CN1809357B

CN1809357B - Small molecule Toll-like receptor (TLR) antagonists

Info

Publication number: CN1809357B
Application number: CN200480017064.7A
Authority: CN
Inventors: 格雷森·B·利普福德; 亚历山德拉·福斯巴赫; 查尔斯·M·泽普
Original assignee: Coley Pharmaceutical GmbH; Coley Pharmaceutical Group Inc
Current assignee: Coley Pharmaceutical GmbH; Coley Pharmaceutical Group Inc
Priority date: 2003-06-20
Filing date: 2004-06-18
Publication date: 2010-12-22
Anticipated expiration: 2024-06-18
Also published as: CN1809357A; ZA200510028B

Abstract

The invention provides methods and compositions useful for modulating signaling through Toll-like receptors. The methods involve contacting a TLR-expressing cell with a small molecule having a core structure including at least two rings. Certain of the compounds are 4-primary amino quinolines. Many of the compounds and methods are useful specifically for inhibiting immune stimulation involving at least one of TLR9, TLR8, TLR7, and TLR3. The methods may have use in the treatment of autoimmunity, inflammation, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer, and immunodeficiency.

Description

Small molecule Toll-like receptor body (TLR) antagonist

Invention field

Relate generally to field of immunology of the present invention.More specifically, the present invention relates to change the compositions and the method for immunologic function.More specifically, the present invention relates to influence by Toll sample receptor (TLR) numerator mediated immunostimulating compositions and method.

Background of invention

Stimulating immune system comprises stimulates innate immunity and/or adaptive immunity, is a kind of complicated phenomenon, may cause protectiveness or disadvantageous physiology consequence to the host.In recent years, to the interest increase of innate immunity mechanism, innate immunity is considered to start and support adaptive immunity.Part is owing to the pattern recognition receptor protein family that recent findings is called the high conservative of Toll sample receptor (TLR) is considered to participate in innate immunity, this interest thereby higher as the receptor of pathogen-associated molecular pattern (PAMP).Therefore the compositions and the method that are used to regulate innate immunity are very interesting, be used for following treatment of diseases method because they can influence, comprise autoimmune, inflammation, allergy, asthma, transplant rejection, graft versus host disease (GvCD), infection, cancer and immunodeficiency.

Have a lot of reports to describe the immunostimulation of some types of nuclear acid molecule recently, these nucleic acid molecules comprise CpG nucleic acid and double-stranded RNA.It should be noted that nearest report Toll sample receptor 9 (TLR9) recognizing bacterial dna and CpG DNA.Hemmi?H?et?al.(2000)Nature?408：740-5；Bauer?S?et?al.(2001)Proc?NatlAcad?Sci?USA?98：9237-42。Recently reported also that the immunocomplex that contains IgG and nucleic acid can stimulate TLR9 and participate in B cell-stimulating in some autoimmune disease.Leadbetter?EA?et?al.(2002)Nature?416：595-8。

Chloroquine is considered to not only can be used as anti-malarial agents but also useful as anti-inflammatory agents.Although its mechanism of action is also not clear, chloroquine has been used to effectively treat the various autoimmune disease, comprises rheumatoid arthritis (RA) and systemic lupus erythematosus (sle) (SLE).Summarize visible Wallace DJ (1996) Lupus 5 Suppl 1:S59-64.Recently Macfarlane and colleague have described and it is reported and can suppress the multiple chloroquine (4-quinolin-2-ylamine) that immune system stimulates and the micromolecule analog and the derivant of atabrine (9-aminoacridine).U.S. Patent No. 6,221,882; U.S. Patent No. 6,479,504; U.S. Patent No. 6,521,637; PCT openly applies for PCT/US00/16723 (WO 00/76982); Openly apply for PCT/US98/13820 (WO 99/01154) with PCT.These micromolecular inhibitors of Macfarlane and colleague report immunne response even effect that also can blocking immunity zest DNA when using with nanomolar concentration.U.S. Patent No. 6,221,882B1.Macfarlane passes through to change the 4-quinolin-2-ylamine relevant with chloroquine and atabrine of limited quantity and the substituent group on the 9-aminoacridine core texture with the colleague, has studied a large amount of chemical compounds.

Summary of the invention

The present invention part is based on applicant's following discovery, promptly multiple micromolecule, and some is known but be different from those that people such as people such as Maclarlane and Tobe describe, can change the immunostimulation signal transmission of TLR mediation.A lot of this chemical compounds suppress the transmission of TLR signal, and can be used as the immunostimulation inhibitor.Compositions described herein and method can be used for suppressing in vitro and in vivo immunostimulation.Therefore these compositionss and method will can be used for the various clinical application, comprise that described disease comprises inflammation and autoimmune disease as treatment and medicine and the method for not expecting the immunocompetence relevant disease.The present composition also can be used for preparing the method for medicine, and described medicine is used for the treatment of and the disease of not expecting that immunocompetence is relevant, comprises multiple inflammation and autoimmune disease.

Wonderful discovery is compared with those relevant with chloroquine and atabrine that people such as Macfarlane describes, and has similar substituent group but the molecule of different core structure is potent immune regulative micromolecule.Not retrained by any theory or mechanism, think that the micromolecule that the present invention describes influences immunostimulation by interacting with TLR.More specifically, think that a lot of micromolecule of the present invention suppresses immunostimulation by the TLR antagonism.Particularly, think that a lot of micromolecule of the present invention suppresses immunostimulation by the TLR9 antagonism.

The present invention is provided for changing the method that the signal of TLR mediation transmits in some aspects.Whenever hope change the TLR mediation, when suitable TLR part or TLR signal are transmitted signal that agonist replys and transmit, the inventive method all is useful.For example, described method can be used for treating any following multiple disease, comprises autoimmune, inflammation, allergy, asthma, transplant rejection, graft versus host disease (GvHD), infection, sepsis, cancer and immunodeficiency.Usually, the method that is used for the treatment of the disease that comprises autoimmune, inflammation, allergy, asthma, transplant rejection and GvHD will utilize suppress the TLR mediation, suitable TLR part or TLR signal are transmitted the micromolecule that signal that agonist replys transmits.Usually, the method that is used for the treatment of the disease that comprises infection, cancer and immunodeficiency will be utilized and strengthen the micromolecule that the signal TLR mediation, that suitable TLR part is replied transmits.In some cases, described method can be used for suppressing or promote the TLR mediation, TLR part or TLR signal are transmitted the signal transmission that agonist is replied.In some cases, described method can be used for suppressing that TLR mediates, TLR part or TLR signal is transmitted the immunostimulating signal transmission that agonist is replied.In some cases, described method can be used for suppressing or promoting the immunostimulation of TLR mediation among the patient.In some cases, described method can be used for suppressing the immunostimulation of TLR mediation among the patient.In some cases, described method can be used for suppressing relevant the replying of immunostimulatory nucleic acid among the patient.

The present invention is provided for changing the new small molecule compositions that the signal of TLR mediation transmits in some aspects.Whenever hope change the TLR mediation, when suitable TLR part or TLR signal are transmitted signal that agonist replys and transmit, the present composition all is useful.For example, described micromolecule can be used for treating the method for any following multiple disease, comprises autoimmune, inflammation, allergy, asthma, transplant rejection, GvHD, infection, sepsis, cancer and immunodeficiency.Usually, the method that is used for the treatment of the disease that comprises autoimmune, inflammation, allergy, asthma, transplant rejection and GvHD will utilize suppress the TLR mediation, suitable TLR part or TLR signal are transmitted the micromolecule that signal that agonist replys transmits.Usually, the method that is used for the treatment of the disease that comprises infection, cancer and immunodeficiency will be utilized and strengthen the micromolecule that the signal TLR mediation, that suitable TLR part is replied transmits.In some cases, described molecule can be used for suppressing or promote the TLR mediation, TLR part or TLR signal are transmitted the method that signal that agonist replys transmits.In some cases, described micromolecule can be used for suppressing the TLR mediation, TLR part or TLR signal are transmitted the method that immunostimulating signal that agonist replys transmits.In some cases, described micromolecule can be used for suppressing or promoting the immunostimulating method of TLR mediation among the patient.In some cases, described micromolecule can be used for suppressing the immunostimulating method of TLR mediation among the patient.In some cases, described micromolecule can be used for suppressing relevant the replying of immunostimulatory nucleic acid among the patient.

As feature of the present invention, the inventive method can with other medicament combined administration to obtain immunostimulating synergism to the TLR mediation.More specifically, also thereby directly influence cell such as the antigen presenting cell (APC) of being with TLR although medicament described herein has been found the direct TLR of influence, these medicaments can be united use with other medicament of non-APC immunocyte of influence such as T lymphocyte (T cell).Such method is effectively introduced the immunomodulating intervention two kinds of levels: innate immunity and acquired immunity.Because innate immunity is considered to start and supports acquired immunity, combinatorial interventions be have synergitic.

In one aspect of the invention, provide and influence the method that signal TLR mediation, that the TLR part is replied transmits.Comprise according in this respect method the cell of expressing TLR contact with the formula I chemical compound of effective dose, with the signal transmission that suppresses or promotion TLR mediates replys the TLR part,

Wherein 2 is five Yuans to seven Yuans homoatomic rings or heterocycle, and wherein each among X, Y and the Z independently is selected from carbon atom (C), nitrogen-atoms (N), oxygen atom (O) and sulphur atom (S), and wherein B2 is optional comprises at least one atom that is selected from C, N, O and S; Wherein the 1 and 2 optional B3 bridgings of pass through form five Yuans to seven Yuans rings (3), and wherein B3 chooses wantonly comprises at least one atom that is selected from C, N, O and S; Wherein when A was carbon, (3) were not pyridines; 2 optional unsaturated bonds that comprise wherein; Wherein (3) are optional comprises unsaturated bond; When R2 exists, be hydrogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group wherein, optional is connected with Z through N, O or S; When R3, R4, R5, R6, R7 and R8 exist, independently be mutually hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group wherein, what each was optional connects through N, O or S; Wherein A is the atom that is selected from C, N, O and S; Wherein A ', A " and A " ' independently be mutually R9 ,-NR9R10 ,-OR9 or-CR9R10, wherein R9 is hydrogen atom, hydroxyl, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what wherein R10 was optional does not exist or hydrogen atom, low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group.

The method of the immunostimulating signal transmission that suppresses the TLR mediation is provided in one aspect of the invention.In this respect method relates to the cell that will express TLR and contact with the formula I chemical compound that as above provides of effective dose according to the present invention, the immunostimulating signal transmission that mediates with inhibition TLR, the TLR part is replied.

In one aspect of the invention, provide the immunostimulating method that influences TLR mediation among the patient.The formula I chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, and to suppress or to promote the immunostimulation of TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

One aspect of the present invention provides the immunostimulating method that suppresses TLR mediation among the patient.The formula I chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, and to suppress the immunostimulation of TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

One aspect of the present invention provides the method that suppresses immunostimulatory nucleic acid associated responses among the patient.The formula I chemical compound that as above provides from effective dose to the patient of this treatment of needs that use is provided in this respect method according to the present invention, to suppress the immunostimulatory nucleic acid associated responses among the described patient.

In one embodiment, described patient does not need the symptom with formula I compounds for treating.

In an embodiment of the aforementioned any aspect according to the present invention, A is a nitrogen, and (3) are five-membered rings.In one embodiment, described chemical compound is a formula II chemical compound,

Wherein A is selected from C and N; R1 is hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S.In multiple specific embodiments, described chemical compound is the chemical compound 455,470,564,568,593,607 listed in the following table 5, among 612-614, the 619-621,636,685,875,878,890,904,918,939,944,1039,1050,1132,1241 and 1243 any one.

In an embodiment of the aforementioned any aspect according to the present invention, A is a nitrogen and (3) are six membered rings, and B3 is an atom that is selected from C, N, O and S.In one embodiment, A is a nitrogen, the 3rd, and six membered ring, B3 are S.In one embodiment, described chemical compound is the formula III chemical compound,

Wherein R1 is hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S, and wherein X is selected from N, O and S.X is S in certain embodiments.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 6 53,64,125,149,313,399,529,576,693,797,840 and 842.

In an embodiment of the aforementioned any aspect according to the present invention, A is a carbon, and (3) are six membered rings, and B3 is C or S.In one embodiment, described chemical compound is a formula IV chemical compound,

Wherein X is C or S, and R1 is hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 7 43,294,340,346,348,413,491,917,1015,1042,1158,1287 and 1337.

In an embodiment of the aforementioned any aspect according to the present invention, A is a nitrogen, and (3) are seven Yuans rings, and B3 comprises two carbon atoms.In one embodiment, described chemical compound is a formula V chemical compound,

Wherein X and Y independently are mutually C, N, O or S; R1 is hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 8 72,74,99,109,343,488,1013 and 1352.

In an embodiment of the aforementioned any aspect according to the present invention, 1 and 2 without the B3 bridging, and A is carbon and A ' is-OR9.In one embodiment, described chemical compound is a formula VI chemical compound,

Wherein R1 is hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S; R9 replaces or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group.R9 and R4 are connected to form lactone in some embodiments.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 9 65,229,239,306,386,707,793,957,970,974,1161 and 1308.

In an embodiment of the aforementioned any aspect according to the present invention, 1 and 2 without the B3 bridging, and A is carbon and A ' is-NR9R10.In one embodiment, described chemical compound is a formula VII chemical compound,

Wherein R1 is hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 10 133,267,312,457 and 510.

In an embodiment of the aforementioned any aspect according to the present invention, 1 and 2 without the B3 bridging, and A is carbon and A ' is-CR9R10.In one embodiment, described chemical compound is formula VIII,

Wherein R1 is hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 11 93,108,138,144,267,270,308,381,560,778,799,822,833,884,965 and 1187.

One aspect of the present invention provides influences the method that the signal TLR mediation, that the TLR part is replied transmits.In this respect method relates to the cell that will express TLR and contact with the formula IX chemical compound of effective dose according to the present invention, with the signal transmission that suppresses or promotion TLR mediates, the TLR part is replied,

Wherein R2 replaces or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S; Wherein R5, R6, R7 and R8 independently are mutually hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S; Wherein R9 is hydrogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group; Wherein R10 is hydrogen atom, low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 12 751,858,2000 and 2001.

The method of the immunostimulating signal transmission that suppresses the TLR mediation is provided in one aspect of the invention.In this respect method relates to the cell that will express TLR and contact with the formula IX chemical compound that as above provides of effective dose according to the present invention, the immunostimulating signal transmission that mediates with inhibition TLR, the TLR part is replied.

