-ylmethyl)
benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7a 8a ##STR00034##
N-(4-Methoxybenzyl)-6-((1-methoxypropan-2-yl)oxy)-1-(4-(pyrrolidin-1-
ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 8a 9g
##STR00035##
6-Butoxy-1-(3-fluoro-4-(pyrrolidin-1-ylmethyl)benzyl)-N-(4-methoxy-
benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 9g 10a ##STR00036##
N.sup.6-Butyl-N.sup.4-(4-methoxybenzyl)-1-(4-(pyrrolidin-1-ylmethyl)benzy-
l)- 1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine 10a 11a ##STR00037##
Methyl 4-((4-methoxybenzyl)amino)-1-(4-(pyrrolidin-1-ylmethyl)
benzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-carboxylate 11a 11b
##STR00038##
4-((4-Methoxybenzyl)amino)-N-propyl-1-(4-(pyrrolidin-1-ylmethyl)
benzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-carboxamide 11lb 12b
##STR00039##
1-(4-((4-Methoxybenzyl)amino)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-
1H-pyrazolo[3,4-d]pyrimidin-6-yl)pentan-1-one 12b
[0062] In another aspect, the present invention relates to a method
for preparing the compound of formula (I-E), comprising a step
of:
##STR00040##
[0063] subjecting a compound of formula (I-C) and a compound of
formula (I-D) to a nucleophilic substitution reaction under an
alkaline condition to obtain the compound of formula (I-E);
[0064] wherein:
[0065] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0066] X is halogen, preferably chlorine;
[0067] ring A, G, L.sup.1-L.sup.2, X.sup.1, R.sup.1.about.R.sup.3
and n are as defined in formula (I-E).
[0068] In another aspect, the present invention relates to a method
for preparing the compound of formula (I), comprising a step
of:
##STR00041##
[0069] removing the protecting group of the compound of formula
(I-E) under an acidic condition to obtain the compound of formula
(I);
[0070] wherein:
[0071] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0072] ring A, G, X.sup.1, L.sup.1.about.L.sup.2,
R.sup.1.about.R.sup.3 and n are as defined in formula (I).
[0073] In another aspect, the present invention relates to a
compound of formula (II-B):
##STR00042##
[0074] or a tautomer, mesomer, racemate, enantiomer, diastereomer
thereof, or mixture thereof, or a pharmaceutically acceptable salt
thereof,
[0075] wherein:
[0076] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0077] X is halogen, preferably chlorine;
[0078] G, L.sup.2, R.sup.2, R.sup.6.about.R.sup.7 and n are as
defined in formula (II).
[0079] In another aspect, the present invention relates to a
compound of formula (II-C):
##STR00043##
[0080] or a tautomer, mesomer, racemate, enantiomer, diastereomer
thereof, or mixture thereof, or a pharmaceutically acceptable salt
thereof,
[0081] wherein:
[0082] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0083] G, L.sup.1.about.L.sup.2, R.sup.1.about.R.sup.2,
R.sup.6.about.R.sup.7 and n are as defined in formula (II).
[0084] In another aspect, the present invention relates to a method
for preparing the compound of formula (II-C), comprising a step
of:
##STR00044##
[0085] subjecting a compound of formula (II-B) and a compound of
formula (I-D) to a nucleophilic substitution reaction under an
alkaline condition to obtain the compound of formula (II-C);
[0086] wherein:
[0087] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0088] X is halogen, preferably chlorine;
[0089] G, L.sup.1.about.L.sup.2, R.sup.1.about.R.sup.2,
R.sup.6.about.R.sup.7 and n are as defined in formula (II). In
another aspect, the present invention relates to a method for
preparing the compound of formula (II), comprising a step of:
##STR00045##
[0090] removing the protecting group of the compound of formula
(II-C) under an acidic condition to obtain the compound of formula
(II);
[0091] wherein:
[0092] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0093] G, L.sup.1.about.L.sup.2, R.sup.1.about.R.sup.2,
R.sup.6.about.R.sup.7 and n are as defined in formula (II).
[0094] In another aspect, the present invention relates to a method
for preparing the compound of formula (III), comprising a step
of:
##STR00046##
[0095] removing the protecting group of the compound of formula
(III-C) under an acidic condition to obtain the compound of formula
(III);
[0096] wherein:
[0097] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0098] L.sup.1, R.sup.1.about.R.sup.2, s and n are as defined in
formula (III).
[0099] In another aspect, the present invention relates to a
pharmaceutical composition, comprising a therapeutically effective
amount of the compound of formula (I), or a tautomer, mesomer,
racemate, enantiomer, diastereomer thereof, or mixture thereof, or
a pharmaceutically acceptable salt thereof, and one or more
pharmaceutically acceptable carriers, diluents or excipients.
[0100] The present invention further relates to a use of the
compound of formula (I), or a tautomer, mesomer, racemate,
enantiomer, diastereomer thereof, or mixture thereof, or a
pharmaceutically acceptable salt thereof, or the pharmaceutical
composition comprising the same, in the preparation of a medicament
for activating TLR7.
[0101] The present invention further relates to a use of the
compound of formula (I), or a tautomer, mesomer, racemate,
enantiomer, diastereomer thereof, or mixture thereof, or a
pharmaceutically acceptable salt thereof, or the pharmaceutical
composition comprising the same, in the preparation of a medicament
for treating an infection caused by a virus selected from the group
consisting of dengue virus, yellow fever virus, West Nile virus,
Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin
virus, Murray Valley encephalitis virus, St. Louis encephalitis
virus, Omsk hemorrhagic fever virus, bovine viral disarrhea virus,
Zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza virus.
[0102] The present invention further relates to a use of the
compound of formula (I), or a tautomer, mesomer, racemate,
enantiomer, diastereomer thereof, or mixture thereof, or a
pharmaceutically acceptable salt thereof, or the pharmaceutical
composition comprising the same, in the preparation of a medicament
for treating or preventing melanoma, non-small cell lung carcinoma,
hepatocellular carcinoma, basal cell carcinoma, renal cell
carcinoma, myeloma, allergic rhinitis, asthma, COPD, ulcerative
colitis and hepatic fibrosis.
[0103] The present invention further relates to the compound of
formula (I), or a tautomer, mesomer, racemate, enantiomer,
diastereomer thereof, or mixture thereof, or a pharmaceutically
acceptable salt thereof, or a pharmaceutical composition comprising
the same, for use as a medicament.
[0104] The present invention further relates to the compound of
formula (I), or a tautomer, mesomer, racemate, enantiomer,
diastereomer thereof, or mixture thereof, or a pharmaceutically
acceptable salt thereof, or the pharmaceutical composition
comprising the same, for use in activating TLR7.
[0105] The present invention further relates to the compound of
formula (I), or a tautomer, mesomer, racemate, enantiomer,
diastereomer thereof, or mixture thereof, or a pharmaceutically
acceptable salt thereof, or the pharmaceutical composition
comprising the same, for use in treating an infection caused by a
virus selected from the group consisting of dengue virus, yellow
fever virus, West Nile virus, Japanese encephalitis virus,
tick-borne encephalitis virus, Kunjin virus, Murray Valley
encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic
fever virus, bovine viral disarrhea virus, Zika virus, HIV, HBV,
HCV, HPV, RSV, SARS and influenza virus.
[0106] The present invention further relates to the compound of
formula (I), or a tautomer, mesomer, racemate, enantiomer,
diastereomer thereof, or mixture thereof, or a pharmaceutically
acceptable salt thereof, or the pharmaceutical composition
comprising the same, for use in treating or preventing melanoma,
non-small cell lung carcinoma, hepatocellular carcinoma, basal cell
carcinoma, renal cell carcinoma, myeloma, allergic rhinitis,
asthma, COPD, ulcerative colitis or hepatic fibrosis.
[0107] The present invention further relates to a method for
activating TLR7, comprising administering to a patient in need
thereof a therapeutically effective amount of the compound of
formula (I) of the present invention, or a tautomer, mesomer,
racemate, enantiomer, diastereomer thereof, or mixture thereof, or
a pharmaceutically acceptable salt thereof, or the pharmaceutical
composition comprising the same.
[0108] The present invention further relates to a method for
treating an infection caused by a virus selected from the group
consisting of dengue virus, yellow fever virus, West Nile virus,
Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin
virus, Murray Valley encephalitis virus, St. Louis encephalitis
virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus,
Zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza virus,
comprising administering to a patient in need thereof a
therapeutically effective amount of the compound of formula (I) of
the present invention, or a tautomer, mesomer, racemate,
enantiomer, diastereomer thereof, or mixture thereof, or a
pharmaceutically acceptable salt thereof, or the pharmaceutical
composition comprising the same.
[0109] The present invention further relates to a method for
treating or preventing melanoma, non-small cell lung carcinoma,
hepatocellular carcinoma, basal cell carcinoma, renal cell
carcinoma, myeloma, allergic rhinitis, asthma, COPD, ulcerative
colitis and hepatic fibrosis, comprising administering to a patient
in need thereof a therapeutically effective amount of the compound
of formula (I) of the present invention, or a tautomer, mesomer,
racemate, enantiomer, diastereomer thereof, or mixture thereof, or
a pharmaceutically acceptable salt thereof, or the pharmaceutical
composition comprising the same.
[0110] Pharmaceutical compositions containing the active ingredient
can be in a form suitable for oral administration, for example, a
tablet, troche, lozenge, aqueous or oily suspension, dispersible
powder or granule, emulsion, hard or soft capsule, or syrup or
elixir. Oral compositions can be prepared according to any known
method in the art for the preparation of pharmaceutical
composition. Such composition can contain one or more ingredients
selected from the group consisting of sweetening agents, flavoring
agents, coloring agents and preservatives, in order to provide a
pleasing and palatable pharmaceutical preparation. Tablets contain
the active ingredient in admixture with nontoxic pharmaceutically
acceptable excipients suitable for the manufacture of tablets.
[0111] An aqueous suspension contains the active ingredient in
admixture with excipients suitable for the manufacture of an
aqueous suspension. The aqueous suspension can also contain one or
more preservative such as ethylparaben or n-propylparaben, one or
more coloring agents, one or more flavoring agents, and one or
sweetening agents.
[0112] An oil suspension can be formulated by suspending the active
ingredient in a vegetable oil. The oil suspension can contain a
thickener. The aforementioned sweetening agents and flavoring
agents can be added to provide a palatable formulation.
[0113] The active ingredient in admixture with the dispersants or
wetting agents, suspending agent or one or more preservatives can
be prepared as a dispersible powder or granule suitable for the
preparation of an aqueous suspension by adding water. Suitable
dispersants or wetting agents and suspending agents are exemplified
by those already mentioned above. Additional excipients, such as
sweetening agents, flavoring agents and coloring agents, can also
be added. These compositions can be preserved by adding an
antioxidant such as ascorbic acid.
[0114] The pharmaceutical composition of the present invention can
also be in the form of an oil-in-water emulsion.
[0115] The pharmaceutical composition can be in the form of sterile
injectable aqueous solution. The acceptable vehicles and solvents
that can be employed are water, Ringer's solution and isotonic
sodium chloride solution. The sterile injectable formulation can be
a sterile injectable oil-in-water micro-emulsion in which the
active ingredient is dissolved in the oil phase. For example, the
active ingredient is dissolved in a mixture of soybean oil and
lecithin, the oil solution is then added into a mixture of water
and glycerol and processed to form a micro-emulsion. The injectable
solution or micro-emulsion can be injected into a patient's
bloodstream by local bolus injection. Alternatively, it can be
advantageous to administer the solution and micro-emulsion in such
a way as to maintain a constant circulating concentration of the
compound of the present invention. In order to maintain such a
constant concentration, a continuous intravenous delivery device
can be utilized. An example of such a device is Deltec
CADD-PLUS..TM. 5400 intravenous injection pump.
[0116] The pharmaceutical composition can be in the form of a
sterile injectable aqueous or oily suspension for intramuscular and
subcutaneous administration. Such a suspension can be formulated
with suitable dispersants or wetting agents and suspending agents
as described above according to known techniques. The sterile
injectable formulation can also be a sterile injectable solution or
suspension prepared in a nontoxic parenterally acceptable diluent
or solvent. Moreover, sterile fixed oils can readily be used as a
solvent or suspending medium.
[0117] The compound of the present invention can be administered in
the form of a suppository for rectal administration. These
pharmaceutical compositions can be prepared by mixing the drug with
a suitable non-irritating excipient that is solid at normal
temperature, but liquid in the rectum, thereby melting in the
rectum to release the drug. Such materials include cocoa butter,
glycerin gelatin, hydrogenated vegetable oils, mixtures of
polyethylene glycols with various molecular weights and fatty acid
esters of poly ethylene glycols.
[0118] It is well known to those skilled in the art that the dosage
of a drug depends on a variety of factors including but not limited
to, the following factors: activity of a specific compound, age of
the patient, weight of the patient, general health of the patient,
behavior of the patient, diet of the patient, administration time,
administration route, excretion rate, drug combination and the
like. In addition, the optimal treatment, such as treatment mode,
daily dose of the compound of formula (I) or the type of
pharmaceutically acceptable salt thereof can be verified by
conventional therapeutic regimens.
DETAILED DESCRIPTION OF THE INVENTION
[0119] Unless otherwise stated, the terms used in the specification
and claims have the meanings described below.
[0120] The term "alkyl" refers to a saturated aliphatic hydrocarbon
group, which is a straight or branched chain group comprising 1 to
20 carbon atoms, preferably an alkyl having 1 to 12 carbon atoms.
Non-limiting examples include methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl,
1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl,
1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1
-ethyl-2-methyl propyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl,
1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl,
2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl,
2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl,
4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl,
2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl,
2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl,
2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl,
3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl,
4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl,
n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl,
2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and
various branched isomers thereof. More preferably, an alkyl group
is a lower alkyl having 1 to 6 carbon atoms, and non-limiting
examples include methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl,
1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl,
2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methyl propyl,
1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl,
2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl,
3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like.
The alkyl group can be substituted or unsubstituted. When
substituted, the substituent group(s) can be substituted at any
available connection point. The substituent group(s) is preferably
one or more groups independently optionally selected from the group
consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl,
heterocyclyl, aryl, heteroaryl, cycloalkoxy, heteroalkoxy,
cycloalkylthio, heterocyclylthio, oxo, --OR.sup.5, --C(O)R.sup.5,
--S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7.
[0121] The term "alkylene" refers to a saturated linear or branched
aliphatic hydrocarbon group having two residues derived from the
removal of two hydrogen atoms from the same carbon atom or two
different carbon atoms of the parent alkane. The linear or branched
alkylene has 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms,
and more preferably 1 to 6 carbon atoms. Non-limiting examples of
alkylene groups include, but are not limited to, methylene
(--CH.sub.2--), 1,1-ethylene(--CH(CH.sub.3)--), 1,2-ethylene
(--CH.sub.2CH.sub.2)--, 1,1-propylene(--CH(CH.sub.2CH.sub.3)--),
1,2-propylene(--CH.sub.2CH(CH.sub.3)--),
1,3-propylene(--CH.sub.2CH.sub.2CH.sub.2--),
1,4-butylene(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and the like.
The alkylene group can be substituted or unsubstituted. When
substituted, the substituent group(s) can be substituted at any
available connection point. The substituent group(s) is preferably
one or more groups independently optionally selected from the group
consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl,
heterocyclyl, aryl, heteroaryl, cycloalkoxy, heteroalkoxy,
cycloalkylthio, heterocyclylthio, oxo, --OR.sup.5, --C(O)R.sup.5,
--S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7.
[0122] The term "alkenyl" refers to a hydrocarbon group formed by
the removal of one or more hydrogen atoms in an olefin molecule.
The alkenyl group can be substituted or unsubstituted. When
substituted, the substituent group(s) is preferably one or more
groups independently selected from the group consisting of
hydrogen, alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl,
cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl,
--OR.sup.5, --C(O)R.sup.5, --S(O).sub.mR.sup.5, --NR.sup.6R.sup.7
and --C(O)NR.sup.6R.sup.7.
[0123] The term "alkynyl" refers to a hydrocarbon group containing
a carbon-carbon triple bond in the molecule. The alkynyl group can
be substituted or unsubstituted. When substituted, the substituent
group(s) is preferably one or more groups independently selected
from the group consisting of hydrogen, alkyl, alkoxy, halogen,
haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl,
heterocyclyl, aryl, heteroaryl, --OR.sup.5, --C(O)R.sup.5,
--S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7.
[0124] The term "cycloalkyl" refers to a saturated or partially
unsaturated monocyclic or polycyclic hydrocarbon group having 3 to
20 carbon atoms, preferably 3 to 12 carbon atoms, preferably 3 to
10 carbon atoms, and more preferably 3 to 6 carbon atoms.
Non-limiting examples of monocyclic cycloalkyl include cyclopropyl,
cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,
cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and
the like. Polycyclic cycloalkyl includes a cycloalkyl having a
spiro ring, fused ring or bridged ring.
[0125] The term "amino protecting group" refers to a group which
prevents an amino group from reaction when other parts of the
molecular are subject to a reaction, and can be easily removed.
Non-limiting examples include tert-butoxycarbonyl, acetyl, benzyl,
allyl and p-methoxybenzyl and the like. These groups can be
optionally substituted by one to three substituent groups selected
from the group consisting of halogen, alkoxy and nitro. The amino
protecting group is preferably p-methoxybenzyl.
[0126] The term "heterocyclyl" refers to a 3 to 20 membered
saturated or partially unsaturated monocyclic or polycyclic
hydrocarbon substituent group, wherein one or more ring atoms are
heteroatoms selected from the group consisting of N, O, and
S(O).sub.m(wherein m is an integer of 0 to 2), but excluding
--O--O--, --O--S-- or --S--S-- in the ring, with the remaining ring
atoms being carbon atoms. Preferably, the heterocyclyl has 3 to 12
ring atoms wherein 1 to 4 atoms are heteroatoms, more preferably 3
to 10 ring atoms wherein 1 to 4 atoms are heteroatoms, and more
preferably 5 to 6 ring atoms wherein 1 to 3 atoms are heteroatoms.
Non-limiting examples of monocyclic heterocyclyl include
pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridyl,
piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl,
homopiperazinyl and the like. Polycyclic heterocyclyl includes a
heterocyclyl having a spiro ring, fused ring or bridged ring.
[0127] The ring of heterocyclyl can be fused to the ring of aryl,
heteroaryl or cycloalkyl, wherein the ring bound to the parent
structure is heterocyclyl. Non-limiting examples include:
##STR00047##
[0128] The heterocyclyl can be optionally substituted or
unsubstituted. When substituted, the substituent group(s) is
preferably one or more group(s) independently optionally selected
from the group consisting of alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano,
cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy,
heteroalkoxy, cycloalkylthio, heterocyclylthio, oxo, --OR.sup.5,
--C(O)R.sup.5, --S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7.
[0129] The term "aryl" refers to a 6 to 14 membered all-carbon
monocyclic ring or polycyclic fused ring (i.e. each ring in the
system shares an adjacent pair of carbon atoms with another ring in
the system) having a conjugated n-electron system, preferably 6 to
10 membered aryl, for example, phenyl and naphthyl. The ring of
aryl can be fused to the ring of heteroaryl, heterocyclyl or
cycloalkyl, wherein the ring bound to the parent structure is aryl
ring. Non-limiting examples include:
##STR00048##
[0130] The aryl can be substituted or unsubstituted. When
substituted, the substituent group(s) is preferably one or more
group(s) independently optionally selected from the group
consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl,
heterocyclyl, aryl, heteroaryl, cycloalkoxy, heteroalkoxy,
cycloalkylthio, heterocyclylthio, --OR.sup.5, --C(O)R.sup.5,
--S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7.
[0131] The term "heteroaryl" refers to a 5 to 14 membered
heteroaromatic system having 1 to 4 heteroatoms selected from the
group consisting of O, S and N. The heteroaryl is preferably 5 to
10 membered heteroaryl, more preferably 5 or 6 membered heteroaryl,
for example, furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl,
pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl,
tetrazolyl, and the like. The ring of heteroaryl can be fused to
the ring of aryl, heterocyclyl or cycloalkyl, wherein the ring
bound to the parent structure is heteroaryl ring. Non-limiting
examples include:
##STR00049##
[0132] The heteroaryl can be optionally substituted or
unsubstituted. When substituted, the substituent group(s) is
preferably one or more group(s) independently selected from the
group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl,
heterocyclyl, aryl, heteroaryl, cycloalkoxy, heteroalkoxy,
cycloalkylthio, heterocyclylthio, --OR.sup.5, --C(O)R.sup.5,
--S(O).sub.mR.sup.5, --NR.sup.6R.sup.7 and
--C(O)NR.sup.6R.sup.7.
[0133] The term "alkoxy" refers to an --O-(alkyl) or an
--O-(unsubstituted cycloalkyl) group, wherein the alkyl is as
defined above. Non-limiting examples of alkoxy include methoxy,
ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy,
cyclopentyloxy, cyclohexyloxy. The alkoxy can be optionally
substituted or unsubstituted. When substituted, the substituent
group(s) is preferably one or more group(s) independently selected
from the group consisting of halogen, alkyl, alkoxy, haloalkyl,
hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl,
heterocyclyl, aryl and heteroaryl.
[0134] The term "haloalkyl" refers to an alkyl group substituted by
one or more halogens, wherein the alkyl is as defined above. The
term "hydroxy" refers to an --OH group.
[0135] The term "hydroxyalkyl" refers to an alkyl group substituted
by hydroxy(s), wherein the alkyl is as defined above.
[0136] The term "halogen" refers to fluorine, chlorine, bromine or
iodine.
[0137] The term "amino" refers to a --NH.sub.2 group.
[0138] The term "cyano" refers to a --CN group.
[0139] The term "nitro" refers to a --NO.sub.2 group.
[0140] The term "oxo" refers to an .dbd.O group.
[0141] "Optional" or "optionally" means that the event or
circumstance described subsequently can, but need not, occur, and
such a description includes the situation in which the event or
circumstance does or does not occur. For example, "the heterocyclyl
optionally substituted by an alkyl" means that an alkyl group can
be, but need not be, present, and such a description includes the
situation of the heterocyclyl being substituted by an alkyl and the
heterocyclyl being not substituted by an alkyl.