In one aspect of the invention, provide the immunostimulating method that influences TLR mediation among the patient.The formula IX chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, to suppress or to promote the immunostimulation of the TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

One aspect of the present invention provides the immunostimulating method that suppresses TLR mediation among the patient.The formula IX chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, and to suppress the immunostimulation of the TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

An aspect of of the present present invention provides the method that suppresses immunostimulatory nucleic acid associated responses among the patient.The formula IX chemical compound that as above provides from effective dose to the patient of this treatment of needs that use is provided in this respect method according to the present invention, to suppress the immunostimulatory nucleic acid associated responses among the described patient.

In one embodiment, described patient does not need the symptom with formula IX compounds for treating.

One aspect of the present invention provides influences the method that the signal TLR mediation, that the TLR part is replied transmits.In this respect method relates to the cell that will express TLR and contact with the formula X chemical compound of effective dose according to the present invention, with the signal transmission that suppresses or promotion TLR mediates, the TLR part is replied,

Wherein R1, R2, R3, R4, R5, R6, R7 and R8 independently are mutually hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 13 431,583,586,792 and 830.

The method of the immunostimulating signal transmission that suppresses the TLR mediation is provided in one aspect of the invention.In this respect method relates to the cell that will express TLR and contact with the formula X chemical compound that as above provides of effective dose according to the present invention, the immunostimulating signal transmission that mediates with inhibition TLR, the TLR part is replied.

In one aspect of the invention, provide the immunostimulating method that influences TLR mediation among the patient.The formula X chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, to suppress or to promote the immunostimulation of the TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

One aspect of the present invention provides the immunostimulating method that suppresses TLR mediation among the patient.The formula X chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, and to suppress the immunostimulation of the TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

An aspect of of the present present invention provides the method that suppresses immunostimulatory nucleic acid associated responses among the patient.The formula X chemical compound that as above provides from effective dose to the patient of this treatment of needs that use is provided in this respect method according to the present invention, to suppress the immunostimulatory nucleic acid associated responses among the described patient.

In one embodiment, described patient does not need the symptom with formula X compounds for treating.

One aspect of the present invention provides influences the method that the signal TLR mediation, that the TLR part is replied transmits.In this respect method relates to the cell that will express TLR and contact with the formula XI chemical compound of effective dose according to the present invention, with the signal transmission that suppresses or promotion TLR mediates, the TLR part is replied,

Wherein each among R1, R2, R5, R6, R7, R8, R11 and the R12 is hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S; X is C, N, O or S.In some embodiments, R2 comprises cycloalkyl, benzyl or phenyl.X is N in some embodiments.R11 and R12 connect into the tap bolt group of five Yuans or six membered ring in some embodiments.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 14 891,926,1137,1213,1248,1320 and 1322.

The method of the immunostimulating signal transmission that suppresses the TLR mediation is provided in one aspect of the invention.In this respect method relates to the cell that will express TLR and contact with the formula XI chemical compound that as above provides of effective dose according to the present invention, the immunostimulating signal transmission that mediates with inhibition TLR, the TLR part is replied.

In one aspect of the invention, provide the immunostimulating method that influences TLR mediation among the patient.The formula XI chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, to suppress or to promote the immunostimulation of the TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

One aspect of the present invention provides the immunostimulating method that suppresses TLR mediation among the patient.The formula XI chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, and to suppress the immunostimulation of TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

An aspect of of the present present invention provides the method that suppresses immunostimulatory nucleic acid associated responses among the patient.The formula XI chemical compound that as above provides from effective dose to the patient of this treatment of needs that use is provided in this respect method according to the present invention, to suppress the immunostimulatory nucleic acid associated responses among the described patient.

In one embodiment, described patient does not need the symptom with formula XI compounds for treating.

One aspect of the present invention provides influences the method that the signal TLR mediation, that the TLR part is replied transmits.In this respect method relates to the cell that will express TLR and contact with the formula XII chemical compound of effective dose according to the present invention, with the signal transmission that suppresses or promotion TLR mediates, the TLR part is replied,

Wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 and R11 independently are mutually hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 15 101,600 and 609.

The method of the immunostimulating signal transmission that suppresses the TLR mediation is provided in one aspect of the invention.In this respect method relates to the cell that will express TLR and contact with the formula XII chemical compound that as above provides of effective dose according to the present invention, the immunostimulating signal transmission that mediates with inhibition TLR, the TLR part is replied.

In one aspect of the invention, provide the immunostimulating method that influences TLR mediation among the patient.The formula XII chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, to suppress or to promote the immunostimulation of TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

One aspect of the present invention provides the immunostimulating method that suppresses TLR mediation among the patient.The formula XII chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, and to suppress the immunostimulation of TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

An aspect of of the present present invention provides the method that suppresses immunostimulatory nucleic acid associated responses among the patient.The formula XII chemical compound that as above provides from effective dose to the patient of this treatment of needs that use is provided in this respect method according to the present invention, to suppress the immunostimulatory nucleic acid associated responses among the described patient.

In one embodiment, described patient does not need the symptom with formula XII compounds for treating.

One aspect of the present invention provides influences the method that the signal TLR mediation, that the TLR part is replied transmits.In this respect method relates to the cell that will express TLR and contact with the formula XIII chemical compound of effective dose according to the present invention, with the signal transmission that suppresses or promotion TLR mediates, the TLR part is replied,

Wherein 2 ' is five Yuans wherein each among X, Y and the Z independently is selected from C, N, O and S to seven element heterocycles, and wherein B2 ' is optional comprises at least one atom that is selected from C, N, O and S; 2 ' the optional unsaturated bond that comprises wherein; Wherein R4, R5, R6, R7 and R8 independently are mutually hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S; Wherein R9 is hydrogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group; Wherein R10 is hydrogen atom, low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group; A wherein " be hydrogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 16 990,1003,1091,1142,1185,1212,1217,1224,1244 and 1334.

The method of the immunostimulating signal transmission that suppresses the TLR mediation is provided in one aspect of the invention.In this respect method relates to the cell that will express TLR and contact with the formula XIII chemical compound that as above provides of effective dose according to the present invention, the immunostimulating signal transmission that mediates with inhibition TLR, the TLR part is replied.

In one aspect of the invention, provide the immunostimulating method that influences TLR mediation among the patient.The formula XIII chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, to suppress or to promote the immunostimulation of TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or has, and the risk that this degree of exempting from stimulates takes place.

One aspect of the present invention provides the immunostimulating method that suppresses TLR mediation among the patient.The formula XIII chemical compound that as above provides from effective dose to the patient that use is provided in this respect method according to the present invention, and to suppress the immunostimulation of TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

An aspect of of the present present invention provides the method that suppresses immunostimulatory nucleic acid associated responses among the patient.The formula XIII chemical compound that as above provides from effective dose to the patient of this treatment of needs that use is provided in this respect method according to the present invention, to suppress the immunostimulatory nucleic acid associated responses among the described patient.

Relate to formula XIII chemical compound aforementioned aspect each in, A in one embodiment " be the substituted alkyl that is selected from ring amino, alkylamino, dialkyl amido, furyl, phenyl, thienyl, azabicyclo octyl group, azabicyclo heptyl and combination in any thereof.In one embodiment, described ring amino is piperazinyl (piperazino), piperidyl (piperidino), pyrrolidinyl (pyrrolidino), imidazole radicals, pyridine radicals or morpholinyl (morpholino).

In one embodiment, described patient does not need the symptom with formula XIII compounds for treating.

One aspect of the present invention provides influences the method that the signal TLR mediation, that the TLR part is replied transmits.Limit relates to the cell that will express TLR according to the present invention's method in this respect and contact with the formula XIV or the formula XV chemical compound of effective dose, with the signal transmission that suppresses or promotion TLR mediates, the TLR part is replied,

Wherein R1, R2, R3, R5, R6, R7 and R8 independently are mutually hydrogen atom, halogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group, and what each was optional connects through N, O or S; Wherein R9 is hydrogen atom, replacement or unsubstituted low alkyl group, aryl, aralkyl, heterocycle or alkyl heterocycle group.In multiple specific embodiments, described chemical compound is any one in the chemical compound of listing in the following table 17 807,1163 and 1367.

The method of the immunostimulating signal transmission that suppresses the TLR mediation is provided in one aspect of the invention.In this respect method relates to the cell that will express TLR and contact with the formula XIV that as above provides or the formula XV chemical compound of effective dose according to the present invention, the immunostimulating signal transmission that mediates with inhibition TLR, the TLR part is replied.

In one aspect of the invention, provide the immunostimulating method that influences TLR mediation among the patient.Formula XIV that as above provides from effective dose to the patient or the formula XV chemical compound of using is provided in this respect method according to the present invention, to suppress or to promote the immunostimulation of TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

One aspect of the present invention provides the immunostimulating method that suppresses TLR mediation among the patient.Formula XIV that as above provides from effective dose to the patient or the formula XV chemical compound of using is provided in this respect method according to the present invention, to suppress the immunostimulation of TLR mediation among the described patient, described patient has the immunostimulation of TLR mediation or this immunostimulating risk of generation is arranged.

An aspect of of the present present invention provides the method that suppresses immunostimulatory nucleic acid associated responses among the patient.Formula XIV that as above provides from effective dose to the patient of this treatment of needs or the formula XV chemical compound of using is provided in this respect method according to the present invention, to suppress the immunostimulatory nucleic acid associated responses among the described patient.

In one embodiment, described patient does not need the symptom with formula XIV or formula XV compounds for treating.

The present invention aforesaid aspect each in, in some embodiments, R2 replaces or unsubstituted phenyl, naphthyl, phenanthryl, styryl, azabicyclo-octane or azabicycloheptane base.In some embodiments, R2 is phenyl, naphthyl, phenanthryl, styryl, azabicyclo-octane or the azabicycloheptane base that is replaced by the one or more substituent groups that are selected from alkyl, alkoxyl, alkoxyalkyl, ester group, alkylamino, dialkyl amido, ring amino, halogen atom and combination in any thereof.In some embodiments, R2 is by being selected from phenyl, naphthyl, phenanthryl, styryl, azabicyclo-octane or the azabicycloheptane base that the amino one or more substituent groups of alkylamino, dialkyl amido and ring replace.In some embodiments, described ring amino is piperazinyl, piperidyl, pyrrolidinyl, imidazole radicals, pyridine radicals or morpholinyl.

The present invention aforesaid aspect each in, in some embodiments, R9 is the substituted alkyl that is selected from ring amino, alkylamino, dialkyl amido, furyl, phenyl, thienyl, azabicyclo octyl group, azabicyclo heptyl and combination in any thereof.In some embodiments, described ring amino is piperazinyl, piperidyl, pyrrolidinyl, imidazole radicals, pyridine radicals or morpholinyl.

The present invention aforesaid aspect each in, in some embodiments, each of R4 and R5 is a hydrogen atom.Each of R5 and R8 is a hydrogen atom in some embodiments.Each of R4, R5 and R8 is a hydrogen atom in some embodiments.R10 is a hydrogen atom in some embodiments.

The present invention aforesaid aspect each in, in one embodiment, described TLR is TLR9.In one embodiment, the part of described TLR is an immunostimulatory nucleic acid.In one embodiment, described immunostimulatory nucleic acid is a CpG nucleic acid.

The present invention aforesaid aspect each in, in one embodiment, described TLR is TLR8.In one embodiment, the part of described TLR is the native ligand of TLR8.In one embodiment, the part of described TLR is resiquimod (R848).

The present invention aforesaid aspect each in, in one embodiment, described TLR is TLR7.In one embodiment, the part of described TLR is the native ligand of TLR7.In one embodiment, the part of described TLR is R848.

The present invention aforesaid aspect each in, in one embodiment, described TLR is TLR3.In one embodiment, the part of described TLR is a double-stranded RNA.In one embodiment, the part of described TLR is poly (I:C).

The present invention part is recognized potential contact the between the biological activity of antimalarial active and relevant micromolecule inhibition TLR9 of known metabolite of chloroquine based on the applicant.For chloroquine, main metabolite is singly to remove ethyl chloroquine and two ethyl chloroquine that goes.

The chloroquine list removes the two ethyls (11) that go of ethyl (1)

Singly go the ethyl chloroquine people intravital half life than chloroquine long 1.5 times (649 hours with 432 hours).de?Vries?PJ?etal.(1994)Drug?Invest?8：143-9。There are not to obtain two data of removing the ethyl chloroquine.In addition, the major metabolite of hydroxychloroquine is the ethyl hydroxychloroquine, singly removes ethyl chloroquine and two ethyl chloroquine that goes.In malaria treatment, the chloroquine and the usefulness of singly removing the anti-plasmodium falciparum of ethyl chloroquine about equally, and two nearly half activity of ethyl chloroquine of going.Aderounmu?AF(1984)Ann?Trop?Med?Parasitol.78(6)：581-5。Because side chain is a chirality, R and S chloroquine have different activities in addition, preferred (S)-(+)-singly the go ethyl chloroquine that generates.Ansari?AM?et?al.(1994)J?PharmSci.83(7)：1040-2。Therefore the applicant proposes to suppose that chloroquine and hydroxychloroquine go the derive substantive part of its curative effect of ethyl metabolite from life-prolonging, and this relation can extend to and the combining of TLR9.

Macfarlane empirical tests chloroquine, hydroxychloroquine, singly remove ethyl chloroquine and two ethyl chloroquine that goes.The IC50 of these chemical compounds is in its test: chloroquine, 1.1 * 10 ^-7M; Hydroxychloroquine, 4.1 * 10 ^-7M; Singly remove the ethyl chloroquine, 7.08 * 10 ^-7M; With two ethyl chloroquine, 18.6 * 10 gone ^-7M.Therefore lose an ethyl from these data from chloroquine and make usefulness reduce about 7 times, lose two ethyls and then make usefulness reduce about 18 times.

The present invention relates to and relevant compositions and the method for some 4-primary amino radical quinoline compound that is used to suppress transmission of TLR9 signal and immune activation in some aspects.The present invention also relates to compositions and the method relevant in some aspects with some quinazoline compound, described chemical compound of some of them and 4-primary amino radical quinoline compound structurally associated of the present invention, they also can be used for suppressing transmission of TLR9 signal and immune activation.