[0142] "Substituted" refers to one or more hydrogen atoms in a
group, preferably up to 5, more preferably 1 to 3 hydrogen atoms,
independently substituted by a corresponding number of
substituents. It goes without saying that the substituents only
exist in their possible chemical position. The person skilled in
the art is able to determine whether the substitution is possible
or impossible by experiments or theory without paying excessive
efforts. For example, the combination of amino or hydroxy having
free hydrogen and carbon atoms having unsaturated bonds (such as
olefinic) may be unstable.
[0143] A "pharmaceutical composition" refers to a mixture of one or
more of the compounds described herein or
physiologically/pharmaceutically acceptable salts or prodrugs
thereof with other chemical components, and other components such
as physiologically/pharmaceutically acceptable carriers and
excipients. The purpose of the pharmaceutical composition is to
facilitate administration of a compound to an organism, which is
conducive to the absorption of the active ingredient so as to show
biological activity.
[0144] A "pharmaceutically acceptable salt" refers to a salt of the
compound of the present invention, which is safe and effective in
mammals and has the desired biological activity.
[0145] m and R.sup.5 to R.sup.7 are as defined in the compound of
formula (I).
[0146] Synthesis Method of the Compound of the Present
Invention
[0147] In order to achieve the object of the present invention, the
present invention employs the following technical solutions:
##STR00050##
[0148] in the first step, a compound of formula (I-A) and a
compound of formula (I-B) are subjected to a nucleophilic
substitution reaction under an alkaline condition to obtain a
compound of formula (I-C);
[0149] in the second step, the compound of formula (I-C) and a
compound of formula (I-D) are subjected to a nucleophilic
substitution reaction under an alkaline condition to obtain a
compound of formula (I-E);
[0150] in the third step, the protecting group of the compound of
formula (I-E) is removed under an acidic condition to obtain the
compound of formula (I);
[0151] wherein:
[0152] M is hydrogen or a metal ion, wherein the metal ion is
preferably sodium ion;
[0153] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0154] X is halogen, preferably chlorine;
[0155] ring A, G, L.sup.1.about.L.sup.2, R.sup.1.about.R.sup.3 and
n are as defined in formula (I).
[0156] The reagent that provides an alkaline condition includes
organic bases and inorganic bases. The organic bases include, but
are not limited to, triethylamine, N,N-diisopropylethylamine,
n-butyllithium, lithium diisopropylamide, lithium
bis(trimethylsilyl)amine, potassium acetate, sodium tert-butoxide,
potassium tert-butoxide and sodium n-butoxide. The inorganic bases
include, but are not limited to, sodium hydride, potassium
phosphate, sodium carbonate, potassium carbonate, potassium
acetate, cesium carbonate, sodium hydroxide and lithium
hydroxide.
[0157] The reagent that provides an acidic condition includes, but
is not limited to, hydrogen chloride, a solution of hydrogen
chloride in 1,4-dioxane, trifluoroacetic acid, formic acid, acetic
acid, hydrochloric acid, sulfuric acid, methanesulfonic acid,
nitric acid, phosphoric acid, p-toluenesulfonic acid, Me.sub.3SiCl,
and TMSOTf.
[0158] The above reactions are preferably carried out in a solvent.
The solvent used includes, but is not limited to, acetic acid,
methanol, ethanol, n-butanol, toluene, tetrahydrofuran,
dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl
sulfoxide, 1,4-dioxane, water and N,N-dimethylformamide.
##STR00051##
[0159] in the first step, a compound of formula (I-A) and a
compound of formula (II-A) are subjected to a nucleophilic
substitution reaction under an alkaline condition to obtain a
compound of formula (II-B);
[0160] in the second step, the compound of formula (II-B) and a
compound of formula (I-D) are subjected to a nucleophilic
substitution reaction under an alkaline condition to obtain a
compound of formula (II-C);
[0161] in the third step, the protecting group of the compound of
formula (II-C) is removed under an acidic condition to obtain the
compound of formula (II);
[0162] wherein:
[0163] M is hydrogen or a metal ion, wherein the metal ion is
preferably sodium ion;
[0164] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0165] X is halogen, preferably chlorine;
[0166] G, L.sup.1.about.L.sup.2, R.sup.6.about.R.sup.7 and n are as
defined in formula (II).
[0167] The reagent that provides an alkaline condition includes
organic bases and inorganic bases. The organic bases include, but
are not limited to, triethylamine, N,N-diisopropylethylamine,
n-butyllithium, lithium diisopropylamide, lithium
bis(trimethylsilyl)amine, potassium acetate, sodium tert-butoxide,
potassium tert-butoxide and sodium n-butoxide. The inorganic bases
include, but are not limited to, sodium hydride, potassium
phosphate, sodium carbonate, potassium carbonate, potassium
acetate, cesium carbonate, sodium hydroxide and lithium
hydroxide.
[0168] The reagent that provides an acidic condition includes, but
is not limited to, hydrogen chloride, a solution of hydrogen
chloride in 1,4-dioxane, trifluoroacetic acid, formic acid, acetic
acid, hydrochloric acid, sulfuric acid, methanesulfonic acid,
nitric acid, phosphoric acid, p-toluenesulfonic acid, Me.sub.3SiCl,
and TMSOTf.
[0169] The above reactions are preferably carried out in a solvent.
The solvent used includes, but is not limited to, acetic acid,
methanol, ethanol, n-butanol, toluene, tetrahydrofuran,
dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl
sulfoxide, 1,4-dioxane, water and N,N-dimethylformamide.
##STR00052##
[0170] in the first step, a compound of formula (I-A) and a
compound of formula (III-A) are subjected to a nucleophilic
substitution reaction under an alkaline condition to obtain a
compound of formula (III-B);
[0171] in the second step, the compound of formula (III-B) and a
compound of formula (I-D) are subjected to a nucleophilic
substitution reaction under an alkaline condition to obtain a
compound of formula (III-C);
[0172] in the third step, the protecting group of the compound of
formula (III-C) is removed under an acidic condition to obtain the
compound of formula (III);
[0173] wherein:
[0174] M is hydrogen or a metal ion, wherein the metal ion is
preferably sodium ion;
[0175] W is an amino protecting group, preferably
tert-butoxycarbonyl, acetyl, benzyl, allyl or p-methoxybenzyl;
[0176] X is halogen, preferably chlorine;
[0177] L.sup.1, R.sup.1.about.R.sup.2, s and n are as defined in
formula (III).
[0178] The reagent that provides an alkaline condition includes
organic bases and inorganic bases. The organic bases include, but
are not limited to, triethylamine, N,N-diisopropylethylamine,
n-butyllithium, lithium diisopropylamide, lithium
bis(trimethylsilyl)amine, potassium acetate, sodium tert-butoxide,
potassium tert-butoxide and sodium n-butoxide. The inorganic bases
include, but are not limited to, sodium hydride, potassium
phosphate, sodium carbonate, potassium carbonate, potassium
acetate, cesium carbonate, sodium hydroxide and lithium
hydroxide.
[0179] The reagent that provides an acidic condition includes, but
is not limited to, hydrogen chloride, a solution of hydrogen
chloride in 1,4-dioxane, trifluoroacetic acid, formic acid, acetic
acid, hydrochloric acid, sulfuric acid, methanesulfonic acid,
nitric acid, phosphoric acid, p-toluenesulfonic acid, Me.sub.3SiCl,
and TMSOTf.
[0180] The above reactions are preferably carried out in a solvent.
The solvent used includes, but is not limited to, acetic acid,
methanol, ethanol, n-butanol, toluene, tetrahydrofuran,
dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl
sulfoxide, 1,4-dioxane, water and N,N-dimethylformamide.
Preferred Embodiments
[0181] The present invention will be further described with
reference to the following examples, but the examples should not be
considered as limiting the scope of the present invention.
EXAMPLES
[0182] The structures of the compounds were identified by nuclear
magnetic resonance (NMR) and/or mass spectrometry (MS). NMR shifts
(6) are given in 10.sup.-6 (ppm). NMR was determined by a Bruker
AVANCE-400 machine. The solvents for determination were
deuterated-dimethyl sulfoxide (DMSO-d.sub.6), deuterated-chloroform
(CDCl.sub.3) and deuterated-methanol (CD.sub.3OD), and the internal
standard was tetramethylsilane (TMS).
[0183] MS was determined by a FINNIGAN LCQAd (ESI) mass
spectrometer (manufacturer: Thermo, type: Finnigan LCQ advantage
MAX).
[0184] High performance liquid chromatography (HPLC) was determined
on Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC
e2695-2489 high pressure liquid chromatographs.
[0185] Chiral HPLC was determined on an Agilent HPLC 1260 DAD high
performance liquid chromatography.
[0186] High performance liquid preparation was carried out on
Waters 2767, Waters 2767--SQ Detecor2, Shimadzu LC-20AP and
Gilson-281 preparative chromatographs.
[0187] Chiral preparation was carried out on a Shimadzu LC-20AP
preparative chromatography.
[0188] CombiFlash rapid preparation instrument used was Combiflash
Rf200 (TELEDYNE ISCO).
[0189] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate
was used as the thin-layer silica gel chromatography (TLC) plate.
The dimension of the silica gel plate used in TLC was 0.15 mm to
0.2 mm, and the dimension of the silica gel plate used in product
purification was 0.4 mm to 0.5 mm.
[0190] Yantai Huanghai 200 to 300 mesh silica gel was generally
used as a carrier for column chromatography.
[0191] The average kinase inhibition rates and IC.sub.50 values
were determined by a NovoStar ELISA (BMG Co., Germany).
[0192] The known starting materials of the present invention can be
prepared by the known methods in the art, or can be purchased from
ABCR GmbH & Co. KG, Acros Organnics, Aldrich Chemical Company,
Accela ChemBio Inc., or Dari chemical Company, etc.
[0193] Unless otherwise stated, the reactions were carried out
under an argon atmosphere or nitrogen atmosphere.
[0194] Argon atmosphere or nitrogen atmosphere means that a
reaction flask is equipped with an argon or nitrogen balloon (about
1 L).
[0195] Hydrogen atmosphere means that a reaction flask is equipped
with a hydrogen balloon (about 1 L).
[0196] Pressurized hydrogenation reactions were performed on a Parr
3916EKX hydrogenation instrument and a Qinglan QL-500 hydrogen
generator or HC2--SS hydrogenation instrument.
[0197] In hydrogenation reactions, the reaction system was
generally vacuumed and filled with hydrogen, with the above
operation was repeated three times.
[0198] CEM Discover-S 908860 type microwave reactor was used in
microwave reactions.
[0199] Unless otherwise stated, the solution refers to an aqueous
solution.
[0200] Unless otherwise stated, the reaction temperature is room
temperature from 20.degree. C. to 30.degree. C.
[0201] The reaction process in the examples was monitored by thin
layer chromatography (TLC). The developing solvent used in the
reactions, the elution system in column chromatography and the
developing solvent system in thin layer chromatography for
purification of the compounds included: A: dichloromethane/methanol
system, and B: n-hexane/ethyl acetate system. The ratio of the
volume of the solvent was adjusted according to the polarity of the
compounds, and a small quantity of alkaline reagent such as
triethylamine or acidic reagent such as acetic acid can also be
added for adjustment.