Find shockingly that according to the present invention 4-primary amino radical quinoline compound of the present invention is that highly active TLR9 signal transmits activity inhibitor and active similar with quinazoline compound.Find shockingly also that according to the present invention although they have the structure and the biological similarity of height, compare with described quinoline compound, described quinazoline molecules shows the toxicity in vivo spectrum of remarkable improvement.

One aspect of the present invention provides new replacement 4-primary amino radical quinoline component and pharmaceutically acceptable hydrate and the salt with structural formula XVI,

Wherein

X does not exist or aryl, alkyl, heterocyclic radical or styryl;

R ₁And R ₂Independently be mutually hydrogen atom or replacement or unsubstituted alkyl, alkane thiazolinyl or aryl, wherein R ₁And R ₂Randomly be combined to form heterocycle;

R ₃Be hydrogen atom, halogen atom, or alkyl, alkane thiazolinyl, aryl, heterocyclic radical, nitro, cyano group, carboxyl, ester group, ketone, amino, acylamino-, hydroxyl, alkoxyl, sulfydryl, sulfo-, sulfoxide, sulfone or sulfonamido, wherein R ₁And R ₃Randomly be combined to form heterocycle;

Y does not exist or oxygen atom, sulphur atom, CR ₈R ₉Or NR ₁₀, R wherein ₈, R ₉And R ₁₀Independently be mutually hydrogen atom or replacement or not substituted alkyl, alkane thiazolinyl or aryl;

L is alkyl or alkane thiazolinyl or the aryl that contains 1 to 10 carbon; And

R ₄, R ₅, R ₆And R ₇Independently be mutually hydrogen atom, halogen atom, or alkyl, alkane thiazolinyl, aryl, heterocyclic radical, nitro, cyano group, carboxyl, ester group, ketone, amino, acylamino-, hydroxyl, alkoxyl, sulfydryl, sulfo-, sulfoxide, sulfone or sulfonamido, wherein R ₄, R ₅, R ₆And R ₇Close on mutually arbitrarily to randomly being combined to form heterocycle or carbocyclic ring.

In one embodiment, R ₅And R ₆Independently be mutually halogen atom or alkoxyl.In one embodiment, R ₅And R ₆Independently be mutually chlorine atom or methoxyl group.

X does not exist or aryl in one embodiment;

NR ₁R ₂Be heterocyclic amine or NR ₈(CH ₂) _nNR ₉R ₁₀, wherein n is 2 to 6 integer, comprises 2 and 6, R ₈, R ₉And R ₁₀Independently be mutually hydrogen atom or alkyl;

R ₃It is hydrogen atom;

Y is aryl or NR ₁₁, R wherein ₁₁Be hydrogen atom or aryl or alkyl;

L does not exist or C ₂-C ₆Alkyl; And

R ₄, R ₅, R ₆And R ₇Independently be mutually hydrogen atom, halogen atom or alkoxyl.

In one embodiment, X does not exist or aryl;

NR ₁R ₂Be to replace or unsubstituted piperazinyl or morpholinyl or NR ₈(CH ₂) _nNR ₉R ₁₀, wherein n is 2 to 6 integer, comprises 2 and 6, R ₈Be hydrogen atom, R ₉And R ₁₀It independently is mutually alkyl;

R ₃It is hydrogen atom;

Y is NH;

L is C ₂-C ₆Alkyl; And

In one embodiment, X is a phenyl;

NR ₁R ₂Be connected to described phenyl X para-position

Y is NH;

L is-(CH ₂) _n-, wherein n is 2 to 6 integer, comprises 2 and 6; And

R ₃, R ₄, R ₅, R ₆And R ₇In each be hydrogen atom.

In aforesaid each embodiment, what described component was optional is pharmaceutically acceptable hydrate or salt form.

One aspect of the present invention provides the method for inhibition through the signal transmission of TLR.The cell that in this respect method relates to expressive function TLR according to the present invention contacts with salt with its pharmaceutically acceptable hydrate of replacement 4-primary amino radical quinoline component with structural formula XVII of effective dose, suppressing signal transmission through TLR,

Wherein

X does not exist or nitrogen, oxygen or sulphur atom or SO or SO ₂Group;

R ₁Be hydrogen atom or replacement or unsubstituted aryl, alkyl, heterocycle or styryl;

R ₂Be hydrogen atom, halogen atom, or alkyl, alkane thiazolinyl, aryl, heterocyclic radical, nitro, cyano group, carboxyl, ester group, ketone, amino, acylamino-, hydroxyl, alkoxyl, sulfydryl, sulfo-, sulfoxide, sulfone or sulfonamido, wherein R ₁And R ₂Randomly be combined to form heterocycle or carbocyclic ring;

Y does not exist or oxygen atom, sulphur atom, CR ₇R ₈Or NR ₉, R wherein ₇, R ₈And R ₉Independently be mutually hydrogen atom or replacement or not substituted alkyl, alkane thiazolinyl or aryl;

There is not or contain alkyl or the alkane thiazolinyl or the aryl of 1 to 10 carbon in L; And

R ₃, R ₄, R ₅And R ₆Independently be mutually hydrogen atom, halogen atom, or alkyl, alkane thiazolinyl, aryl, heterocyclic radical, nitro, cyano group, carboxyl, ester group, ketone, amino, acylamino-, hydroxyl, alkoxyl, sulfydryl, sulfo-, sulfoxide, sulfone or sulfonamido, wherein R ₃, R ₄, R ₅And R ₆Close on mutually arbitrarily to randomly being combined to form heterocycle or carbocyclic ring.

One aspect of the present invention provides the method for inhibition through the signal transmission of TLR.In this respect method relates to the immunocyte with expressive function TLR according to the present invention

(a) contact with the TLR signal agonist of effective dose lacking to replace under the 4-primary amino radical quinoline component, stinging the signal transmission of menstruating on time after pregnancy TLR, and

(b) with effective dose have as defined above that the replacement 4-primary amino radical quinoline component of structural formula XVII contacts, to replace the signal transmission that TLR under the 4-primary amino radical quinoline component replys TLR signal agonist and compare with lacking, suppress the signal transmission that TLR replys TLR signal agonist.

In one embodiment, described TLR is TLR9, and described TLR signal agonist is a TLR9 signal agonist.Described in one embodiment TLR signal agonist is CpG DNA, and it can be oligodeoxynucleotide (ODN).

In one embodiment, described TLR signal agonist is an immunocomplex.In one embodiment, described TLR signal agonist is the immunocomplex that comprises nucleic acid.In one embodiment, to be included nucleic acid be the immunocomplex of self DNA to described TLR signal agonist.In one embodiment, to be included nucleic acid be the immunocomplex of self RNA to described TLR signal agonist.

One aspect of the present invention provides the method for inhibition to the immunne response of antigenicity substance.In this respect method relates to the immunocyte with expressive function Toll sample receptor according to the present invention

(a) contact with the antigenicity substance of effective dose lacking to replace under the 4-primary amino radical quinoline component, with the immunne response of stimulation to described antigenicity substance, and

(b) contact with the replacement 4-primary amino radical quinoline component that has as above-mentioned structural formula XVII of effective dose,, suppress immunne response described antigenicity substance the immunne response of described antigenicity substance is compared with lacking to replace under the 4-primary amino radical quinoline component.

In one embodiment, described immunne response is that innate immunity is replied.In one embodiment, described immunne response comprises adaptive immune response.

In one aspect of the invention, provide the treatment patient method of autoimmune disease.In this respect method relates to the replacement 4-primary amino radical quinoline component as above-mentioned structural formula XVII of having from effective dose to the patient who suffers from autoimmune disease that use according to the present invention, to treat described autoimmune disease.In one embodiment, described autoimmune disease is selected from systemic lupus erythematosus (sle), rheumatoid arthritis, inflammatory bowel, xerodermosteosis, polymyositis, vasculitis, wegener granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter syndrome, arthritic psoriasis and behcet syndrome.In a specific embodiments, described autoimmune disease is a systemic lupus erythematosus (sle).In a specific embodiments, described autoimmune disease is a rheumatoid arthritis.In one embodiment, described patient is the people.In one embodiment, described autoimmune disease is the disease relevant with immunocomplex.

One aspect of the present invention provides new quinazoline component and pharmaceutically acceptable hydrate and the salt with structural formula XVIII of the present invention,

Wherein

X does not exist or aryl, alkyl, heterocyclic radical or styryl, if X is a phenyl, and NR ₁R ₂Be heterocyclic part or diamidogen;

R ₁And R ₂Independently be mutually hydrogen atom or alkyl, alkane thiazolinyl or aryl, wherein R ₁And R ₂Randomly be combined to form heterocycle;

Y is oxygen atom, sulphur atom, CR ₉R ₁₀Or NR ₁₁, R wherein ₉, R ₁₀And R ₁₁Independently be mutually hydrogen atom or alkyl, alkane thiazolinyl or aryl, wherein R ₉, R ₁₀And R ₁₁In any one optional and R ₃Or R ₄Be combined to form and replace or unsubstituted heterocycle;

L is alkyl or alkane thiazolinyl or the aryl that contains 1 to 10 carbon;

R ₃And R ₄Independently be mutually hydrogen atom or alkyl, alkane thiazolinyl or aryl, wherein R ₃And R ₄Randomly be combined to form heterocycle; And

R ₅, R ₆, R ₇And R ₈Independently be mutually hydrogen atom, halogen atom, or alkyl, alkane thiazolinyl, aryl, heterocyclic radical, nitro, cyano group, carboxyl, ester group, ketone, amino, acylamino-, hydroxyl, alkoxyl, sulfydryl, sulfo-, sulfoxide, sulfone or sulfonamido, wherein R ₅, R ₆, R ₇And R ₈Close on mutually arbitrarily to randomly being combined to form heterocycle.

In one embodiment, R ₆And R ₇Independently be mutually halogen atom or alkoxyl.

In one embodiment, R ₆And R ₇Independently be mutually chlorine atom or methoxyl group.

X does not exist or aryl in one embodiment;

NR ₁R ₂Be heterocyclic amine or NR ₉(CH ₂) _nNR ₁₀R ₁₁, wherein n is 2 to 6 integer, comprises 2 and 6, R ₉, R ₁₀And R ₁₁Independently be mutually hydrogen atom or alkyl;

Y is aryl or NR ₁₂, R wherein ₁₂Be hydrogen atom or aryl or alkyl;

L does not exist or C ₂-C ₆Alkyl;

R ₃And R ₄Independently be mutually hydrogen atom or alkyl, wherein R ₃And R ₄Randomly be combined to form heterocycle; And

R ₅, R ₆, R ₇And R ₈Independently be mutually hydrogen atom, halogen atom or alkoxyl.

In one embodiment, X does not exist or aryl;

NR ₁R ₂Be to replace or unsubstituted piperazinyl or morpholinyl or NR ₉(CH ₂) _nNR ₁₀R ₁₁, wherein n is 2 to 6 integer, comprises 2 and 6, R ₉Be hydrogen atom, R ₁₀And R ₁₁It independently is mutually alkyl;

Y is NH;

L is C ₂-C ₆Alkyl;

In one embodiment, X is an aryl;

NR ₁R ₂Be to replace or unsubstituted piperazinyl or morpholinyl;

Y is NH;

L is C ₂-C ₆Alkyl;

R ₃And R ₄Independently be mutually methyl or ethyl or R ₃And R ₄Randomly be combined to form morpholinyl; And

In one embodiment, X is a phenyl;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄It all is methyl; And

R ₅, R ₆, R ₇And R ₈In each be hydrogen atom.

In one embodiment, X is a phenyl;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄Be combined to form morpholinyl; And

R ₅, R ₆, R ₇And R ₈In each be hydrogen atom.

In one embodiment, X does not exist;

NR ₁R ₂Be to replace or unsubstituted piperazinyl or morpholinyl;

Y is NH;

L is C ₂-C ₆Alkyl;

R ₃And R ₄Independently be mutually methyl or ethyl, or R ₃And R ₄Randomly be combined to form morpholinyl; And

In one embodiment, X does not exist;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄It all is methyl;

R ₆And R ₇It all is methoxyl group; And

R ₅And R ₈It all is hydrogen atom.

In one embodiment, X does not exist;

NR ₁R ₂It is the N-phenylpiperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄It all is methyl;

R ₆And R ₇It all is methoxyl group; And

R ₅And R ₈It all is hydrogen atom.

In one embodiment, X does not exist;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄Be combined to form morpholinyl;

R ₆And R ₇It all is methoxyl group; And

R ₅And R ₈It all is hydrogen atom.

One aspect of the present invention provides the method for inhibition through the signal transmission of TLR.The cell that in this respect method relates to expressive function TLR according to the present invention contacts with salt with the quinazoline component with structural formula XIX and the pharmaceutically acceptable hydrate thereof of effective dose, suppressing signal transmission through TLR,

Wherein

X replaces or unsubstituted aryl, alkyl, heterocycle or styryl, and is optional through nitrogen, oxygen or sulphur atom or through SO or SO ₂Group is connected with quinazoline;

Y does not exist or oxygen atom, sulphur atom, CR ₉R ₁₀Or NR ₁₁, R wherein ₉, R ₁₀And R ₁₁Independently be mutually hydrogen atom or alkyl, alkane thiazolinyl or aryl, wherein R ₉, R ₁₀And R ₁₁In any one optional and R ₃Or R ₄Be combined to form heterocycle;

L does not exist or hydrogen atom, the alkyl that contains 1 to 10 carbon or alkane thiazolinyl or aryl;

R ₅, R ₆, R ₇And R ₈Independently be mutually hydrogen atom, halogen atom, or alkyl, alkane thiazolinyl, aryl, heterocyclic radical, nitro, cyano group, carboxyl, ester group, ketone, amino, acylamino-, hydroxyl, alkoxyl, sulfydryl, sulfo-, sulfoxide, sulfone or sulfonamido, wherein R ₅, R ₆, R ₇And R ₈Close on mutually arbitrarily to randomly being combined to form heterocycle or carbocyclic ring.

(a) contact with the TLR signal agonist of effective dose lacking under the quinazoline component, with the signal transmission of thorn menstruating on time after pregnancy TLR, and

(b) with effective dose have as defined above that the quinazoline component of structural formula XIX contacts, with lack the quinazoline component under the TLR signal transmission of replying TLR signal agonist compare, suppress the signal transmission that TLR replys TLR signal agonist.

In one embodiment, described TLR signal agonist is the immunocomplex that comprises nucleic acid.

(a) contact with the antigenicity substance of effective dose lacking under the quinazoline component, stimulating immunne response to described antigenicity substance, and

(b) contact with the quinazoline component that has as above-mentioned structural formula XIX of effective dose, with lack the quinazoline component under the immunne response of described antigenicity substance is compared, suppress immunne response to described antigenicity substance.

In one aspect of the invention, provide the treatment patient method of autoimmune disease.In this respect method relates to the quinazoline component as above-mentioned structural formula XIX of having from effective dose to the patient who suffers from autoimmune disease that use according to the present invention, with the treatment autoimmune disease.In one embodiment, described autoimmune disease is selected from systemic lupus erythematosus (sle), rheumatoid arthritis, inflammatory bowel, xerodermosteosis, polymyositis, vasculitis, wegener granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter syndrome, arthritic psoriasis and behcet syndrome.In a specific embodiments, described autoimmune disease is a systemic lupus erythematosus (sle).In a specific embodiments, described autoimmune disease is a rheumatoid arthritis.In one embodiment, described patient is the people.In one embodiment, described autoimmune disease is as the above-mentioned disease relevant with immunocomplex.

Description of drawings

Representative molecular compound 613 (hydrochloric acid yageine), 878 (3-HMCs) and 470 (norharman) of Fig. 1 expression II chemical compound to reply CpG ODN 2006 (5 '-TCGTCGTTTTGTCGTTTTGTCGTT-3 '; SEQ ID NO:1) the concentration dependent inhibitory action that TLR9 signal transmits.

Fig. 2 is a pair of figure of specific quinoline of expression and the toxicity in vivo test result of quinazoline compound in mice.Fig. 2 A represents the variation (gram) of average weight, and Fig. 2 B represents that using 1mg and 4mg (being respectively open and closed triangle), Compound C MZ 203-34 with dimethyl sulfoxide (DMSO) contrast (closed circle), Compound C MZ 203-43 (being also referred to as 203-43 herein) uses that 1mg and 4mg (being respectively open and closed rhombus) and Compound C MZ 203-49 use 1mg and 4mg (being respectively open and closed del) handles 5 days average weight of mice.Each processed group N=3.It is ill and put to death at the 3rd day to accept the group of 4mg CMZ 203-43.

Fig. 3 is a block diagram, and the expression intraperitoneal is used the overall of mice that the specific quinoline of single dose and quinazoline compound measure after 5 days and broken up numeration of leukocyte.Each processed group N=3.

Fig. 4 is a block diagram, represents with the interior inhibitory action of body of after PBS contrast or quinoline compound (203-43) or multiple indicated quinazoline compound (203-34,203-49 or the 203-51) pretreatment CpG ODN 2006 being induced IP-10 (pg/ml).Every group of N=5.

Fig. 5 is a block diagram, represents with the interior inhibitory action of body of after PBS contrast or quinoline compound (203-43) or multiple indicated quinazoline compound (203-34,203-49 or the 203-51) pretreatment CpG ODN 2006 being induced TNF-α (pg/ml).Every group of N=5.

The specific embodiment

The invention provides the new compositions and the method that suppress immunne response, described immunne response comprises the immunne response that participates in clinical disease, and described clinical disease is characterised in that autoimmune disease and immunocomplex relevant disease.Except new component disclosed herein, the quinazoline that also comprises 4-primary amino radical quinoline and structural similarity, the present invention also provides the method that the known component in these and other compounds is used to suppress immunne response, described immunne response comprises the immunne response that participates in clinical disease, and described clinical disease is characterised in that autoimmune disease and immunocomplex relevant disease.

Discovery when the present invention's part is screened the micromolecular compound storehouse based on the applicant in the testing in vitro that the TLR9 signal transmits, the transmission of described TLR9 signal is replying immunostimulating CpG oligodeoxynucleotide (ODN).Observe, some micromolecule in the described storehouse can effectively change the TLR9 signal transmission that CpG ODN is replied.Based on results of preliminary screening, select additional compounds to screen again.Importantly, the micromolecule of so identifying be different from Macfarlane and colleague described those.

Have been found that according to the present invention some micromolecule with some minimum characteristics can regulate the transmission of TLR signal, for example have PAMP or other TLR part or as replying to them.Some described micromolecule are potent inhibitors that the TLR signal transmits, thereby can be used for suppressing the immunostimulation of TLR mediation.A lot of but non-all described micromolecular minimum characteristics can be summarized as follows: comprise the core texture of at least two rings, they can merge or not merge, and wherein at least one ring contains nitrogen-atoms and/or has the side chain of nitrogen atom.Some described molecule even still effective when not having nitrogen-atoms.

Observe, the tricyclic structure of fusion such as atabrine (quinacrine) are typically renderd a service a strong order of magnitude than two ring structures such as the chloroquine that merge as the immunostimulation inhibitor of TLR mediation.For example, it is reported the EC of atabrine ₅₀Be about 8nM, wherein EC ₅₀Be to exist the half maximum that CpG-ODN takes in the thymus pyrimidine effect to WEHI 231 B cells under the anti-surperficial IgM condition to suppress needed concentration.U.S. Patent No. 6,479,504B1.Have been found that also that according to the present invention the fusion two nucleolus core structures that connect nitrogenous ring substituents can have same strong effectiveness with fusion tricyclic structure such as atabrine.Concrete, these chemical compounds can show the EC of low nanomole scope ₅₀In certain embodiments, described nitrogenous ring substituents is tertiary amine capped C ₂-C ₆Alkyl as Methylethyl amine, diethylamine, dimethylamine, or contains the ring of at least one nitrogen.

Be not subjected to the restriction of any concrete theory or mechanism, the inventor thinks that the core texture and the side chain substituents that contain ring all have contribution to affinity and curative effect.The inventor thinks, has the substituent molecule of high-affinity core texture and low-affinity and has the low-affinity core texture and the substituent molecule of high-affinity is effective equally.

Observed for many years and be used to influence that in fact some common type medicine of purpose has the immunosuppressant of comprising side effect outside the immune system.For example, reported that phenothiazine chlorpromazine (phenothiazinechlorpromazine) can suppress the messenger RNA expression of interleukin-22 (IL-2), tumor necrosis factor (TNF-α) and interferon gamma (IFN-γ).Schleuning?MJ?et?al.(1989)Eur?J?Immunol?19：1491-6。In other research, the report chlorpromazine makes Cavia porcellus reduce the contact hypersensitivity of dinitrochlorobenzene.Descotes?J?et?al.(1982)JNeuroimmunol?2：21-5。The acute high dose of report is used the inductive TNF-α of tricyclics thing imipramine inhibition lipopolysaccharide (LPS) in another research increases, but influences very little or not influence to inductive interleukin-11 β of LPS (IL-1 β) or interleukin-11 0 (IL-10).Dredge?K?et?al.(1999)Int?J?Immunopharm?21：663-73。

Histamine is the allergy immediately extensively admitted and the medium of inflammation, and they mainly are the immunological phenomena of mastocyte and basophil mediation.The multiple-effect effect of histamine is by the relevant histamine receptor mediation of three types of films, and they are histamine H1-receptor (H1R), histamine H2-receptor (H2R) and histamine H 3 receptor (H3R).The pharmacology inhibitor of these receptors is known, comprises pyrilamine and tripelennamine (H1R); Cimetidine, famotidine and ranitidine (H2R); With sulfur third miaow amine and clobenpropit (H3R).Report strengthens replying of T cell and B cell antigen receptor mediation through the signal transmission of H1R recently.Banu?Y?et?al.(1999)J?Exp?Med?189：673-82。It is reported and derive from H1R disappearance (H1R ^-/-) T cell and the B cell of mice have normal response to LPS.People such as Banu report that also using H2R specific antagonists famotidine further suppresses H1R ^-/-The mediation of T cell and B cell antigen receptor replys in the mice.Jutel and colleague are reported in their the T cell research CD recently ⁴⁺The Th1 subgroup of T cell is preferentially expressed H1R, and the Th2 cell is preferentially expressed H2R.They report that also histamine strengthens the Th1-type by triggering H1R and replys H1R ^-/-Th1 cytokine IFN-γ level is suppressed and Th2 cytokine IL-4 and IL-13 level increase in the mice.Jutel?M?et?al.(2001)Nature?413：420-5。

Comparatively speaking, for dendritic cell (DC), Mazzoni and other people have been reported among the DC of the cells of monocytic origin that LPS stimulates histamine and have suppressed IL-12 and produce and stimulate the IL-10 secretion, cause the transformation to the polar immunne response of Th2 from Th1.Mazzoni?A?et?al.(2001)J?Clin?Invest?108：1865；Caron?G?et?al.(2001)J?Immunol?166：6000-6。Recently people such as Mazzoni reports that also they observe: to be exposed to CpG ODN or the replying of the influenza virus that lives in, histamine discharges I type IFN and TNF-α by the main generation person's Plasmacytoid type DC (pDC) that acts on H2R and suppress IFN-α.Mazzoni?A?et?al.(2003)J?Immunol?170：2269-73。At last, report that recently histamine acts on monocyte/macrophage by H2R and suppresses nadph oxidase, the key enzyme that oxygen-derived free radicals forms causes conservation of nature to kill and wound (NK) cell and inductive dysfunction of T cell resistance oxygen-derived free radicals and apoptosis.Hellstrand?K(2002)Semin?Oncol?29(3?Suppl?7)：35-40。

Definition

Unless otherwise defined, all technology used herein and scientific terminology and one skilled in the art of the present invention conventional understand have an equivalent.

As used herein, the antigen-specific immune response of term " adaptive immune response " expression any kind.Adaptive immune response relates to lymphocyte, is also referred to as specific immune response in this area, it is characterized in that immunological memory, thus to for the second time or be exposed to antigen afterwards and have than being exposed to stronger the replying of described antigen for the first time.The term adaptive immune response comprises body fluid (antibody) immunity and cell-mediated (cell) immunity.

As used herein, " allergy " expression is to the acquired allergy of material (allergen).Allergic disease comprises eczema, allergic rhinitis, pollinosis (hay fever), asthma, rubella (urticaria) and food allergy, and other specificity allergic disease.

As used herein, any material of adaptability (specificity) immunne response is induced in term " antigenicity substance " expression.Antigen is typically can be by the bonded any material of T cell antigen receptor, antibody or B cell antigen receptor specificity.Antigenicity substance includes but not limited to peptide, protein, carbohydrate, fat, phospholipid, nucleic acid, autacoid and hormone.Antigenicity substance further specifically comprises the antigen that ranges allergen, cancer antigen and microbial antigen.

As used herein, " asthma " represents a kind of respiratory system disease, it is characterized in that inflammation, the air flue stenosis is narrow and air flue increases the reactivity of inhalation (inhalatio).Although be not limited to this, asthma is often relevant with atopy or allergic conditions.For example by be exposed to allergen, be exposed to cold air, respiratory tract infection and fatigue, asthma can take place suddenly.

As used herein, term " autoimmune disease " and " autoimmune disease " and " autoimmune " expression immunology of equal value mediate to the tissue that derives from the host or the acute or chronic injury of organ.This term comprises cell and antibody-mediated autoimmune phenomena, and organ specificity and the non-specific autoimmune of organ.Autoimmune disease comprises insulin-dependent diabetes, rheumatoid arthritis, systemic lupus erythematosus (sle), multiple sclerosis, arteriosclerosis and inflammatory bowel.Autoimmune disease also includes but not limited to ankylosing spondylitis, autoimmune hemolytic anemia, behcet syndrome, goodpasture's syndrome, Graves disease, barre-Guillaian syndrome, chronic lymphocytic thyroiditis, the special property sent out thrombocytopenia, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polymyositis/dermatomyositis, the sclerosis of constitutional gallbladder, psoriasis, sarcoidosis, sclerosing cholangitis, xerodermosteosis, systemic sclerosis (scleroderma and CREST syndrome), aortic arch syndrome (takayasu ' s arteritis), temporal arteritis (temporal arteritis) and wegener granulomatosis.Autoimmune disease also comprises the disease that some is relevant with immunocomplex.

As used herein, term " cancer " and " tumor " of equal value are illustrated in the disease of the unusual replicating cell that has detectable host source among the patient.Cancer can be pernicious or non-malignant cancer.Cancer or tumor include but not limited to cancer of biliary duct; The brain cancer, breast carcinoma; Cervical cancer; Choriocarcinoma; Colon cancer; Carcinoma of endometrium; The esophageal carcinoma; Gastric cancer; Last Intradermal tumor, leukemia; Lymphoma; Hepatocarcinoma; Pulmonary carcinoma (for example minicell and nonsmall-cell lung cancer); Melanoma; Neuroblastoma; Oral cancer; Ovarian cancer; Cancer of pancreas; Carcinoma of prostate; Rectal cancer; Renal carcinoma; Sarcoma; Skin carcinoma; Carcinoma of testis; Thyroid carcinoma; And other cancer and sarcoma.Cancer can be former or metastatic.

As used herein, term " CpG DNA " expression contains the immunostimulatory nucleic acid of cytosine-guanine (CG) dinucleotide, and C residue does not wherein methylate.CpG nucleic acid is extensively described in following document immunoregulatory effect: United States Patent (USP), as U.S. Patent No. 6,194,388; 6,207,646; 6,239,116 and 6,218,371; With disclosed international patent application, as WO98/37919, WO98/40100, WO98/52581 and WO99/56755.The full content of these patents and publication application is introduced herein as a reference.Whole immunostimulatory nucleic acid can be unmethylated or part is unmethylated, but the C at least of described 5 '-CG-3 ' must not methylate.

CpG DNA comprises naturally occurring immunostimulatory nucleic acid, as in DNA of bacteria and plasmid, finding, and synthetic oligodeoxynucleotide (ODN).

In one embodiment, described CpG DNA is CpG ODN, its base sequence is 5 '-(ODN 2006 for TCGTCGTTTTGTCGTTTTGTCGTT-3 '; SEQ ID NO:1).