Example 1
6-Butoxy-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-
-amine
##STR00053## ##STR00054##
[0202] Step 1
6-Chloro-N-(4-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
1c
[0203] 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine la (120 mg, 0.63
mmol), 4-methoxybenzylamine 1b (87.1 mg, 0.63 mmol) and
triethylamine (64.13 mg, 0.63 mmol) were dissolved in 2 mL of
tetrahydrofuran, and the reaction solution was stirred at room
temperature for 1 hour. The reaction was stopped, and the reaction
solution was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography with elution system A
to obtain the title compound 1c (140 mg, yield: 76.1%).
[0204] MS m/z (ESI): 290.2 [M+1]
Step 2
6-Chloro-N-(4-methoxybenzyl)-1
-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazol[3,4-d
]pyrimidin-4-amine 1e
[0205] Compound 1c (140 mg, 0.48 mmol),
1-(4-(chloromethyl)benzyl)pyrrolidine 1d (101.34 mg, 0.48 mmol,
prepared according to the method disclosed in the patent
application "WO2002012224") and potassium carbonate (66.79 mg, 0.48
mmol) were dissolved in 2 mL of N,N-dimethylformamide. The reaction
was stopped after stirring at room temperature for 16 hours. The
reaction solution was concentrated under reduced pressure, and the
residue was purified by silica gel column chromatography with
elution system A to obtain the title compound 1e (70 mg, yield:
31.3%).
[0206] MS m/z (ESI): 463.2 [M+1]
Step 3
6-Butoxy
--N-(4-methoxybenzyl)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyra-
zolo[3,4-d ]pyrimidin-4-amine 1f
[0207] Compound 1e (70 mg, 0.15 mmol), sodium n-butoxide (0.3 mL,
0.60 mmol) and 1 mL of n-butanol were added to a microwave tube
successively, heated to 160.degree. C. and stirred for 1.5 hours.
The reaction was stopped, and the reaction solution was
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography with elution system A to obtain
the title compound 1f (40 mg, yield: 52.8%).
[0208] MS m/z (ESI): 501.2 [M+1]
Step 4
6-Butoxy-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-
-amine 1
[0209] Compound 1f (40 mg, 0.08 mmol) and 2 mL of trifluoroacetic
acid were added to a reaction flask, heated to reflux, and stirred
for 24 hours. The reaction was stopped, and the reaction solution
was concentrated under reduced pressure and added with 1 mL of
ammonia in methanol. The residue was purified by thin layer
chromatography with developing solvent system A to obtain the title
compound 1 (15 mg, yield: 46.0%).
[0210] MS m/z (ESI): 381.2 [M+1]
[0211] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.98 (s, 1H), 7.41
(d, 2H), 7.36 (d, 2H), 5.48 (s, 2H), 4.39 (t, 2H), 4.13 (s, 2H),
3.12-3.08 (m, 4H), 2.02-1.98 (m, 4H), 1.80-1.76 (m, 2H), 1.55-1.49
(m, 2H), 1.01 (t, 3H).
Example 2
1-(4-(Azetidin-1-ylmethyl)benzyl)-6-butoxy-1H-pyrazolo[3,4-d]pyrimidin-4-a-
mine2
##STR00055## ##STR00056##
[0212] Step 1
Methyl 4-(azetidin-1-ylmethyl)benzoate 2c
[0213] Methyl 4-(bromomethyl)benzoate 2a (1.0 g, 4.37mmo1),
azetidine 2b (299 mg, 5.24 mmol) and triethylamine (529 mg, 5.24
mmol) were dissolved in 10 mL of tetrahydrofuran, and the reaction
solution was stirred at room temperature for 16 hours. The reaction
solution was added with water (100 mL), and extracted with ethyl
acetate (100 mL). The organic phase was washed with saturated
sodium chloride solution (100 mL), dried over anhydrous sodium
sulfate and filtered. The filtrate was concentrated under reduced
pressure to obtain the crude title compound 2c (880 mg), which was
used directly in the next step without purification.
[0214] MS m/z (ESI): 206.1 [M+1]
Step 2
4-(Azetidin-1-ylmethyl)phenylcarbinol 2d
[0215] The crude compound 2c (880 mg, 0.33 mmol) was dissolved in
10 mL of diethyl ether, lithium aluminum hydride (326 mg, 8.57
mmol) was added at 0.degree. C., and the reaction solution was
stirred at 0.degree. C. for 2 hours. Then 0.3 mL of water, 0.3 mL
of 15% sodium hydroxide solution and 0.9 mL of water were added
successively to quench the reaction. The reaction solution was
filtered, and the filtrate was concentrated under reduced pressure
to obtain the crude title compound 2d (700 mg), which was used
directly in the next step without purification.
[0216] MS m/z (ESI): 178.3 [M+1]
Step 3
1-(4-(Chloromethyl)benzyl)azetidine 2e
[0217] The crude compound 2d (700 mg, 3.95 mmol) was dissolved in
10 mL of dichloromethane, thionyl chloride (0.58 mL, 7.90 mmol) was
added at 0.degree. C., and the reaction solution was stirred at
room temperature for 3 hours. The reaction solution was
concentrated under reduced pressure, and the resulting residue was
added with saturated sodium carbonate solution (50 mL), and
extracted with dichloromethane (100 mL.times.2). The organic phases
were combined, dried over anhydrous sodium sulfate and filtered.
The filtrate was concentrated under reduced pressure to obtain the
crude title compound 2e (720 mg), which was used directly in the
next step without purification.
[0218] MS m/z (ESI): 197.2 [M+1]
Step 4
1-(4-(Azetidin-1-ylmethyl)benzyl)-6-chloro-N-(4-methoxybenzyl)-1H-pyrazolo-
[3,4-d]pyrimidin-4-amine 2f
[0219] Compound 1c (600 mg, 2.07 mmol), the crude compound 2e (405
mg, 2.07 mmol) and potassium carbonate (286 mg, 2.07 mmol) were
dissolved in 10 mL of N,N-dimethylformamide, and the reaction
solution was stirred at room temperature for 16 hours. The reaction
solution was concentrated under reduced pressure, and the resulting
residue was purified by silica gel column chromatography with
elution system A to obtain the title compound 2f (300 mg, yield:
32.3%).
[0220] MS m/z (ESI): 449.2 [M+1]
Step 5
1-(4-(Azetidin-1-ylmethyl)benzyl)-6-butoxy-N-(4-methoxybenzyl)-1H-pyrazolo-
[3,4-d]pyrimidin-4-amine 2g
[0221] Compound 2f (150 mg, 0.33 mmol), sodium n-butoxide (0.7 mL,
1.40 mmol) and 2 mL of n-butanol were added to a microwave tube
successively, heated to 160.degree. C. and stirred for 1.5 hours.
The reaction solution was cooled to room temperature, and
concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography with elution system A
to obtain the title compound 2g (60 mg, yield: 36.9%).
[0222] MS m/z (ESI): 487.3 [M+1]
Step 6
1-(4-(Azetidin-1-ylmethyl)benzyl)-6-butoxy-1H-pyrazolo[3,4-d]pyrimidin-4-a-
mine 2
[0223] Compound 2g (60 mg, 0.12 mmol) and 2 mL of trifluoroacetic
acid were added to a reaction flask, heated to reflux, and stirred
for 24 hours. The reaction solution was cooled to room temperature,
and concentrated under reduced pressure. The reaction mixture was
added with a solution of 7 N ammonia in methanol (1 mL), and
concentrated under reduced pressure. The resulting residue was
purified by high performance liquid chromatography (Waters-2767,
elution system: 10 mmoL/L ammonium bicarbonate, water,
acetonitrile) to obtain the title compound 2 (15 mg, yield:
33.2%).
[0224] MS m/z (ESI): 367.2 [M+1]
[0225] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.97 (s, 1H),
7.34-7.26 (m, 4H), 5.44 (s, 2H), 4.39 (t, 2H), 3.77 (s, 2H), 3.47
(t, 4H), 2.22-2.18 (m, 2H), 1.80-1.76 (m, 2H), 1.55-1.49 (m, 2H),
1.01 (t, 3H).
Example 3
6-Butoxy-1-(4-(piperidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4--
amine 3
##STR00057## ##STR00058##
[0226] Step 1
1-(4-(Chloromethyl)benzyl)piperidine 3b
[0227] 4-(Piperidin-1-ylmethyl)phenylcarbinol 3a (1.17 g, 5.70
mmol, prepared according to the known method disclosed in "Journal
of Medicinal Chemistry, 2003, 46(8), 1523-1530") was dissolved in
20 mL of dichloromethane, thionyl chloride (0.83 mL, 11.4mmol) was
added at 0.degree. C., and the reaction solution was stirred at
room temperature for 3 hours. The reaction solution was warmed up
to room temperature, and concentrated under reduced pressure. The
reaction mixture was added with saturated sodium carbonate solution
(50 mL), and extracted with dichloromethane (100 mL.times.2). The
organic phases were combined, dried over anhydrous sodium sulfate
and filtered. The filtrate was concentrated under reduced pressure
to obtain the crude title compound 3b (1.2 g), which was used
directly in the next step without purification.
[0228] MS m/z (ESI): 224.2 [M+1]
Step 2
6-Chloro-N-(4-methoxy
benzyl)-1-(4-(piperidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4--
amine 3c
[0229] Compound 1c (1.5 g, 5.18 mmol), the crude compound 3b (1.16
g, 5.18 mmol) and potassium carbonate (716 mg, 5.18 mmol) were
dissolved in 20 mL of N,N-dimethylformamide, and the reaction
solution was stirred at room temperature for 16 hours. The reaction
solution was concentrated under reduced pressure, and the resulting
residue was purified by silica gel column chromatography with
elution system A to obtain the title compound 3c (400 mg, yield:
16.2%).
[0230] MS m/z (ESI): 477.3 [M+1]
Step 3
6-Butoxy-N-(4-methoxyb
enzyl)-1-(4-(piperidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-a-
mine 3d
[0231] Compound 3c (100 mg, 0.21 mmol), sodium n-butoxide (0.2 mL,
0.80 mmol) and 1 mL of n-butanol were added to a microwave tube
successively, heated to 160.degree. C. and stirred for 1.5 hours.
The reaction solution was cooled to room temperature, and
concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography with elution system A
to obtain the title compound 3d (50 mg, yield: 46.3%).
[0232] MS m/z (ESI): 515.3 [M+1]
Step 4
6-Butoxy
-1-(4-(piperidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-
-amine 3
[0233] Compound 3d (60 mg, 0.12 mmol) and 2 mL of trifluoroacetic
acid were added to a reaction flask. The reaction solution was
heated to reflux, and stirred for 24 hours. The reaction solution
was cooled to room temperature, and concentrated under reduced
pressure. The reaction mixture was added with a solution of 7 N
ammonia in methanol (1 mL), and concentrated under reduced
pressure. The resulting residue was purified by thin layer
chromatography with developing system A to obtain the title
compound 3 (20 mg, yield: 49.5%).
[0234] MS m/z (ESI): 395.3 [M+1]
[0235] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.99 (s, 1H),
7.47-7.38 (m, 4H), 5.49 (s, 2H), 4.39 (t, 2H), 4.18 (s, 2H),
3.09-3.00 (m, 4H), 1.81-1.76 (m, 6H), 1.68-1.62 (m, 2H), 1.55-1.49
(m, 2H), 1.00 (t, 3H).
Example 4
[0236] 6-Butoxy-1-(3
-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
4
##STR00059##
Step 1
6-Chloro-N-(3-methoxybenzyl)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazo-
lo[3,4-d 1pyrimidin-4-amine 4b
[0237] Compound 1c (1.0g, 3.45 mmol),
1-(3-(chloromethyl)benzyl)pyrrolidine 4a (724 mg, 3.45 mmol,
prepared according to the method disclosed in the patent
application "W02016040419") and potassium carbonate (377 mg, 3.45
mmol) were dissolved in 10 mL of N,N-dimethylformamide. The
reaction was stopped after stirring at room temperature for 16
hours. The reaction solution was concentrated under reduced
pressure, and the resulting residue was purified by silica gel
column chromatography with elution system A to obtain the title
compound 4b (300 mg, yield: 18.7%).
[0238] MS m/z (ESI): 463.2 [M+1]
Step 2
6-Butoxy-N-(3-methoxybenzyl)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazo-
lo[3,4-d ]pyrimidin-4-amine 4c
[0239] Compound 4b (300 mg, 0.65 mmol), sodium n-butoxide (1.3 mL,
2.60 mmol) and 2 mL of n-butanol were added to a microwave tube
successively, heated to 160.degree. C. and stirred for 1.5 hours.
The reaction solution was cooled to room temperature, and
concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography with elution system A
to obtain the title compound 4c (140 mg, yield: 43.1%).
[0240] MS m/z (ESI): 501.2 [M+1]
Step 3
6-Butoxy-1-(3 -(pyrroli
din-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 4
[0241] Compound 4c (140 mg, 0.08 mmol) and 2 mL of trifluoroacetic
acid were added to a reaction flask, heated to reflux, and stirred
for 24 hours. The reaction solution was cooled to room temperature,
and concentrated under reduced pressure. The reaction mixture was
added with a solution of 7 N ammonia in methanol (1 mL), and
concentrated under reduced pressure. The resulting residue was
purified by high performance liquid chromatography (Waters-2767,
elution system: 10 mmoL/L ammonium bicarbonate, water,
acetonitrile) to obtain the title compound 4 (60 mg, yield:
56.3%).
[0242] MS m/z (ESI): 381.2 [M+1]
[0243] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.98 (s, 1H),
7.35-725 (m, 4H), 5.47 (s, 2H), 4.39 (t, 2H), 3.81 (s, 2H),
2.76-2.70 (m, 4H), 1.98-1.93 (m, 4H), 1.79-1.76 (m, 2H), 1.55-1.50
(m, 2H), 1.01 (t, 3H).
Example 5
1-(3-(Azetidin-1-ylmethyl)benzyl)-6-butoxy-1H-pyrazolo[3,4-d]pyrimidin-4-a-
mine 5
##STR00060## ##STR00061##
[0244] Step 1
Methyl 3-(azetidin-1-ylmethyl)benzoate 5b
[0245] Methyl 3-(bromomethyl)benzoate 5a (1.0 g, 4.37mmo1),
azetidine 2b (299 mg, 5.24 mmol) and triethylamine (529 mg, 5.24
mmol) were dissolved in 10 mL of tetrahydrofuran, and the reaction
solution was stirred at room temperature for 16 hours. The reaction
solution was added with water (100 mL), and extracted with ethyl
acetate (100 mL). The organic phase was washed with saturated
sodium chloride solution (100 mL), dried over anhydrous sodium
sulfate and filtered. The filtrate was concentrated under reduced
pressure to obtain the crude title compound 5b (840 mg), which was
used directly in the next step without purification.
[0246] MS m/z (ESI): 206.1 [M+1]
Step 2
3-(Azetidin-1-ylmethyl)phenylcarbinol 5c
[0247] The crude compound 5b (840 mg, 4.09 mmol) was dissolved in
10 mL of diethyl ether, lithium aluminum hydride (310 mg, 8.19
mmol) was added at 0.degree. C., and the reaction solution was
stirred at 0.degree. C. for 2 hours. Then 0.3 mL of water, 0.3 mL
of 15% sodium hydroxide solution and 0.9 mL of water were added
successively to quench the reaction. The reaction solution was
filtered, and the filtrate was concentrated under reduced pressure
to obtain the crude title compound 5c (700 mg), which was used
directly in the next step without purification.
[0248] MS m/z (ESI): 178.3 [M+1]
Step 3
1-(3-(Chloromethyl)benzyl)azetidine 5d
[0249] The crude compound 5c (700 mg, 3.95 mmol) was dissolved in
10 mL of dichloromethane, thionyl chloride (0.58 mL, 7.90 mmol) was
added at 0.degree. C., and the reaction solution was stirred at
room temperature for 3 hours. The reaction solution was
concentrated under reduced pressure, added with saturated sodium
carbonate solution (50 mL), and extracted with dichloromethane (100
mL.times.2). The organic phases were combined, dried over anhydrous
sodium sulfate and filtered. The filtrate was concentrated under
reduced pressure to obtain the crude title compound 5d (700 mg),
which was used directly in the next step without purification.
[0250] MS m/z (ESI): 197.2 [M+1]
Step 4
1-(3-(Azetidin-1-ylmethyl)benzyl)-6-chloro-N-(4-methoxybenzyl)-1H-pyrazolo-
[3,4-d]pyrimidin-4-amine 5e
[0251] Compound 1c (300 mg, 1.04 mmol), the crude compound 5d (203
mg, 1.04 mmol) and potassium carbonate (144 mg, 1.04 mmol) were
dissolved in 5 mL of N,N-dimethylformamide, and the reaction
solution was stirred at room temperature for 16 hours. The reaction
solution was concentrated under reduced pressure, and the resulting
residue was purified by silica gel column chromatography with
elution system A to obtain the title compound 5e (30 mg, yield:
6.5%).
[0252] MS m/z (ESI): 449.2 [M+1]
Step 5
1-(3-(Azetidin-1-ylmethyl)benzyl)-6-butoxy-N-(4-methoxybenzyl)-1H-pyrazolo-
[3,4-d]pyrimidin-4-amine 5f
[0253] Compound 5e (50 mg, 0.11 mmol), sodium n-butoxide (0.2 mL,
0.40 mmol) and 1 mL of n-butanol were added to a microwave tube
successively, heated to 160.degree. C. and stirred for 1.5 hours.
The reaction solution was cooled to room temperature, and
concentrated under reduced pressure. The resulting residue was
purified by thin layer chromatography with developing system A to
obtain the title compound 5f (35 mg, yield: 64.8%).
[0254] MS m/z (ESI): 487.3 [M+1]
Step 6
1-(3-(Azetidin-1-ylmethyl)benzyl)-6-butoxy-1H-pyrazolo[3,4-d]pyrimidin-4-a-
mine 5
[0255] Compound 5f (35 mg, 0.07 mmol) and 1 mL of trifluoroacetic
acid were added to a reaction flask. The reaction solution was
heated to reflux, and stirred for 24 hours. The reaction solution
was cooled to room temperature, and concentrated under reduced
pressure. The reaction mixture was added with a solution of 7 N
ammonia in methanol (1 mL), and concentrated under reduced
pressure. The resulting residue was purified by high performance
liquid chromatography (Waters-2767, elution system: 10 mmoL/L
ammonium bicarbonate, water, acetonitrile) to obtain the title
compound 5 (2.0 mg, yield: 7.9%).
[0256] MS m/z (ESI): 367.2 [M+1]
[0257] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.98 (s, 1H),
7.30-7.28 (m, 1H), 7.22-7.19 (m, 3H), 5.45 (s, 2H), 4.39 (t, 2H),
3.60 (s, 2H), 3.28 (t, 4H), 2.12-2.09 (m, 2H), 1.80-1.76 (m, 2H),
1.55-1.49 (m, 2H), 1.00 (t, 3H).
Example 6
6-Butoxy -1-(3 -(piperi
din-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 6
##STR00062## ##STR00063##
[0258] Step 1
1-(3-(Chloromethyl)benzyl)piperidine 6b
[0259] 3-(Piperidin-1-ylmethyl)phenylcarbinol 6a (1.7 g, 8.28 mmol,
prepared according to the known method disclosed in "Bioorganic
& Medicinal Chemistry, 2004, 12(10), 2727-2736") was dissolved
in 20 mL of dichloromethane, thionyl chloride (1.2 mL, 16.56 mmol)
was added at 0.degree. C., and the reaction solution was stirred at
room temperature for 3 hours. The reaction solution was
concentrated under reduced pressure, added with saturated sodium
carbonate solution (50 mL), and extracted with dichloromethane (100
mL.times.2). The organic phases were combined, dried over anhydrous
sodium sulfate and filtered. The filtrate was concentrated under
reduced pressure to obtain the crude title compound 6b (1.7 g),
which was used directly in the next step without purification.
[0260] MS m/z (ESI): 224.2 [M+1]
Step 2
6-Chloro-N-(3-methoxy
benzyl)-1-(4-(piperidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4--
amine 6c
[0261] Compound 1c (300 mg, 1.04 mmol), the crude compound 6b (232
mg, 1.04 mmol) and potassium carbonate (144 mg, 1.04 mmol) were
dissolved in 5 mL of N,N-dimethylformamide. The reaction was
stopped after stirring at room temperature for 16 hours. The
reaction solution was concentrated under reduced pressure, and the
resulting residue was purified by silica gel column chromatography
with elution system A to obtain the title compound 6c (50 mg,
yield: 10.1%).
[0262] MS m/z (ESI): 477.3 [M+1]
Step 3
6-Butoxy-N-(3-methoxybenzyl)-1-(4-(piperi
din-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 6d
[0263] Compound 6c (50 mg, 0.10 mmol), sodium n-butoxide (0.2 mL,
0.40 mmol) and 1 mL of n-butanol were added to a microwave tube
successively, heated to 160.degree. C. and stirred for 1.5 hours.
The reaction solution was cooled to room temperature, and
concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography with elution system A
to obtain the title compound 6d (30 mg, yield: 55.5%).
[0264] MS m/z (ESI): 515.3 [M+1]
Step 4
6-Butoxy-1-(3-(piperidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4--
amine 6
[0265] Compound 6d (30 mg, 0.06 mmol) and 2 mL of trifluoroacetic
acid were added to a reaction flask, heated to reflux, and stirred
for 24 hours. The reaction was stopped, and the reaction solution
was concentrated under reduced pressure and added with a solution
of 7 N ammonia in methanol (1 mL). The reaction solution was
concentrated under reduced pressure, and the residue was purified
by thin layer chromatography with developing system A to obtain the
title compound 6 (7.0 mg, yield: 29.2%).
[0266] MS m/z (ESI): 395.3 [M+1]
[0267] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.99 (s, 1H),
738-7.31 (m, 4H), 5.48 (s, 2H), 4.38 (t, 2H), 3.86 (s, 2H),
2.87-2.80 (m, 4H), 1.79-1.75 (m, 2H), 1.71-1.68 (m, 4H), 1.54-1.40
(m, 4H), 1.00 (t, 3H).
Example 7
6-(2-Methoxyethoxy)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]-
pyrimidin-4-amine 7
##STR00064##
[0268] Step 1
N-(4-Methoxybenzyl)-6-(2-methoxyethoxy)-1-(4-(pyrrolidin-1-ylmethyl)benzyl-
)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7a
[0269] Compound 1e (90 mg, 0.19 mmol), sodium 2-methoxyethanol (0.3
mL, 0.60 mmol) and 1 mL of 2-methoxyethanol were added to a
microwave tube successively, heated to 160.degree. C. and stirred
for 1.5 hours. The reaction solution was cooled to room
temperature, and concentrated under reduced pressure. The resulting
residue was purified by silica gel column chromatography with
elution system A to obtain the title compound 7a (30 mg, yield:
30.7%).