CpG ODN further is divided into following at least three types according to 26S Proteasome Structure and Function, they all be included in as used herein in the term CpG dna bound: B-class CpG ODN, as ODN 2006, the immunostimulating CpG ODN that comprises original description, characteristic activates B cell and NK cell, but do not induce or only a little less than induce I type interferon (as IFN-α) expression.A-class CpG ODN as describing in the open PCT International Application No. WO 01/22990, wherein has the CpG motif, comprise mosaic type di-phosphate ester/phosphorothioate backbone, and distinctive activation NK cell, induce Plasmacytoid type dendritic cell to express a large amount of IFN-α, but do not activate or the only weak B of activation cell.The example of A-class CpG ODN is 5 '-G ^*G ^*G_G_G_A_C_G_A_T_C_G_T_C_G ^*G ^*G ^*G ^*G ^*G-3 ' (ODN 2216, SEQ ID NO:2), wherein " ^*" represent thiophosphate, " _ " represents di-phosphate ester.Among the C-class CpGODN CpG is arranged, comprise whole phosphorothioate backbone, comprise the palindrome or the nearly palindrome zone of being rich in GC, and can activate the B cell, induce the IFN-alpha expression.C-class CpG ODN has been described in for example laid-open U.S. Patents application 2003/0148976.The example of C-class CpG ODN is 5 '-(ODN 2395 for TCGTCGTTTTCGGCGCGCGCCG-3 '; SEQ ID NO:3).As for the summary of described multiple CpG ODN, also see Vollmer J et al. (2004) Eur J Immunol 34:251-62.

As used herein, " cytokine " expression acts on immunocyte by specific receptor influences the multiple soluble protein of the state of activation of described immunocyte and function or in the glycoprotein any one.Cytokine comprises interferon, interleukin, tumor necrosis factor, transforming growth factor, colony stimulating factor (CSF), chemotactic factor etc.The various kinds of cell factor influences innate immunity, acquired immunity or influences both simultaneously.Cytokine specifically includes but not limited to IFN-α, IFN-β, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-18, TNF-α, TNF-β, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony stimutaing factor (GM-CSF).Chemotactic factor specifically includes but not limited to IL-8, IP-10, I-TAC, RANTES, MIP-1 α, MIP-1 β, Gro-α, Gro-β, Gro-γ, MCP-1, MCP-2 and MCP-3.

Most ripe CD4 ⁺T helper cell can range two kinds of cytokines relevant, intersect a kind of in the subgroup of regulating or the phenotype: Th1 or Th2.Th1 cell and IL-2, IL-3, IFN, GM-CSF are relevant with high-level TNF-α.Th2 cell and IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF are relevant with low-level TNF-α.The Th1 subgroup promotes that simultaneously cell-mediated immunity and feature are the humoral immunization that immunoglobulin class converts IgG2a in mice.Th1 replys also can be relevant with delayed hypersensitivity and autoimmune disease.The Th2 subgroup mainly induce humoral immunization and in mice induction of immunity globulin type conversion become IgE and IgG1.Reply relevant antibody isotype with Th1 and generally have good neutralization and opsonizing capacity, and to reply relevant antibody isotype much more relevant with allergic response with Th2.

Shown the typing that several factor affecting Th1 or Th2 spectrum are arranged.The best regulatory factor that characterizes is a cytokine.IL-12 and be positive Th1 regulatory factor and negative Th2 regulatory factor.IL-12 promotes IFN-γ to generate, and IFN-γ provides positive feedback to IL-12.Showing in setting up Th2 cytokine spectrum needs IL-4 and IL-10, and they are also reduced the Th1 cytokine and produce; Acting on of IL-4 surpasses IL-12 in some cases.IL-13 shows can the mode similar to IL-4 suppress the inductive monocytes inflammatory cytokine of LPS, comprises IL-12 and TNF-α.

As used herein, " effective dose " is expressed as and reaches or promote expected result and essential or sufficient any amount.Effective dose is the treatment effective dose in some cases.The treatment effective dose is for promotion in the patient or reaches the essential or sufficient any amount of expectation biological effect.Effective dose to any concrete application can change according to these factors, the order of severity of these factors such as disease to be treated or disease, the particular agent of using, individual size or disease or disease.Those of ordinary skills can empirical definite particular agent effective dose and needn't be through overtesting.

As used herein, " transplant rejection " expression immune-mediated, to deriving from the tissue beyond the host or super acute, the acute or chronic injury of organ.Therefore term comprises cell and antibody-mediated repulsion, and allograft and xenograft repulsion.

As used herein, term " immunocyte " expression belongs to immune cell.Immunocyte comprises the specialization form of T lymphocyte (T cell), bone-marrow-derived lymphocyte (B cell), NKT (NK) cell, granulocyte, neutrophil(e) cell, macrophage, mononuclear cell, dendritic cell and any above cell, as Plasmacytoid type dendritic cell, plasma cell, NKT, t helper cell and cytotoxic T lymphocyte (CTL).

As used herein, term " immunocomplex " expression comprise antibody and with the bonded antigenic any conjugate of antibody specificity.In one embodiment, described antigen is autoantigen.

As used herein, term " immunocomplex that contains nucleic acid " expression comprise antibody and with the bonded any conjugate that contains antigen nucleic acid of antibody specificity.The described immunocomplex that contains nucleic acid can comprise chromatin, ribosome, micronucleus protein, histone, nucleosome, DNA, RNA or its combination in any.Described antibody can but must not need specificity to be attached to the described nucleic acid compositions that contains antigen nucleic acid.

As used herein, term " disease relevant with immunocomplex " representation feature is any disease of immunocomplex generation and/or tissue deposition, include but not limited to immunocomplex disease (as cryoglobulinemia), behcet syndrome, autoimmunity glomerulonephritis that systemic lupus erythematosus (sle) (SLE) is relevant with relevant connective tissue disease, rheumatoid arthritis, hepatitis C and hepatitis B, and with have the relevant angiopathy of the anti-LDL immunocomplex of LDL/.

As used herein, the immunne response that " immunodeficiency " expression patient's immune system could not work orderly or strengthen the patient for example will help eliminating the disease or the disease of patient's tumor or cancer (as brain, lung (as minicell and non-small cell), ovary, breast, prostate, colon tumor, and other cancer and sarcoma) or infection.Described immunodeficiency can be acquired or geneogenous.

As used herein, measurable reply relevant among " the immunostimulatory nucleic acid associated responses among the patient " expression patient with use immunostimulatory nucleic acid to the patient.These are replied and include but not limited to produce cytokine, chemotactic factor, somatomedin or immunoglobulin; Express immunocyte surface activation mark; Th1/Th2 deflection (skewing); With the clinical disease activity.

As used herein, term " infection " and of equal value " infectious disease " but expression infectious organism or material are present in patient's blood or the disease condition in normal sterile tissue or the normal sterile region with detection limit.Infectious organism and material comprise virus, antibacterial, fungus and parasite.Term comprises acute and chronic infection, and sepsis.

As used herein, term " innate immunity is replied " expression is to the immunne response of any kind of the relevant molecular pattern (PAMP) of some pathogen.Innate immunity is also referred to as the nature or the natural immunity in this area, relate generally to neutrophil(e) cell, granulocyte, mononuclear phagocyte, dendritic cell, NKT cell and NK cell.Innate immunity is replied and can be included but not limited to that I type interferon produces (as IFN-α), neutrophil's activation, macrophage activation, endocytosis, opsonic action, complement activation and combination in any thereof.

As used herein, term " self DNA " expression derives from genomic any DNA of host patient.In one embodiment, self DNA comprises the complementary DNA (cDNA) that derives from host patient.Self DNA comprises the DNA of complete sum degraded.

As used herein, term " self RNA " expression derives from genomic any RNA of host patient.In one embodiment, self RNA comprises the messenger RNA (mRNA) that derives from host patient.In one embodiment, self RNA comprises the ribosomal RNA (rRNA) that derives from host patient.Self RNA comprises the DNA of complete sum degraded.

As used herein, term " patient " expression vertebrates.In one embodiment, the patient is a mammal.In one embodiment, the patient is the people.In other embodiments, the patient is non-human Spine animal, includes but not limited to non-human primates, laboratory animal, domestic animal, domestic animal and non-domestic animal.

As used herein, " has the immunostimulation of TLR mediation or the patient that TLR mediation immunostimulation risk takes place arranged " that expression is exposed to or the patient of the risky PAMP of being exposed to or other TLR part.

As used herein, term " Toll sample receptor " and of equal value " TLR " expression identification pathogen associated molecular pattern (PAMP) and as any member of the mammal pattern recognition receptor family of at least ten kinds of high conservatives of key signal transmitting element in the innate immunity.TLR polypeptide common characteristic structure is rich in interior (endochylema) domain of cell that the multiple extracellular of leucine (endochylema is outer) domain, membrane spaning domain and participation TLR signal transmit comprising having.TLR includes but not limited to people TLR.

The nucleic acid of current known all ten kinds of people TLR and aminoacid sequence can obtain from public database such as GenBank.Similar, the nucleic acid of the multiple TLR of a lot of inhuman species and aminoacid sequence also can obtain from the public database that comprises GenBank.For example, the nucleic acid of people TLR9 (hTLR9) and aminoacid sequence can find to be respectively GenBank registration number AF245704 (coding region nucleotide 145-3243) and AAF78037.The nucleic acid of mouse TLR 9 (mTLR9) and aminoacid sequence can find to be respectively GenBank registration number AF348140 (coding region nucleotide 40-3138) and AAK29625.The people TLR9 albumen of inferring contains 1032 aminoacid and has 75.5% whole aminoacid concordance with mouse TLR 9.As other TLR albumen, people TLR9 contains the extracellular and is rich in Toll/ interleukin-11 R (TIR) domain in leucic repetition (LRR) and the cytoplasm.It also has signal peptide (residue 1-25) and membrane spaning domain (residue 819-836).

The nucleic acid of people TLR8 (hTLR8) and aminoacid sequence can find to be respectively GenBank registration number AF245703 (coding region nucleotide 49-3174) and AAF78036.The nucleic acid of mice TLR8 (mTLR8) and aminoacid sequence can find to be respectively GenBank registration number AY035890 (coding region nucleotide 59-3157) and AAK62677.

The nucleic acid of people TLR7 (hTLR7) and aminoacid sequence can find to be respectively GenBank registration number AF240467 (coding region nucleotide 135-3285) and AAF60188.The nucleic acid of mice TLR7 (mTLR7) and aminoacid sequence can find to be respectively GenBank registration number AY035889 (coding region nucleotide 49-3201) and AAK62676.

The nucleic acid of people TLR3 (hTLR3) and aminoacid sequence can find to be respectively GenBank registration number NM 003265 (coding region nucleotide 102-2816) and NP 003256.The nucleic acid of mice TLR3 (mTLR3) and aminoacid sequence can find to be respectively GenBank registration number AF355152 (coding region nucleotide 44-2761) and AAK26117.

HTLR1 is a wide expression, and hTLR2, hTLR4 and hTLR5 are present in mononuclear cell, polymorphonuclear cell and the dendritic cell.Muzio?M?et?al.(2000)J?Leukoc?Biol?67：450-6。Nearest publication report hTLR1, hTLR6, hTLR7, hTLR9 and hTLR10 are present in the human B cell.People TLR7 and hTLR9 are present in the Plasmacytoid type dendritic cell (pDC), and marrow sample dendritic cell are expressed hTLR7 and hTLR8, but do not express hTLR9.Yet people TLR8 demonstration is not expressed in pDC.

As the member of short inflammatory interleukin-1 receptor (IL-1R) family, TLR has homology in it is called born of the same parents' intracellular domain of Toll/IL-1R homology (TIR) domain.PCT openly applies for PCT/US98/08979 and PCT/US01/16766.Similar with TNF (tumor necrosis factor) receptor associated factor 6 (TRAF6) by the general demonstration of signal pass through mechanism in the cell of TLR mediation to MyD88 with pivotal role.Wesche?H?et?al.(1997)Immunity7：837-47；Medzhitov?R?et?al.(1998)Mol?Cell?2：253-8；Adachi?O?et?al.(1998)Immunity9：143-50；Kawai?T?et?al.(1999)Immunity?11：115-22)；Cao?Z?et?al.(1996)Nature383：443-6；Lomaga?MA?et?al.(1999)Genes?Dev?13：1015-24。Signal transduction between known MyD88 and the TRAF6 relates to the member of serine-threonine kinase IL-1 receptor-associated kinase (IRAK) family, comprises IRAK-1 and IRAK-2 at least.Muzio?M?et?al.(1997)Science?278：1612-5。

Briefly, MyD88 is considered to play the linkers effect between the TIR of TLR or IL-1R domain and IRAK (comprise among IRAK-1, IRAK-2, IRAK-4 and the IRAK-M at least any one).MyD88 comprises C end Toll homeodomain and N end death domain.The Toll homeodomain of MyD88 is in conjunction with the TIR domain of TLR or IL-1R, and the death domain of MyD88 is in conjunction with the death domain of serine kinase IRAK.IRAK and TRAF6 interact, and described TRAF6 is as the inlet that enters at least two approach, and one causes activating transcription factor NF-κ B, and another causes activating activity factor albumen-1 (AP-1) transcription factor family member Jun and Fos.Activate NF-κ B and relate to activation MAP3 kinases (MAPK) family member TAK-1, and the I kappa b kinase.I kappa b kinase phosphorylation I κ B causes its degraded and NF-κ B to insert in the nuclear.Activate Jun and Fos and be considered to relate to map kinase kinases (MAPKK) and map kinase ERK, p38 and JNK/SAPK.NF-κ B and AP-1 participate in transcribing of multiple crucial immune response gene, comprise regulating the various kinds of cell factor and costimulatory molecules gene transcription.See Aderem A et al. (2000) Nature 406:782-7;

H et al. (1999) EMBO J 18:6973-82.

As used herein, term " TLR part " and of equal value " part of TLR " and " the TLR signal transmits agonist " expression according to formula I-XIX described herein or according to beyond the molecule outside 4-primary amino radical quinoline of the present invention or the quinazoline molecules, directly or indirectly interact by the TLR domain outside the TIR domain and induce the molecule of the signal transmission that TLR mediates with TLR.In one embodiment, the TLR part is a native ligand, i.e. the TLR part of occurring in nature discovery.In one embodiment, the TLR part is represented the molecule outside the TLR native ligand, as the molecule by the mankind's activity preparation.In one embodiment, described TLR is TLR9, and TLR signal agonist is a CpG nucleic acid.