[0270] MS m/z (ESI): 503.3 [M+1]
Step 2
6-(2-Methoxyethoxy)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]-
pyrimidin-4-amine 7
[0271] Compound 7a (30 mg, 0.06 mmol) and 5 mL of trifluoroacetic
acid were added to a reaction flask, heated to 100.degree. C., and
stirred for 2 hours. The reaction solution was cooled to room
temperature, and concentrated under reduced pressure. The resulting
residue was purified by high performance liquid chromatography
(Waters-2767, elution system: 10 mmoL/L ammonium bicarbonate,
water, acetonitrile) to obtain the title compound 7 (5 mg, yield:
19.7%).
[0272] MS m/z (ESI): 383.2 [M+1]
[0273] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.96 (s, 1H),
7.30-7.28 (d, 2H), 7.25-7.23 (d, 2H), 5.42 (s, 2H), 4.51-4.48 (t,
2H), 3.74-3.72 (t, 2H), 3.65 (s, 2H), 3.39 (s, 3H), 2.57(s, 4H),
1.81-1.78 (m, 4H).
Example 8
6-((1-Methoxypropan-2-yl)oxy)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyraz-
olo[3,4-d ]pyrimidin-4-amine 8
##STR00065##
[0274] Step 1
N-(4-Methoxybenzyl)-6-((1-methoxypropan-2-yl)oxy)-1-(4-(pyrrolidin-1-ylmet-
hyl)benzyl)-1H-pyrazolo[3,4-dlpyrimidin-4-amine 8a
[0275] Compound 1e (200 mg, 0.43 mmol), 2-methoxy-1-methyl-ethoxy
sodium (96.9 mg, 0.86 mmol) and 5 mL of propylene glycol methyl
ether were added to a microwave tube successively, heated to
160.degree. C. and stirred for 1.5 hours. The reaction solution was
cooled to room temperature, and concentrated under reduced
pressure. The resulting residue was purified by silica gel column
chromatography with elution system A to obtain the title compound
8a (150 mg, yield: 67.2%).
[0276] MS m/z (ESI): 517.3 [M+11
Step 2
6-((1-Methoxypropan-2-yl)oxy)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyraz-
olo[3,4-d ]pyrimidin-4-amine 8
[0277] Compound 8a (80 mg, 0.15 mmol) and 5 mL of trifluoroacetic
acid were added to a reaction flask, heated to 80.degree. C., and
stirred for 1 hour. The reaction solution was cooled to room
temperature, and concentrated under reduced pressure. The resulting
residue was purified by high performance liquid chromatography
(Waters-2767, elution system: 10 mmoL/L ammonium bicarbonate,
water, acetonitrile) to obtain the title compound 8 (20 mg, yield:
32.6%).
[0278] MS m/z (ESI): 397.2 [M+1]
[0279] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.95 (s, 1H),
7.35-7.33 (d, 2H), 7.29-7.27 (d, 2H), 5.43 (m, 3H), 3.83 (s, 2H),
3.60-3.52 (m, 2H), 3.37 (s, 3H), 2.76(s, 4H), 1.87 (s, 4H),
1.34-1.32 (t, 3H).
Example 9
6-Butoxy-1-(3-fluoro-4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]py-
rimidin-4-amine 9
##STR00066## ##STR00067##
[0280] Step 1
3-Fluoro-4-(pyrrolidin-1-ylmethyl)benzonitrile 9c
[0281] 4-(Bromomethyl)-3-fluorobenzonitrile 9a (1 g, 4.67 mmol),
pyrrolidine 9b (332 mg, 4.67 mmol) and N,N-diisopropylethylamine
(1.21 g, 9.34 mmol) were dissolved in 10 mL of acetonitrile. After
stirring for 2 hours, the reaction solution was concentrated under
reduced pressure to obtain the crude title compound 9c (1 g), which
was used directly in the next step without purification.
[0282] MS m/z (ESI): 205.4 [M+1]
Step 2
3-Fluoro-4-(pyrroli din-1-ylmethyl)benzoic acid 9d
[0283] The crude compound 9c (1 g, 4.9 mmol) was dissolved in a
mixed solvent of 5 mL of sulfuric acid, 5 mL of water and 10 mL of
acetic acid. The reaction was stopped after stirring at 90.degree.
C. for 16 hours. The reaction solution was cooled to room
temperature, and concentrated under reduced pressure. The residue
was added with methanol, and filtered to remove insoluble matters.
The filtrate was concentrated under reduced pressure to obtain the
crude title compound 9d (1 g), which was used directly in the next
step without purification.
[0284] MS m/z (ESI): 224.4 [M+1]
Step 3
(3-F luoro-4-(py rroli din-1-ylmethyl)phenyl)methanol 9e
[0285] The crude compound 9d (1 g, 4.48 mmol) was dissolved in 20
mL of tetrahydrofuran. The reaction solution was cooled to
0.degree. C., added with lithium aluminum hydride (607 mg,
17.9mmo1) and stirred for 3 hours. Then 1 mL of water, 1 mL of 2N
sodium hydroxide solution and 3 mL of water were added successively
to quench the reaction. The reaction solution was filtered, and the
filtrate was collected and concentrated under reduced pressure to
obtain the crude title compound 9e (820 mg), which was used
directly in the next step without purification.
[0286] MS m/z (ESI): 210.4 [M+1]
Step 4
6-Chl
oro-1-(3-fluoro-4-(pyrrolidin-1-ylmethyl)benzyl)-N-(4-methoxybenzyl)-
-1H-pyrazolo[3,4-d]pyrimidin-4-amine 9f
[0287] The crude compound 9e (100 mg, 0.48 mmol), compound 1c
(141.34 mg, 0.48 mmol) and triphenylphosphine (192 mg, 0.73 mmol)
were dissolved in 10 mL of 1,4-dioxane, and diisopropyl
azodicarboxylate (148 mg, 0.73 mmol) was then added dropwise. The
reaction solution was warmed up to 85.degree. C., and stirred for 4
hours. The reaction solution was cooled to room temperature, and
concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography with elution system A
to obtain the title compound 9f (90 mg, yield: 38.3%).
[0288] MS m/z (ESI): 481.4 [M+1]
Step 5
6-Butoxy-1-(3-fluoro-4-(pyrrolidin-1-ylmethyl)benzyl)-N-(4-methoxybenzyl)--
1H-pyrazolo[3,4-d]pyrimi din-4-amine 9g
[0289] Compound 9f (90 mg, 0.19 mmol), sodium n-butoxide (18 mg,
0.18 mmol) and 5 mL of n-butanol were added to a microwave tube
successively, heated to 160.degree. C. and stirred for 1.5 hours.
The reaction was stopped, and the reaction solution was cooled to
room temperature and concentrated under reduced pressure. The
resulting residue was purified by silica gel column chromatography
with elution system A to obtain the title compound 9g (35 mg,
yield: 36.1%).
[0290] MS m/z (ESI): 519.5 [M+1]
Step 6
6-Butoxy-1-(3-fluoro-4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]py-
rimidin-4-amine 9
[0291] Compound 9g (35 mg, 0.07 mmol) and 10 mL of trifluoroacetic
acid were added to a sealed tube, heated to 100.degree. C., and
stirred for 1 hour. The reaction was stopped, and the reaction
solution was cooled to room temperature and concentrated under
reduced pressure. The resulting residue was purified by high
performance liquid chromatography (Waters-2767, elution system: 10
mmoL/L ammonium bicarbonate, water, acetonitrile) to obtain the
title compound 9 (20 mg, yield: 74.3%).
[0292] MS m/z (ESI): 399.5 [M+1]
[0293] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.97 (s, 1H),
7.37-7.33 (m, 1H), 7.07-6.99 (m, 2H), 5.42 (s, 2H), 4.38-4.35 (t,
2H), 3.68 (s, 2H), 2.56 (s, 4H), 1.79-1.73(m, 6H), 1.52-1.46 (m,
2H), 0.99-0.96 (t, 3H).
Example 10
N.sup.6-Butyl-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimi-
dine-4,6-diamine 10
##STR00068##
[0294] Step 1
N.sup.6-Butyl-N.sup.4-(4-methoxybenzyl)-1-(4-(pyrroli
din-1-ylmethyl)benzyl)-1H-pyrazolo[3 ,4-d ]pyrimidine-4,6-diamine
10a
[0295] Compound 1e (50 mg, 0.11 mmol), n-butylamine (23.7 mg, 0.32
mmol) and N,N-diisopropylethylamine (41.9 mg, 0.32 mmol) were added
to 5 mL of n-butanol successively. The reaction solution was warmed
up to 120.degree. C. and stirred under microwave for 1 hour. The
reaction solution was cooled to room temperature, and concentrated
under reduced pressure to obtain the crude title compound 1Oa (20
mg), which was used directly in the next step without
purification.
Step 2
N.sup.6-Butyl-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimi-
dine-4,6-diamine 10
[0296] The crude compound 10a (20 mg, 0.04 mmol) and 5 mL of
trifluoroacetic acid were added to a reaction flask, heated to
100.degree. C., and stirred overnight. The reaction was stopped,
and the reaction solution was cooled to room temperature and
concentrated under reduced pressure. The resulting residue was
purified by high performance liquid chromatography (Waters-2767,
elution system: 10 mmoL/L ammonium bicarbonate, water,
acetonitrile) to obtain the title compound 10 (15.2 mg, a yellow
solid, yield: 62.5%).
[0297] MS m/z (ESI): 380.3 [M+1]
[0298] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.04 (s, 1H),
7.49-7.47 (d, 2H), 7.42-7.40 (d, 2H), 5.44 (s, 2H), 4.34 (s, 2H),
3.49-3.45 (m, 4H), 3.15 (s, 2H), 2.13(s, 2H), 1.93 (s, 2H),
1.65-1.60 (m, 2H), 1.45-1.39 (m, 2H), 0.98-0.94 (t, 3H).
Example 11
4-Amino-N-propyl-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyr-
imidine-6-carboxamide 11
##STR00069##
[0299] Step 1
Methyl
4-((4-methoxybenzyl)amino)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-p-
yrazolo[3,4-d]pyrimidine-6-carboxylate 11a
[0300] Compound 1e (200 mg, 0.43 mmol), palladium acetate (2.9 mg,
0.013 mmol), 4,5 -bi s dipheny 1phos phino-9,9-dimethyl oxanthene
(15 mg, 0.026 mmol) and triethylamine (44 mg, 0.4 mmol) were
dissolved in 3 mL of n-butanol and 3 mL of N,N-dimethylformamide.
The reaction system was purged with carbon monoxide three times.
The reaction solution was warmed up to 70.degree. C., and stirred
for 16 hours. The reaction solution was cooled to room temperature,
and concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography with elution system A
to obtain the title compound 1la (150 mg, yield: 71.4%).
[0301] MS m/z (ESI): 487.5 [M+1]
Step 2
4-((4-Methoxy benzyl)amino)-N-propyl-1-(4-(pyrroli
din-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-carboxamide
11b
[0302] Compound 11a (50 mg, 0.1 mmol) and n-propylamine (12 mg, 0.2
mmol) were dissolved in 5 mL of ethanol successively. The reaction
solution was added to a sealed tube, warmed up to 60.degree. C.,
and stirred for 16 hours. The reaction was stopped, and the
reaction solution was cooled to room temperature and concentrated
under reduced pressure to obtain the crude title compound 1lb (20
mg), which was used directly in the next step without
purification.
Step 3
4-Amino-N-propyl-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyr-
imidine-6-carboxamide 11
[0303] The crude compound 1lb (20 mg, 0.04 mmol) and 5 mL of
trifluoroacetic acid were added to a reaction flask, heated to
100.degree. C., and stirred for 12 hours. The reaction solution was
cooled to room temperature, and concentrated under reduced
pressure. The resulting residue was purified by high performance
liquid chromatography (Waters-2767, elution system: 10 mmoL/L
ammonium bicarbonate, water, acetonitrile) to obtain the title
compound 11 (10 mg, yield: 60.3%).
[0304] MS m/z (ESI): 394.5 [M+1]
[0305] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.12 (s, 1H), 7.28
(m, 4H), 5.64 (s, 2H), 3.62 (s, 2H), 3.41-3.37 (t, 2H), 2.55-2.52
(m, 4H), 1.80-1.77 (m, 4H), 1.70-1.64 (m, 2H), 0.98-1.02 (t,
3H).
Example 12
1-(4-Amino-1-(4-pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin--
6-yl)pentan-1-one 12
##STR00070##
[0306] Step 1
4-((4-Methoxybenzyl)amino)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo-
[3,4-d]pyrimidine-6-carbonitrile 12a
[0307] Compound 1e (260 mg, 0.56 mmol),
tris(dibenzylideneacetone)dipalladium (52 mg, 0.056 mmol),
1,1'-bis(diphenylphosphino)ferrocene (31 mg, 0.056 mmol), zinc
cyanide (99 mg, 0.84 mmol) and zinc powder (37 mg, 0.56 mmol) were
suspended in 5 mL of N,N-dimethylacetamide. The reaction solution
was warmed up to 140.degree. C., and stirred for 16 hours under an
argon atmosphere. The reaction solution was cooled to room
temperature, and concentrated under reduced pressure. The resulting
residue was purified by thin layer chromatography with developing
system A to obtain the title compound 12a (160 mg, yield: 63%).
[0308] MS m/z (ESI): 454.5 [M+1]
Step 2
1-(4-((4-Methoxybenzyl)amino)-1-(4-(pyrrolidin-1-ylmethyl)benzyl)-1H-pyraz-
olo[3,4-d]pyrimidin-6-yl)pentan-1-one 12b
Compound 12a (160 mg, 0.35 mmol) was dissolved in 5 mL of
tetrahydrofuran, and a solution of 2 M n-butylmagnesium chloride in
tetrahydrofuran (0.9 mL, 1.77 mmol) was then added at 0.degree. C.
The reaction solution was warmed up to 60.degree. C., and stirred
for 2 hours under an argon atmosphere. The reaction solution was
cooled to room temperature, added with an aqueous solution of
ammonium chloride, and extracted with ethyl acetate. The organic
phases were combined and concentrated under reduced pressure. The
resulting residue was purified by thin layer chromatography with
developing system A to obtain the title compound 12b (150 mg,
yield: 83%).
[0309] MS m/z (ESI): 513.6 [M+1]
Step 3
1-(4-Amino-1-(4-pyrrolidin-1-ylmethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin--
6-yl)pentan-1 -one 12
[0310] Compound 12b (150 mg, 0.29 mmol) was dissolved in 10 mL of
trifluoroacetic acid. The reaction solution was added to a sealed
tube, heated to 110.degree. C., and stirred for 16 hours. The
reaction solution was cooled to room temperature, and concentrated
under reduced pressure. The resulting residue was purified by high
performance liquid chromatography (Waters-2767, elution system: 10
mmoL/L ammonium bicarbonate, water, acetonitrile) to obtain the
title compound 12 (19 mg, yield: 17%).
[0311] MS m/z (ESI): 393.5 [M+1]
[0312] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.01 (s, 1H),
7.35-7.29 (q, 4H), 5.62 (s, 2H), 3.62 (s, 2H), 3.26 (t, 2H), 2.53
(s, 4H), 1.78 (s, 4H), 1.76-1.70 (m, 2H), 1.48-1.42 (m, 2H), 0.98
(t, 3H).
TEST EXAMPLES
Biological Assay
[0313] Test Example 1. Determination of agonistic activity of the
compounds of the present invention on human TLR7
[0314] The activation effect of the compounds of the present
invention on the hTLR7 protein expressed by the HEK-Blue.TM. hTLR7
stably transfected cells was determined by the following
experimental method:
[0315] I. Experimental materials and instruments
[0316] 1. DMEM (Gibco, 10564-029),
[0317] 2. Fetal bovine serum (GIBCO, 10099),
[0318] 3. Penicillin-streptomycin (Gibco, 15140-122),
[0319] 4. Trypan blue solution (Sigma, T8154-100ML),
[0320] 5. Flexstation 3 multi-function microplate reader
(Molec.mu.lar Devices),
[0321] 6. HEK-Blue.TM. HTLR7 cell line (InvivoGen, hkb-hTLR7),
[0322] 7. HEK-Blue detection reagent (InvivoGen, hb-det3).
[0323] II. Experimental Procedures
[0324] A bag of HEK-Blue detection dry powder was dissolved in 50
ml of water free of endotoxin, and the solution was then placed in
an incubator at 37.degree. C. for 10 minutes followed by sterile
filtration to prepare a HEK-Blue detection medium. The compound was
firstly formulated into a 20 mM stock solution, then diluted with
pure DMSO to a maximum concentration of 6.times.10.sup.6 nM, and a
total of 10 points were obtained by a 3-fold gradient dilution.
[0325] The above formulated compound was firstly diluted 20-fold
with the medium, then 20 .mu.l of the diluted compound were added
to each well. The supernate was removed from the HEK-Blue.TM. hTLR7
cells, to which 2-5 ml of pre-warmed PBS were then added. The cells
were placed in an incubator for 1-2 minutes, gently pipetted, and
counted by trypan blue staining. The cells were re-suspended in the
HEK-Blue detection medium to adjust the concentration to
2.2.times.10.sup.5 cells/ml. 180 .mu.l of cells were added to the
above 96-well plate already added with 20 .mu.l of the compounds,
and incubated at 37.degree. C. for 6-16 hours.
[0326] The microplate reader read at a wavelength of 620 nm to
obtain corresponding OD values, and the EC.sub.50 values of the
compounds was calculated by Graphpad Prism.
[0327] The activation effect of the compounds of the present
invention on human TLR7 can be determined by the above test, and
the obtained EC.sub.50 values are shown in Table 1.
TABLE-US-00004 TABLE 1 EC.sub.50 of the compounds of the present
invention on human TLR7 Example No. EC.sub.50 (nM) Emax (%) 1 28
100 2 64 91 3 77 91 4 166 88 6 233 91 7 180 95 8 217 104 9 128 96
10 349 79 11 335 85 12 388 78
[0328] Conclusion: The compounds of the present invention have a
significant activation effect on human TLR7.
[0329] Test Example 2. Determination of agonistic activity of the
compounds of the present invention on human TLR8
[0330] The activation effect of the compounds of the present
invention on the hTLR8 protein expressed by the HEK-Blue.TM. hTLR8
stably transfected cells was determined by the following
experimental method:
[0331] I. Experimental Materials and Instruments
[0332] 1. DMEM (Gibco, 10564-029),
[0333] 2. Fetal bovine serum (GIBCO, 10099),
[0334] 3. Penicillin-streptomycin (Gibco, 15140-122),
[0335] 4. Trypan blue solution (Sigma, T8154-100ML),
[0336] 5. Flexstation 3 multi-function microplate reader
(Molec.sub.illar Devices),
[0337] 6. HEK-Blue.TM. HTLR8 cell line (InvivoGen, hkb-hTLR8),
[0338] 7. HEK-Blue detection reagent (InvivoGen, hb-det3).
[0339] II. Experimental Procedures
[0340] A bag of HEK-Blue detection dry powder was dissolved in 50
ml of water free of endotoxin, and the solution was then placed in
an incubator at 37.degree. C. for 10 minutes followed by sterile
filtration to prepare a HEK-Blue detection medium. The compound was
firstly formulated into a 20 mM stock solution, then diluted with
pure DMSO to a maximum concentration of 6.times.10.sup.6 nM, and a
total of 10 points were obtained by a 3-fold gradient dilution. The
compound was firstly diluted 20-fold with the medium, then 20 .mu.l
of the diluted compound were added to each well.
[0341] The supernate was removed from the HEK-Blue.TM. hTLR8 cells,
to which 2-5 ml of pre-warmed PBS were then added. The cells were
placed in an incubator for 1-2 minutes, gently pipetted, and
counted by trypan blue staining. The cells were re-suspended in the
HEK-Blue detection medium to adjust the concentration to
2.2.times.10.sup.5 cells/ml. 180 .mu.l of cells were added to the
above 96-well plate already added with 20 .mu.l of the compounds,
and incubated at 37.degree. C. for 6-16 hours.
[0342] The microplate reader read at a wavelength of 620 nm to
obtain corresponding OD values, and the EC.sub.50 values of the
compounds was calculated by Graphpad Prism.
[0343] The activation effect of the compounds of the present
invention on human TLR8 can be determined by the above test, and
the obtained EC.sub.50 values are shown in Table 2.
TABLE-US-00005 TABLE 2 EC.sub.50 of the compounds of the present
invention on human TLR8 Example No. EC.sub.50 (.mu.M) Emax (%) 1
>30 8 2 >29 52 3 >24 2 4 >30 28 6 >6 35 7 >30 0 8
>30 2 10 >30 0 11 >30 0 12 >30 5
[0344] Conclusion: The compounds of the present invention have no
activation effect on human TLR8, indicating that the compounds of
the present invention have a high selectivity on TLR7.
[0345] Test Example 3. Determination of the ability of the
compounds of the present invention to stimulate the secretion of
IFN-.alpha. from peripheral blood mononuclear cells (PBMC)
[0346] The ability of the compounds of the present invention to
stimulate the secretion of IFN-a from PBMC was determined by the
following experimental method:
[0347] I. Experimental Materials and Instruments
[0348] 1. RPMI 1640 (Invitrogen,11875),
[0349] 2. FBS (Gibco,10099-141)
[0350] 3. Penicillin-streptomycin (Gibco, 15140-122),
[0351] 4. Ficoll-Paque PREMIUM (GE, 17-5442-02),
[0352] 5. Trypan blue solution (Sigma, T8154-100ML),
[0353] 6. SepMate.TM.-50 (Stemcell, 15460),
[0354] 7. Bright-Line.TM. blood cell counter (Sigma,
Z359629-1EA),
[0355] 8. Human IFN-.alpha. kit (cisbio, 6FHIFPEB),
[0356] 9. PHERAStar multi-function microplate reader (BMG,
PHERAStar).
[0357] II. Experimental Procedures
[0358] The compound was diluted with pure DMSO to a maximum
concentration of 5 mM, and a total of 9 points were obtained by a
4-fold gradient dilution. 4 .mu.l of the compound were then added
to 196 .mu.l of RMPI 1640 medium containing 10% FBS and mixed well.
50 .mu.l of the mixture were taken from each well and added to a
new 96-well plate.