A lot of but non-whole TLR part has been described.For example, reported the TLR2 signal that Peptidoglycan and lipopeptid are replied.Yoshimura?A?et?al.(1999)J?Immunol?163：1-5；Brightbill?HD?et?al.(1999)Science?285：732-6；Aliprantis?AO?et?al.(1999)Science?285：736-9；Takeuchi?O?et?al.(1999)Immunity?11：443-51；Underhill?DM?et?al.(1999)Nature?401：811-5。Reported that TLR4 transmits the signal that lipopolysaccharide (LPS) is replied.Hoshino?K?et?al.(1999)Immunol?162：3749-52；Poltorak?Aet?al.(1998)Science?282：2085-8；Medzhitov?R?et?al.(1997)Nature?388：394-7。The reporter bacterium flagellin is the native ligand of TLR5.Hayashi?F?et?al.(2001)Nature?410：1099-1103。TLR6 has reported the signal that transmission is replied Dan Baijutang with TLR2.Ozinsky?A?et?al.(2000)Proc?NatlAcad?Sci?USA?97：13766-71；Takeuchi?O?et?al.(2001)Int?Immunol?13：933-40。

Reported that recently TLR9 is the receptor of CpG DNA.Hemmi?H?et?al.(2000)Nature?408：740-5；Bauer?S?et?al.(2001)Proc?Natl?Acad?Sci?USA?98：9237-42。CpG DNA comprises having cytosine not by the DNA of bacteria of methylated CG dinucleotide and synthetic DNA, herein other local more detailed description.Marshak-Rothstein and colleague report also that recently they find that the transmission of TLR9 signal can be used as and take place containing replying in some autoimmune disease of IgG and chromatinic immunocomplex.Leadbetter?EA?et?al.(2002)Nature416：595-8。Therefore on broad sense more, when described nucleic acid is present in suitable environment as in as the part of immunocomplex the time, TLR9 shows can transmit the signal that self or non-self nucleic acid (DNA or RNA) are replied.

Report that recently some imidazoquinolie compounds with antiviral activity is the part of TLR7 and TLR8.Hemmi?H?et?al.(2002)Nat?Immunol?3：196-200；Jurk?M?et?al.(2002)Nat?Immunol?3：499。Imidazoquinolie is the potent synthetic activator of immunocyte with antiviral and antitumor characteristic.Use macrophage from wild type and MyD88 deletion form mice, people such as Hemmi are two kinds of imidazoquinolies of report recently, be imiquimod and resiquimod (R848) only induced tumor necrosin (TNF) and il-1 2 (IL-12) and activate NF-κ B in wild-type cell, this is with consistent through the TLR activation.Hemmi?H?et?al.(2002)Nat?Immunol?3：196-200。These imidazoquinolies are replied and do not produce detectable cytokine from the macrophage of the mice of disappearance TLR7 but not other TLR.In addition, described imidazoquinolie induce propagation that spleen B cell dosage relies on and from wild type but not TLR7-/-cell of mice in the activation of signal cascade in the cell.Luciferase analysis confirmation expressing human TLR7 but not TLR2 or TLR4 in HEKC cause resiquimod is replied and activate NF-κ B.Therefore people's such as Hemmi discovery points out these imidazoquinolie compounds is non-natural parts of TLR7, can induce 7 signal transmission through TLR.

Report R848 also is the part of people TLR8 recently.Jurk?M?et?al.(2002)Nat?Immunol?3：499。

The part of report TLR3 comprises poly (I:C) and double-stranded RNA (dsRNA) recently.For the object of the invention, poly (I:C) and double-stranded RNA (dsRNA) range oligonucleotide molecules.By using poly (I:C) to stimulate the nephrocyte of expressing one of multiple TLR, people such as Alexopoulou report that having only the cell of expressing TLR3 to make by activation NF-κ B replys.Alexopoulou?L?et?al.(2001)Nature?413：732-8。People such as Alexopoulou report that also the wild-type cell that stimulates with poly (I:C) activates NF-κ B and produces inflammatory cytokine IL-6, IL-12 and TNF-α, and TLR3 ^-/-The respective acknowledgement of cell is subjected to remarkable inhibition.By comparison, TLR3 ^-/-Cell and wild-type cell equivalence lipopolysaccharide, Peptidoglycan and CpG dinucleotide are made replied.MyD88 ^-/-Cell analysis shows that this joint albumen participates in the inductive cytokine generation of dsRNA and proliferative is replied, although the activation of NF-κ B and map kinase is unaffected, this shows in these cells has replied different approaches.People such as Alexopoulou advise that TLR3 may work in the host anti-virus defence.

As used herein, any cell of natural or artificial functional TLR is expressed in expression " to express the cell of TLR ".Functional TLR is that total length TLR albumen or its can be induced the fragment of replying with the signal of its ligand interaction.Usually, functional TLR will comprise at least total length TLR extracellular domain TLR part binding fragment and can contain the polypeptide such as the interactional TIR domain of the MyD88 fragment of Toll homeodomain at least with another kind.In multiple embodiments, described functional TLR is the total length TLR that is selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10.

In certain embodiments, described functional TLR is the natural expression of cell.

In one embodiment, the natural expressive function TLR of described cell and be to be isolated cells the RPMI 8226 (ATCC CCL-155) from people's multiple myeloma cells.This cell line is set up from peripheral blood when the old man diagnosed out multiple myeloma (IgG λ type) in 61 years old.Matsuoka?Y?et?al.(1967)Proc?Soc?Exp?BiolMed?125：1246-50。Report RPMI 8226 can produce CpG nucleic acid and reply in the past, and this is by inducing IL-6 albumen and IL-12p40mRNA as supporting evidence.Takeshita?F?et?al.(2000)Eur?J?Immunol?30：108-16；Takeshita?F?et?al.(2000)Eur?J?Immunol?30：1967-76。People such as Takeshita use independent described cell line research promoter structure to identify the CpG nucleic acid signal to be transmitted important transcription factor binding site point.Existing known RPMI 8226 cells are made immunostimulatory nucleic acid and are replied and secrete multiple other chemotactic factor and cytokine, comprise IL-8, IL-10 and IP-10.Because this expression of cell lines TLR9, by TLR9 immunostimulatory nucleic acid such as their effect of CpG nucleic acid mediation, it be suitable for the relevant CpG nucleic acid of the present invention as a reference with the cell line of the method for test compounds and relevant other TLR9 part.

(PBMC) is similar to peripheral blood lymphocytes, has observed as the cell surface expression that RPMI8226 cell line raises mark such as CD71, CD86 and HLA-DR of replying that CpG nucleic acid is exposed.This also observes by the flow cytometry to described cell line.Therefore, method provided herein can be organized into uses the cell surface marker of suitably selecting to express as read output signal, to replenish or to replace chemotactic factor or cytokine to produce or other local other read output signal of describing of this paper.

Having been found that also that RPMI 8226 cell lines can produce some micromolecule that comprises imidazoquinolie compounds reply.For example, RPMI 8226 cells are cultivated with imidazoquinolie compounds R848 (resiquimod) and are induced IL-8, IL-10 and IP-10 to produce.Reported that recently R848 mediates its immunostimulation by TLR7 and TLR8.RPMI 8226 replys ability prompting RPMI 8226 cell lines of R848 and also expresses TLR7, as former report to normal person B cell.

Described RPMI cell line can be used unmodified form or modified forms.In one embodiment, RPMI 8226 cells are with reporting the construct transfection.Preferably, described cell is with described report construct stable transfection.Described report construct generally comprises promoter, coded sequence and polyadenylation signal.Described coded sequence can comprise the report sequence that is selected from down group: enzyme (as luciferase, alkali phosphatase, beta galactosidase, chloramphenicol acetyltransferase (CAT), secreting type alkali phosphatase etc.), bioluminescence mark are (as green fluorescent protein (GFP, U.S. Patent No. 5,491,084) etc.), surface expression molecule (as CD25), secretion molecule (as IL-8, IL-12 p40, TNF-α etc.) and well known by persons skilled in the art other can detect protein product.Preferably, described coded sequence coding has quantitatively level or active albumen.

In certain embodiments, described functional TLR manually expresses (comprising expression) by cell, and for example by introduce the expression vector that carries functional TLR coded sequence in cell, wherein said coded sequence is exercisable to be connected on the expressed sequence.As used herein, when coded sequence and expressed sequence with the expression of described coded sequence or transcribe and/or translate the influence that places described expressed sequence or the mode under the control and when covalently bound, just we can say that they are exercisable connections.If induce promoter in the 5 ' expressed sequence cause described coded sequence transcribe and two DNA sequence between connection character not (1) cause introducing ability or (3) that frameshift mutation, (2) disturb described promoter region to instruct described coded sequence to transcribe and disturb corresponding rna transcription originally to translate into proteinic ability, say that then two DNA sequence are exercisable connections.Therefore, if expressed sequence can influence transcribing of coded sequence and make the gained transcript translate into expectation albumen or polypeptide, then described expressed sequence will be exercisable being connected on the described coded sequence.

In some embodiments, the nucleotide sequence of the functional TLR of coded sequence presentation code.In some embodiments, the nucleotide sequence of coded sequence presentation code reporter gene.

Artificial cell of expressing functional TLR can be not express described functional TLR but the cell that is used for the TLR expression vector.For example, people 293 fibroblasts (ATCC CRL-1573) are not expressed TLR3, TLR7, TLR8 or TLR9.Following embodiment is described, and these cells can instantaneous or stable transfection suitable expression vector (or carrier), to produce the cell of expressing TLR3, TLR7, TLR8, TLR9 or its combination in any.Scheme as an alternative, the cell of manually expressing functional TLR can be not compare with there being described TLR expression vector, expresses the cell of described functional TLR when using described TLR expression vector with remarkable higher level.

For the purposes of the inventive method, the cell of manually expressing functional TLR is preferably expressed the stable transfected cells of described functional TLR.This cell also can the suitable report construct of stable transfection.

As used herein, " the signal transmission of TLR mediation " expression participates in the arbitrary portion of the intracellular signal transduction path of TLR and suitable TLR ligand interaction, and connect described TLR or connect aspect, any downstream by MyD88 by MyD88, describe in the 26S Proteasome Structure and Function about the TIR domain of TLR in the above.

As used herein, the signal transmission that causes measurable immunostimulatory response of " the immunostimulating signal of TLR mediation transmits " expression TLR mediation.This term application is in body and external sort signal transmission.

The immunostimulating signal transmission of TLR mediation when as used herein, " immunostimulation of TLR mediation among the patient " expression is in being applied to body.

As used herein, term " treatment " refers to intervene these diseases, disease or disease at disease, disease or when sick, to prevent or to slow down its process, increases the weight of to prevent, to slow down or to stop it, or eliminates described disease, disease or disease.

Immunostimulatory nucleic acid

The nucleic acid molecules that the induction of immunity system cells was activated when as used herein, " immunostimulatory nucleic acid " expression contacted with immune system cell.Immunocyte activates available any appropriate method well known by persons skilled in the art to be determined, include but not limited to measure growth behind the irriate, propagation, differentiation, migration, relevant with immune activation expressed or the secretor product transcribe or expression, cell in the activity etc. of signal transmission path.The example of the activated sign of immunocyte includes but not limited to secrete cytokines (as IL-6), immunoglobulin,exocrine (as IgG), express cell surface antigen (as CD86), inducing cell lytic activity and induced transcription factor and nuclear translocation (as NF-κ B) thereof.

As used about the inventive method herein, " nucleic acid " represents any polymer of two or more independent nucleoside or nucleotide units.Nucleic acid can be single or double-stranded.Typical independent nucleoside or nucleotide units comprise any one or the combination in any in dezyribonucleoside, ribonucleotide, deoxyribonucleotide and the ribonucleotide.The individual core thuja acid of described nucleic acid or nucleoside unit can be that natural existence or non-natural exist.For example independent nucleotide units can comprise deoxyadenine, deoxidation cytosine, deoxy-guanine, thymus pyrimidine and uracil.Except naturally occurring 2 '-deoxidation and 2 '-OH-form, independent nucleoside also comprises the synthetic nucleosides that has the modified base part and/or modify sugar moieties, as is described in hereinafter: Uhlmann E et al. (1990) Chem Rev 90:543-84.Connecting key between individual core thuja acid or the nucleoside unit can be natural existence or not be naturally occurring.For example described connecting key can be phosphodiester bond, phosphorothioate bond, phosphordithiic acid ester bond (phosphorodithiolate), phosphoramidic acid ester bond (phosphoramidate), and peptide bond and other covalent bond that is suitable for being connected adjacent nucleoside or nucleotide units known in the art.The immunostimulatory nucleic acid typical size range is a unit, a 3-4 unit to tens, as 18-40 unit, although longer nucleic acid is also included within the scope of the invention.

In some embodiments, described nucleic acid by 2 to about 100 nucleotide, more typical 4 to about 40 oligonucleotide that nucleotide is formed.All the oligonucleotide of being made up of deoxyribonucleotide is called oligodeoxynucleotide or oligodeoxynucleotide (ODN) of equal value.

Immunostimulatory nucleic acid comprises any member of number of different types immunostimulatory nucleic acid, specifically comprises immunostimulating CpG nucleic acid (CpG nucleic acid), includes but not limited to A, B and C type; With the non-CpG nucleic acid of immunostimulating, include but not limited to methylated CpG nucleic acid, be rich in nucleic acid and poly--G nucleic acid of T.Some of these polytype immunostimulatory nucleic acids can coexist in given nucleic acid molecules.

As used herein, term " CpG nucleic acid " and " CpG ODN " expression of equal value contain cytosine-guanine (CG) dinucleotide and wherein the C residue not by methylated immunostimulatory nucleic acid.CpG nucleic acid extensively has been described in following document to immunoregulatory effect: United States Patent (USP), as U.S. Patent No. 6,194,388; 6,207,646; 6,218,371 and 6,239,116; With disclosed international patent application, as WO98/37919, WO98/40100, WO98/52581 and WO99/56755.The full content of these patents and publication application is introduced herein as a reference.Whole immunostimulatory nucleic acid can be unmethylated or part is unmethylated, but the C at least of described 5 '-CG-3 ' must not methylate.CpG nucleotide sequence of the present invention comprises above broadly described those and U.S. Patent No. 6,207,646B1 and 6,239, those that disclose among the 116B1.

In one embodiment, described CpG nucleic acid has such base sequence: 5 '-(ODN 2006 for TCGTCGTTTTGTCGTTTTGTCGTT-3 '; SEQ ID NO:1).