[0359] All reagents were equilibrated to room temperature. 60 ml of
blood and PBS+2% FBS were added to a 250 ml culture flask, gently
pipetted, mixed well and diluted. 15 ml of lymphocyte separation
solution Ficoll-Paque PREMIUM and then 30 ml of diluted blood were
added to a 50 ml PBMC centrifuge tube SepMateTM-50. The mixture was
centrifuged at 1200 g for 10 minutes at room temperature. The
supernatant was taken and then centrifuged at 300 g for 8 minutes.
The cells were re-suspended in the RMPI 1640 medium containing 10%
FBS and counted, and the number of PBMCs was adjusted to
3.33.times.10.sup.6 cells/ml. 150 .mu.l of the cell solution were
added to the plate added with the compound, and incubated in an
incubator at 37.degree. C., in 5.0% CO.sub.2 for 24 hours.
[0360] The cell culture plate was placed in a centrifuge, and
centrifuged at 1200 rpm for 10 minutes at room temperature. 150
.mu.l of the supernatant were taken from each well. The reagents in
the human IFN-a kit were first equilibrated to normal temperature.
The anti-IFN-.alpha.a-Eu.sup.3+-Cryptate conjugate and the
anti-IFN-.alpha.-d2-conjugate were formulated in the dark according
to the kit instructions, and both of them were mixed well with the
conjugate buffer at a ratio of 1:40. 16 .mu.l of the supernatant
obtained by centrifugation were then added to each well. 2 .mu.l of
anti-IFN-.alpha.-Eu.sup.3+-Cryptate conjugate and
anti-IFN-.alpha.-d2-conjugate formulated just now were then added
to each well. The plate was shaken and mixed well, and incubated in
the dark at room temperature for 3 hours.
[0361] The PHERAStar was read in the HTRF mode. The lowest compound
concentration that stimulated cytokine levels of at least 3 times
higher than the minimum detection limit was defined as the minimal
effective concentration (MEC) value of the compound in the cytokine
stimulation test.
[0362] The ability of the compounds of the present invention to
stimulate the secretion of IFN-a from PBMC was determined by the
above test, and the obtained MEC values are shown in Table 3.
TABLE-US-00006 TABLE 3 MEC of the compounds of the present
invention to stimulate the secretion of IFN-.alpha. from PBMC
Example No. MEC (nM) 1 6 2 23 3 20 4 100 5 41 7 89
[0363] Conclusion: It can be seen from the data of activity of
stimulating the secretion of IFN-.alpha. from PBMC that the
compounds of the present invention have an advantage of lower
effective concentration.
[0364] Test Example 4. Inhibition effect of the compounds of the
present invention on the enzyme activity of midazolam metabolite
site of CYP3A4 in human liver microsomes
[0365] The effect of the compounds of the present invention on the
enzyme activity of midazolam metabolite site of CYP3A4 in human
liver microsomes was determined by the following experimental
method:
[0366] I. Experimental Materials and Instruments
[0367] 1. Phosphate buffer (PBS),
[0368] 2. NADPH (Sigma N-1630),
[0369] 3. Human liver microsome (Corning Gentest),
[0370] 4. ABI QTrap 4000 liquid chromatograph/mass spectrometer (AB
Sciex),
[0371] 5. Inertsil C8-3 column, 4.6.times.5Omm, 5p.m (Dikma
Technologies Inc., USA),
[0372] 6. CYP probe substrate (midazolam/10 .mu.M) and positive
control inhibitor (ketoconazole).
[0373] II. Experimental Procedures
[0374] 100 mM PBS buffer was formulated, which was then used to
formulate 2.5 mg/ml microsome solution and 5 mM NADPH solution. The
5.times. concentration of the compound working solution was diluted
with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 .mu.M). The
5.times. concentration of ketoconazole working solution was diluted
with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 .mu.M).
Dextromethorphan working solution was diluted with PBS to a
concentration of 50 .mu.M.
[0375] 20 .mu.l of 2.5 mg/ml microsome solution, 20 .mu.l of 50
.mu.M testosterone working solution, 20 .mu.l of MgCl.sub.2
solution and 20 .mu.l of the compound working solution (150, 50,
15, 5, 1.5, 0.15, 0.015, 0 .mu.M, different reaction systems for
each concentration) were taken respectively and mixed well. For the
positive control group, the compound was replaced with the same
concentration of ketoconazole. The mixture together with 5 mM NADPH
solution were pre-incubated at 37.degree. C. for 5 minutes. After 5
minutes, 20 .mu.l of NADPH were added to each well, the reaction
was started and incubated for 30 minutes. All the incubated samples
were present in duplicate. After 30 minutes, 250 .mu.l of
acetonitrile containing internal standard were added to all
samples, mixed well, shaken at 800 rpm for 10 minutes, and then
centrifuged at 3700 rpm for 10 minutes. 80 .mu.l of the supernatant
were taken and analyzed by LC-MS/MS.
[0376] The data were calculated by Graphpad Prism to obtain the
IC.sub.50 values of the compounds on the midazolam metabolite site
of CYP3A4.
[0377] IC.sub.50 values of the compounds of the present invention
on the midazolam metabolite site of CYP3A4 in human liver
microsomes.
TABLE-US-00007 Example No. IC.sub.50 (.mu.M) 1 14 2 10 3 7 4 11 6
10 7 >30 12 16
[0378] Conclusion: The compounds of the present invention have a
weak inhibition effect on the midazolam metabolic site of CYP3A4 in
human liver microsome, and show better safety, indicating that the
metabolic drug interaction based on the midazolam metabolic site of
CYP3A4 will not occur.
[0379] Test Example 5. Inhibition effect of the compounds of the
present invention on the enzyme activity of CYP2D6 in human liver
microsomes
[0380] The effect of the compounds of the present invention on the
enzyme activity of CYP2D6 in human liver microsomes was determined
by the following experimental method:
[0381] I. Experimental materials and instruments
[0382] 1. Phosphate buffer (PBS),
[0383] 2. NADPH (Sigma N-1630),
[0384] 3. Human liver microsome (Corning Gentest),
[0385] 4. ABI QTrap 4000 liquid chromatograph/mass spectrometer (AB
Sciex),
[0386] 5. Inertsil C8-3 column, 4.6.times.5Omm, 5 .mu.m (Dikma
Technologies Inc., USA),
[0387] 6. CYP probe substrate (dextromethorphan/10 .mu.M) and
positive control inhibitor (quinidine).
[0388] II. Experimental Procedures
[0389] 100 mM PBS buffer was formulated, which was then used to
formulate 2.5 mg/ml microsome solution and 5 mM NADPH solution. The
5.times. concentration of the compound working solution was diluted
with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 .mu.M). The
5.times. concentration of quinidine working solution was diluted
with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0.mu.M).
Dextromethorphan working solution was diluted with PBS to a
concentration of 50 04.
[0390] 20 .mu.l of 2.5 mg/ml microsome solution, 20 .mu.l of 50
.mu.M testosterone working solution, 20 .mu.l of MgCl.sub.2
solution and 20 .mu.l of the compound working solution (150, 50,
15, 5, 1.5, 0.15, 0.015, 0 .mu.M, different reaction systems for
each concentration) were taken respectively and mixed well. For the
positive control group, the compound was replaced with the same
concentration of quinidine. The mixture together with 5 mM NADPH
solution were pre-incubated at 37.degree. C. for 5 minutes. After 5
minutes, 20 .mu.l of NADPH were added to each well, the reaction
was started and incubated for 30 minutes. All the incubated samples
were present in duplicate. After 30 minutes, 250 .mu.l of
acetonitrile containing internal standard were added to all
samples, mixed well, shaken at 800 rpm for 10 minutes, and then
centrifuged at 3700 rpm for 10 minutes. 80 .mu.l of the supernatant
were taken and analyzed by LC-MS/MS.
[0391] The data were calculated by Graphpad Prism to obtain the
IC.sub.50 values of the compounds on the metabolite site of
CYP2D6.
[0392] IC.sub.50 values of the compounds of the present invention
for no inhibition on CYP2D6 in human liver microsomes.
TABLE-US-00008 Example No. IC.sub.50 (.mu.M) 1 >30 2 >30 3 4
4 16 6 10 7 >30 12 16
[0393] Conclusion: The compounds of the present invention have a
weak inhibition effect on the enzyme activity of CYP2D6 in human
liver microsomes, and show better safety, indicating that the
metabolic drug interaction based on CYP2D6 will not occur.
[0394] Test Example 6. Inhibition effect of the compounds of the
present invention on the enzyme activity of testosterone metabolite
site of CYP3A4 in human liver microsomes
[0395] The effect of the compounds of the present invention on the
enzyme activity of testosterone metabolite site of CYP3A4 in human
liver microsomes was determined by the following experimental
method:
[0396] I. Experimental Materials and Instruments
[0397] 1. Phosphate buffer (PBS),
[0398] 2. NADPH (Sigma N-1630),
[0399] 3. Human liver microsome (Corning Gentest),
[0400] 4. ABI QTrap 4000 liquid chromatograph/mass spectrometer (AB
Sciex),
[0401] 5. Inertsil C8-3 column, 4.6.times.50 mm, 5 .mu.m (Dikma
Technologies Inc., USA),
[0402] 6. CYP probe substrate (testosterone/10 .mu.M) and positive
control inhibitor (ketoconazole).
[0403] II. Experimental Procedures
[0404] 100 mM PBS buffer was formulated, which was then used to
formulate 2.5 mg/ml microsome solution and 5 mM NADPH solution. The
5.times. concentration of the compound working solution was diluted
with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 .mu.M). The
5.times. concentration of ketoconazole working solution was diluted
with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0.mu.M).
Dextromethorphan working solution was diluted with PBS to a
concentration of 50 .mu.M.
[0405] 20 .mu.l of 2.5 mg/ml microsome solution, 20 .mu.l of 50
.mu.M testosterone working solution, 20 .mu.l of MgCl.sub.2
solution and 20 .mu.l of the compound working solution (150, 50,
15, 5, 1.5, 0.15, 0.015, 0 .mu.M, different reaction systems for
each concentration) were taken respectively and mixed well. For the
positive control group, the compound was replaced with the same
concentration of ketoconazole. The mixture together with 5 mM NADPH
solution were pre-incubated at 37.degree. C. for 5 minutes. After 5
minutes, 20 .mu.l of NADPH were added to each well, the reaction
was started and incubated for 30 minutes. All the incubated samples
were present in duplicate. After 30 minutes, 250 .mu.l of
acetonitrile containing internal standard were added to all
samples, mixed well, shaken at 800 rpm for 10 minutes, and then
centrifuged at 3700 rpm for 10 minutes. 80 .mu.l of the supernatant
were taken and analyzed by LC-MS/MS.
[0406] The data were calculated by Graphpad Prism to obtain the
IC.sub.50 values of the compounds on the testosterone metabolite
site of CYP3A4.
[0407] IC.sub.50 values of the compounds of the present invention
on the testosterone metabolite site of CYP3A4 in human liver
microsomes
TABLE-US-00009 Example No. IC.sub.50 (.mu.M) 1 4 2 19 3 3 4 6 6 3 7
>30 12 >30
[0408] Conclusion: The compounds of the present invention have a
weak inhibition on the testosterone metabolite site of CYP3A4 in
human liver microsomes, and show better safety.
* * * * *