CpG nucleic acid further is categorized as following at least three types according to 26S Proteasome Structure and Function, they all be included in the scope of the inventive method: B-class CpG nucleic acid, as ODN 2006, comprise the CpG nucleic acid of describing the earliest, characteristic activates the B cell, but do not induce or only a little less than induce IFN-α expression.A-class CpG nucleic acid, as openly describing among International Application PCT/US00/26527 (WO01/22990), the CpG motif is wherein arranged, di-phosphate ester/the phosphorothioate backbone that comprises heterozygosis, and the distinctive Plasmacytoid type dendritic cell of inducing express a large amount of IFN-α, but do not activate or the only weak B of activation cell.In the C-class oligonucleotide CpG is arranged, comprise the mosaic type skeleton, comprise the palindrome or the nearly palindrome zone of being rich in GC, and can activate the B cell, induce the IFN-alpha expression.These for example have been described in laid-open U.S. Patents application 2003/0148976.

In other embodiments of the present invention, use non-CpG nucleic acid.Non-CpG nucleic acid is the immunostimulatory nucleic acid that does not have the CpG motif in the sequence or have the CpG motif that contains the C residue that methylates.In some cases, described non-CpG nucleic acid still can be immunostimulating because it has other immunostimulating motif, as described here with known in the art those.In one embodiment, described non-CpG nucleic acid is methylated CpG nucleic acid.In some cases, described non-CpG nucleic acid is still immunostimulating, although the C of CpG motif methylates, also is like this when even not having the non-CpG immunostimulating of another kind described herein and known in the art motif.

In one embodiment, described non-CpG nucleic acid is the methylated CpG nucleic acid with following base sequence: 5 '-(ODN 2117 for TZGTZGTTTTGTZGTTTTGTZGTT-3 '; SEQ ID NO:4, wherein Z represents 5-methylcytosine).

Non-CpG nucleic acid comprises the immunostimulatory nucleic acid that is rich in T.The immunostimulatory nucleic acid of the described T of being rich in comprises those that disclose among PCT patent application PCT/US00/26383 openly, and its full content is introduced herein as a reference.In some embodiments, use the long nucleic acid that is rich in T of 24 bases.The nucleic acid that is rich in T is to comprise that the T nucleotide residue surpasses 25% nucleic acid at least one poly T sequence and/or the nucleotide composition.Nucleic acid with poly-T sequence comprises at least 4 T continuously, as 5 '-TTTT-3 '.In some embodiments, the nucleic acid of the described T of being rich in comprises an above polyT sequence.In important embodiment, the nucleic acid of the described T of being rich in can have 2,3,4 or more polyT sequences, as ODN 2006.

Non-CpG nucleic acid also comprises the poly-G immunostimulatory nucleic acid.The immunostimulatory properties of poly-G nucleic acid is described in many pieces of lists of references.Pisetsky?DS?et?al.(1993)Mol?Biol?Reports?18：217-221；Krieger?M?et?al.(1994)Ann?Rev?Biochem?63：601-637；Macaya?RF?et?al.(1993)Proc?Natl?Acad?Sci?USA90：3745-3749；Wyatt?JR?et?al.(1994)Proc?Natl?Acad?Sci?USA?91：1356-1360；Rando?andHogan，1998，In?Applied?Antisense?Oligonucleotide?Technology，Krieg?and?Stein，eds.，pp.335-352；Kimura?Y?et?al.(1994)J?Biochem(Tokyo)116：991-994。

Immunostimulatory nucleic acid of the present invention also can be not have CpG, methylated CpG, be rich in those of T or poly-G motif.

Exemplary immunization zest nucleotide sequence further includes but not limited to disclosing those immunostimulating sequences of describing and list among the PCT patent application WO01/22972.

One aspect of the present invention provides and drops on formula VI and the interior noval chemical compound of formula VII scope that discloses herein.These chemical compounds comprise multiple diarylmethanes TLR9 antagonist, are expressed as CMZ 203-84, CMZ 203-85, CMZ 203-88, CMZ 203-88-1, CMZ 203-89 and CMZ 203-91 herein.The synthetic embodiment 12-17 that is provided in respectively of each in these chemical compounds.

Similar to other chemical compound of formula VI and formula VII, these specific compounds carry out testing in vitro and find to suppress the transmission of TLR9 signal.See embodiment 18.Therefore Compound C MZ 203-84, CMZ 203-85, CMZ 203-88, CMZ 203-88-1, CMZ 203-89 and CMZ 203-91 are considered to can be used for the inventive method.More specifically, one aspect of the present invention provides the method that the signal that the TLR part is replied that influences TLR mediation transmits.Described method comprises the step that the cell that will express TLR contact with following any chemical compound or its combination of effective dose: CMZ203-84, CMZ 203-85, CMZ 203-88, CMZ 203-88-1, CMZ 203-89 and CMZ 203-91, and with the signal transmission that suppresses or promotion TLR mediates replys the TLR part.The method that the signal that the TLR that one aspect of the present invention provides inhibition that the TLR part is replied mediates transmits.Described method comprises the step that the cell that will express TLR contact with following any chemical compound or its combination of effective dose: CMZ 203-84, CMZ 203-85, CMZ203-88, CMZ 203-88-1, CMZ 203-89 and CMZ 203-91, the signal transmission that the TLR part is replied that mediates with inhibition TLR.One aspect of the present invention provides and influences that TLR mediates immunostimulating method among the patient.Described method comprises to having TLR mediation immunostimulation or having the patient that this immunostimulation risk takes place to use following any chemical compound of effective dose or the step of its combination: CMZ 203-84, CMZ 203-85, CMZ 203-88, CMZ203-88-1, CMZ 203-89 and CMZ 203-91, and to suppress or to promote the immunostimulation of TLR mediation among the patient.One aspect of the present invention provides and suppresses that TLR mediates immunostimulating method among the patient.Described method comprises to having TLR mediation immunostimulation or having the patient that this immunostimulation risk takes place to use following any chemical compound of effective dose or the step of its combination: CMZ 203-84, CMZ 203-85, CMZ 203-88, CMZ 203-88-1, CMZ 203-89 and CMZ 203-91, and to suppress the immunostimulation of TLR mediation among the patient.One aspect of the present invention provides the method that suppresses immunostimulatory nucleic acid associated responses among the patient.Comprise that according in this respect method the patient to this treatment of needs uses following any chemical compound of effective dose or the step of its combination: CMZ 203-84, CMZ 203-85, CMZ203-88, CMZ 203-88-1, CMZ 203-89 and CMZ 203-91, to suppress relevant the replying of immunostimulatory nucleic acid among the patient.

One aspect of the present invention provides new replacement 4-primary amino radical quinoline component.Following will stating, these components and other 4-primary amino radical quinoline component have been found and have can be used in the body and the method for vitro inhibition immunne response, comprise the disease relevant with immunocomplex and the method for autoimmune disease for the treatment of.Because they and the similarity of some known anti-malarial agents think that also new replacement 4-primary amino radical quinoline component of the present invention also will can be used for prevention and treatment malaria, and treatment has been described and can have replied other disease that chloroquine is treated.

New replacement 4-primary amino radical quinoline component of the present invention is structural formula XVI chemical compound and pharmaceutically acceptable hydrate and salt,

Wherein

X does not exist or aryl, alkyl, heterocyclic radical or styryl;

R ₃Be hydrogen atom, halogen atom, or alkyl, alkane thiazolinyl, aryl, heterocyclic radical, nitro, cyano group, carboxyl, ester group, ketone, amino, acylamino-, hydroxyl, alkoxyl, sulfydryl, sulfo-, sulfoxide, sulfone or sulfonamido, wherein R ₁And R ₃Randomly be combined to form heterocycle or carbocyclic ring;

L is alkyl or alkane thiazolinyl or the aryl that contains 1 to 10 carbon; With

X does not exist or aryl in one embodiment;

R ₃It is hydrogen atom;

Y is aryl or NR ₁₁, R wherein ₁₁Be hydrogen atom or aryl or alkyl;

L does not exist or C ₂-C ₆Alkyl; And

In one embodiment, X does not exist or aryl;

R ₃It is hydrogen atom;

Y is NH;

L is C ₂-C ₆Alkyl; With

In one embodiment, X is a phenyl;

NR ₁R ₂Be connected to described phenyl X para-position

Y is NH;

L is-(CH ₂) _n-, wherein n is 2 to 6 integer, comprises 2 and 6; And

R ₃, R ₄, R ₅, R ₆And R ₇In each be hydrogen atom.

Representational, non-limiting instance that formula XVI of the present invention replaces 4-primary amino radical quinoline component are chemical compound 101-104,106-109,111,113-116 and the 118-119 that is shown in table 1.

Table 1: replacement 4-primary amino radical quinoline component of the present invention

Have been found that secondary similarly with uncle's 4-aminoquinoline compounds to 4-according to the present invention, 4-primary amino radical quinoline component can be used for suppressing the transmission of TLR9 signal.

Wherein

X does not exist or nitrogen, oxygen or sulphur atom or SO or SO ₂Group;

There is not or contain alkyl or the alkane thiazolinyl or the aryl of 1 to 10 carbon in L; With

The present invention relates to replace 4-primary amino radical quinoline component purposes in this respect with all others in replacement 4-primary amino radical quinoline component can be hydrate or pharmaceutically acceptable salt form.In this respect method can be external or implement in vivo according to the present invention.In addition, express described functional TLR cell can but must not be immunocyte.For the carrier that instructs the described TLR of described cellular expression.In one embodiment, described TLR is TLR9, thereby described method is to suppress the method that signal transmits in the cell of TLR9.

Except chemical compound 101-104, the 106-109,111,113-116 and the 118-119 that are shown in table 1, below other representative and nonrestrictive replacement 4-primary amino radical quinoline compounds, be shown in the chemical compound 105,110,117 and the 120-123 of table 2 with reference to formula XVII, can be used for according to the present invention in this respect the method with all others, these methods relate to the method that replaces 4-primary amino radical quinoline compound of using.Other example table is shown in the following examples.

Table 2. is used for other replacements 4-primary amino radical quinoline component of the inventive method

(b) with effective dose have as defined above that the replacement 4-primary amino radical quinoline component of structural formula XVII contacts, to replace the signal transmission that TLR under the 4-primary amino radical quinoline component replys TLR signal agonist and compare with lacking, suppress the signal transmission that TLR replys TLR signal agonist.The present invention's replacement 4-primary amino radical quinoline component in this respect can be hydrate or pharmaceutically acceptable salt form.In this respect method can be implemented in external or body according to the present invention.

Also according to this aspect of the invention, described in one embodiment TLR is TLR9, described TLR signal agonist is a TLR9 signal agonist, and therefore method in one embodiment is to suppress the method that TLR9 replys the interior signal transmission of cell of TLR9 signal agonist.Described in one embodiment TLR signal agonist is CpGDNA, and CpG ODN for example is as ODN 2006.Described CpG ODN can belong to the CpG ODN of any class, comprises category-A (as ODN 2216), category-B (as ODN 2006) or C class (as ODN 2395).

In one embodiment, described TLR signal agonist is the immunocomplex that comprises nucleic acid.The known immunocomplex of nucleic acid that comprises of persons skilled in the art comprises and the bonded immunoglobulin of nucleic acid, described nucleic acid or exposed, or more common combining with protein or other non-nucleic acid compositions.For example can comprise chromatin, ribosome, micronucleus protein, ribonucleoprotein (RNP), histone, nucleosome protein-dna complex and nucleosome with protein or the bonded nucleic acid of other non-nucleic acid compositions.The anti-nucleic acid of specificity comprises antinuclear antibody (ANA), anti-dsDNA, anti-ssDNA, anti-Sm, anti-RNP, anti-Ro (SS-A), anti-La (SS-B) and anti-histone antibody with the example that contains the clinically important antibody of nucleic acid substances.The immunocomplex that comprises nucleic acid include but not limited to the bonded immunoglobulin of DNA that specifically comprises double-stranded DNA and with the bonded immunoglobulin of nucleosome material, this is two features of systemic lupus erythematosus (sle), and with the bonded immunoglobulin of RNA.

(b) with effective dose have as defined above that the replacement 4-primary amino radical quinoline component of structural formula XVII contacts, the immunne response of described antigenicity substance is compared with lacking to replace under the 4-primary amino radical quinoline component, suppress immunne response to described antigenicity substance.The present invention's replacement 4-primary amino radical quinoline component in this respect can be hydrate or pharmaceutically acceptable salt form.In this respect method can be implemented in external or body according to the present invention.Described in one embodiment immunne response is that innate immunity is replied.Described in one embodiment immunne response comprises adaptive immune response.

The present invention relates in the patient immunocyte in one embodiment with expressive function Toll sample receptor

(a) contact with the antigenicity substance of effective dose lacking to replace under the 4-primary amino radical quinoline component, stimulating the immunne response of described patient to described antigenicity substance, and

(b) with effective dose have as defined above that the replacement 4-primary amino radical quinoline component of structural formula XVII contacts, the immunne response of described antigenicity substance is compared with lacking to replace under the 4-primary amino radical quinoline component, suppress the immunne response of described patient to described antigenicity substance.

In one embodiment, contact with the antigenicity substance of effective dose, relate to lacking the immunocyte that replaces under the 4-primary amino radical quinoline component expressive function Toll sample receptor: lacking under the replacement 4-primary amino radical quinoline component and initiatively using the antigenicity substance of effective dose with the step of stimulation to the immunne response of described antigenicity substance to stimulate step to the immunne response of described antigenicity substance.Can use and use described antigenicity substance so that effectively any effective way or the mode of contact are used described antigenicity substance.In the mode of example, can carry out administration by local or systemic injection, suction, per os absorption, surperficial administration, mucosa delivery or its combination in any.

In one embodiment, contact with the antigenicity substance of effective dose, be passive to the step of the immunne response of described antigenicity substance lacking the immunocyte that replaces under the 4-primary amino radical quinoline component expressive function Toll sample receptor with stimulation.For example in one embodiment, with the immunocyte of expressive function Toll sample receptor and effective dose have as defined above that the replacement 4-primary amino radical quinoline component of structural formula XVII contacts, with lack described replacement 4-primary amino radical quinoline component under the immunne response of described antigenicity substance compared inhibition in the immunne response of described antigenicity substance or before, described immunocyte has contacted or the antigenicity substance of contacted effective dose, to stimulate the immunne response to described antigenicity substance.

Described antigenicity substance can be an allergen.Allergen is the material that can induce allergia or asthma to reply in responsive patient.Allergenic tabulation is huge, can comprise pollen, insecticide venom, animal scurf, dirt, fungal spore and medicine (as penicillin).The allergenic example of natural animal and plant comprises the protein that is specific to the subordinate: dog (domesticated dog), dirt demodicid mite (as the dust demodicid mite); Cat (domestic cat), hogweed (America hogweed); Rye grass (as English ryegrass or annual ryegrass); Cortex Cryptomeriae Fortunei Radicis (Japanese cedar); Chain lattice spore (alternaric bacteria); Folium Et Cacumen Alni Japonicae; Alder (Alnusgultinosa); Birch (wart skin birch); Oak (white oak); Olea europaea (Fructus oleae europaeae); Artemisia (Herba Artemisiae Selengensis); Herba Plantaginis (as buckhorn plantain); Wall pellitory (as common milfoil or Judas wall pellitory); Little Lian (as Groton bug); Apis (as Apis multiforum); Cupressus funebris Endl. (as cupressus sempervirens, dried cypress and cupressus macrocarpa); Juniperus oxycedrus (as Junipers sabinoides, North America Chinese juniper, European perverse cypress and Juniperus asheo; Thuya (as Thuya orientalis); Cupressus funebris (as Japanese cypress); Cockroach (as american cockroach); Roegneria kamoji belongs to (as quackgrass); Secale (as rye (Secale cereale L.)); Triticum (as Semen Tritici aestivi); Orchardgrass (as orchardgrass); The Vulpes thatch belongs to (as meadow fescue); Annual bluegrass (as English grass or Canada blue grass); Avena (as Herba bromi japonici); Holcus (as the floss grass); Anthoxanthum (as XIANGHUANG flower thatch); Oatgrass (as Herba avenae fatuae); Creeping bentgrass belongs to ((as white bent); Kittentails (as timothy grass); Phalaris arundinacea (as reed canary grass); Paspalum (as Paspalum notatum); Sorghum vulgare Pers. (as Sorghumhalepensis); And Brome (as awnless brome).Term " allergy " expression is to the acquired hypersensitivity of material (allergen)." allergy " is to be that allergic patient's immune system is to described allergenic replying to allergen.Allergic disease comprises eczema, allergic rhinitis, pollinosis, bronchial asthma, rubella (urticaria) and food allergy and other atopic diseases.

Described antigenicity substance can be to derive from or be exactly the antigen of infectious microorganism agent, comprises antibacterial, virus, fungus or parasitic antigen.The example of infective bacterial comprises: helicobacter pylori, the Borrelia burgdoyferi bacterium, legionella pneumophilia, the mycobacterium species are (as mycobacterium tuberculosis, the fowl mycobacterium, mycobacterium in the cell, Kansas mycobacterium and Gordon's mycobacterium), staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, monocyte hyperplasia Li Site bacterium, micrococcus scarlatinae (A group streptococcus), streptococcus agalactiae (B group streptococcus), streptococcus (grass green colo(u)r group), Streptococcus faecalis, Streptococcus bovis, streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicity Campylobacter species, the Enterococcus species, hemophilus influenza, Bacillus anthracis, diphtheria corynebacterium, the corynebacterium species, erysipelothrix ruhsiopathiae, bacillus perfringens, clostridium tetani, clostridium perfringen, pneumonia Crewe uncle Salmonella, Pasturella multocida, the Bacteroides species, Fusobacterium nucleatum, Streptobacillus moniliformis, treponema pallidum, Treponema pertenue, leptospira and actinomyces israelii.The example of infective virus comprises: retroviral section (including but not limited to human immunodeficiency virus (HIV)); Picornaviridae (for example poliovirus, hepatitis A virus (HAV), enterovirus, human coxsackievirus, rhinovirus, she coe virus); The Calciviridae Strain of gastroenteritis (as cause); Togaviridae (for example equine encephalitis virus, rubella virus); Flaviviridae (for example dengue virus, encephalitis, yellow fever virus); Coronaviridae (for example coronavirus); Rhabdoviridae (for example vesicular stomatitis virus, rabies virus); Filamentous virus section (for example Ebola virus); Paramyxoviridae Paramyxoviridae (for example parainfluenza virus, mumps virus, Measles virus, respiratory syncytial virus); Orthomyxoviridae family (for example influenza virus); Bunyaviridae (for example Hantaan virus, Bunyavirus, phlebotomus fever virus and Nairo viruses); Arenaviridae (hemorrhagic fever virus); Reoviridae (as reovirus, Orbivirus and rotavirus); Birnavirus section; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (human papillomavirus, polyoma virus); Adenoviridae (most adenovirus); Herpetoviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (smallpox virus, vaccinia virus, poxvirus); And Iridoviridae (as the African swine fever poison); With unfiled virus (cause of disease (1 class=intestinal transmitted of the cause of disease of the cause of disease of spongiform encephalopathy, hepatitis D virus (the deficiency associated virus that is considered to hepatitis B virus), non-A non-B hepatitis for example; 2 classes=intestinal is propagated (being hepatitis C) outward; Norwalk agent and correlated virus, and star virus).The example of infectiousness fungus includes but not limited to neogenesis cryptococcus, Histoplasma capsulatum, Blastomyces coccidioides, Blastomyces dermatitidis, chlamydia trachomatis and Candida albicans.

Described antigenicity substance can be a cancer antigen.Cancer antigen used herein is a kind of chemical compound, as peptide or albumen, with tumor or cancerous cell surface combination, and can arouse immunne response when being expressed in the antigen presenting cell surface in the environment of major histocompatibility complex (MHC) molecule.For instance, cancer antigen or by as Cohen PA et al. (1994) Cancer Res 54:1055-8 as described in antigen and, perhaps pass through recombinant technique or de novo synthesis known antigens as described in the preparation cancerous cell crude extract, partial purification from the cancerous cell preparation.Cancer antigen includes but not limited to recombinant expressed antigen, its immunogenicity part or whole tumor or cancerous cell.Through reorganization or by any other method known in the art, these antigens can separated or preparation.

Term " cancer antigen " and " tumor antigen " are used interchangeably, and therefore expression also can be used in the antigen of target cancer cell by the cancerous cell differential expression.Cancer antigen is the antigen of potential energy obvious stimulation tumour-specific immune response.In these antigens some are encoded by normal cell, although might not express.These antigens can be characterized by in normal cell the antigen of normal reticent (promptly not expressing), the antigen of only expressing in some differential period and the antigen of transient expression, as embryo and tire antigen.Other cancer antigens are by the cell mutation gene code, as oncogene (as the active ras oncogene), antioncogene (as the p53 mutant), from the fusion rotein of inside disappearance or chromosome translocation.The gene code that also has other cancer antigens to carry by viral gene such as RNA and DNA oncovirus.The example of tumor antigen comprises MAGE, MART-l/Melan-A, gp100, DPP IV (DPPIV), ABP (ADAbp), cyclophilin b (cyclophilin b), colorectum related antigen (CRC)--C017-lA/GA733, carcinoembryonic antigen (CEA) and immunogenicity epi-position CAP-1 and CAP-2, etv6, amll, prostate specific antigen (PSA) and immunogenicity epi-position PSA-1 thereof, PSA-2 and PSA-3, prostate specific membrane antigen (PSMA), TXi Baoshouti/CD3-zeta chain, the tumor antigen of MAGE family is (as MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), the tumor antigen of GAGE family is (as GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAG, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tryrosinase, p53, MUC family, HER2/neu, p21ras, RCAS, α-fetoprotein, the E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 ^Pmel117, PRAME, NY-ESO-1, cdc27, apc protein (adenomatous polyposis coli protein, APC), fodrin, Connexin 37, nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and the CT-7 of Ig idiotype (Ig-idiotype), p15, gp75, GM2 and GD2 ganglioside, viral product such as human papilloma virus toxalbumin, Smad family tumor antigen, Imp-1, P1A, EBV coding are with c-erbB-2..

Cancer or tumor and the tumor antigen (but nonexcludability ground) relevant with these tumors comprise acute lymphoblastic leukemia (etv6; Amll; Cyclophilin b), B cell lymphoma (Ig idiotype), glioma (E-cadherin; α-catenin; β-catenin; γ-catenin; Pl20ctn), bladder cancer (p21ras), carcinoma of gallbladder (p21ras), breast carcinoma (MUC family; HER2/neu; C-erbB-2), cervical cancer (p53; P21ras), colon cancer (p21ras; HER2/neu; C-erbB-2; MUC family), colorectal carcinoma (colorectum related antigen (CRC)--C017-1A/GA733; APC), choriocarcinoma (CEA), cell carcinoma (cyclophilin b), gastric cancer (HER2/neu; C-erbB-2; The ga733 glycoprotein), hepatocarcinoma (α-fetoprotein), hodgkin's lymphoma (lmp-1; EBNA-1), pulmonary carcinoma (CEA; MAGE-3; NY-ESO-1), lymphoid cell source leukemia (cyclophilin b), melanoma (p15 albumen, gp75, oncofetal antigen (oncofetal antigen), GM2 and GD2 ganglioside), myeloma (MUC family; P21ras), nonsmall-cell lung cancer (HER2/neu, c-erbB-2), nasopharyngeal carcinoma (lmp-1; EBNA-1), ovarian cancer (MUC family; HER2/neu; C-erbB-2), carcinoma of prostate (prostate specific antigen (PSA) and immunogenicity epi-position PSA-1, PSA-2 and PSA-3; Prostate specific membrane antigen (PSMA); HER2/neu; C-erbB-2, cancer of pancreas (p21ras; MUC family; HER2/neu; C-erbB-2; The ga733 glycoprotein), renal carcinoma (HER2/neu; C-erbB-2), the squamous cell carcinoma of cervix uteri and esophagus (viral product such as human papilloma virus toxalbumin), carcinoma of testis (NY-ESO-1), T chronic myeloid leukemia (HTLV-1 epi-position) and melanoma (Melan-A/MART-1; Cdc27; MAGE-3; P2lras; Gp100 ^Pmel117).

The method of autoimmune disease among the treatment patient is provided in one aspect of the invention.In this respect method relates to the replacement 4-primary amino radical quinoline component as above-mentioned structural formula XVII of having from effective dose to the patient who suffers from autoimmune disease that use according to the present invention, with the treatment autoimmune disease.Replacement 4-primary amino radical quinoline component among the present invention in this respect can be hydrate or pharmaceutically acceptable salt form.In one embodiment, described autoimmune disease is selected from systemic lupus erythematosus (sle), rheumatoid arthritis, inflammatory bowel, xerodermosteosis, polymyositis, vasculitis, wegener granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter syndrome, arthritic psoriasis and behcet syndrome.In a specific embodiments, described autoimmune disease is a systemic lupus erythematosus (sle).In a specific embodiments, described autoimmune disease is a rheumatoid arthritis.In one embodiment, described patient is the people.Described method also can be applicable to the animal model of any autoimmune disease listed above.

In one embodiment, described autoimmune disease is the disease relevant with immunocomplex.The disease relevant with immunocomplex specifically includes but not limited to systemic lupus erythematosus (sle), rheumatoid arthritis, polyarteritis nodosa, post-streptococcal glomerulonephritis, cryoglobulinemia and acute and chronic serum sickness.

Described replacement 4-primary amino radical quinoline component can any appropriate route of administration be applied to the patient, includes but not limited to the outer approach of per os and gastrointestinal tract.Gastrointestinal tract external administration approach includes but not limited in intravenous, intramuscular, intraperitoneal, subcutaneous, intranasal, the lung, transdermal, local topical and mucosa.Gastrointestinal tract external administration approach also comprises and is injected directly into concrete tissue or other injection site, comprises for example lymphoid tissue and inflammation part.

Have been found that the method that can be used for suppressing in external and the body immunne response with the quinazoline compound of quinoline structural similarity of the present invention unexpectedly according to the present invention, comprise the disease relevant and the method for autoimmune disease for the treatment of with immunocomplex.It is unexpected that quinazoline compound according to the present invention and the using method thereof found keep a lot of or whole inhibitive ability of immunity characteristics of corresponding quinoline compound and using method thereof, but have the toxic characteristic of other reduction, like this at least in vivo.

One aspect of the present invention provides novel quinazoline quinoline component.Following will stating, these components and other quinazoline component have been found the method that can be used for suppressing in external and the body immunne response, comprise the disease relevant with immunocomplex and the method for autoimmune disease for the treatment of.Because they and the similarity of some known anti-malarial agents also are considered to novel quinazoline quinoline component of the present invention and also will can be used for prevention and treatment malaria, treatment has been described to treat chloroquine and made other disease of replying.

Novel quinazoline quinoline component of the present invention has structural formula XVIII and pharmaceutically acceptable hydrate and salt,

Wherein

L is alkyl or alkane thiazolinyl or the aryl that contains 1 to 10 carbon;

X does not exist or aryl in one embodiment;

Y is aryl or NR ₁₂, R wherein ₁₂Be hydrogen atom or aryl or alkyl;

L does not exist or C ₂-C ₆Alkyl;

In one embodiment, X does not exist or aryl;

Y is NH;

L is C ₂-C ₆Alkyl;

In one embodiment, X is an aryl;

NR ₁R ₂Be to replace or unsubstituted piperazinyl or morpholinyl;

Y is NH;

L is C ₂-C ₆Alkyl;

In one embodiment, X is a phenyl;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄It all is methyl; And

R ₅, R ₆, R ₇And R ₈In each be hydrogen atom.

In one embodiment, X is a phenyl;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄Be combined to form morpholinyl; And

R ₅, R ₆, R ₇And R ₈In each be hydrogen atom.

In one embodiment, X does not exist;

NR ₁R ₂Be to replace or unsubstituted piperazinyl or morpholinyl;

Y is NH;

L is C ₂-C ₆Alkyl;

In one embodiment, X does not exist;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄It all is methyl;

R ₆And R ₇It all is methoxyl group; And

R ₅And R ₈It all is hydrogen atom.

In one embodiment, X does not exist;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄It all is methyl;

R ₆And R ₇It all is methoxyl group; And

R ₅And R ₈It all is hydrogen atom.

In one embodiment, X does not exist;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄Be combined to form morpholinyl;

R ₆And R ₇It all is methoxyl group; And

R ₅And R ₈It all is hydrogen atom.

Representativeness, the limiting examples of the quinazoline component of formula XVIII of the present invention is the chemical compound 201-214 that lists in table 3.

Table 3. quinazoline component of the present invention

Have been found that according to the present invention some quinazoline compound can be used for suppressing the transmission of TLR9 signal.

One aspect of the present invention provides other novel quinazoline quinoline chemical compounds that can be used for the inventive method.These chemical compounds are expressed as CMZ 203-34, CMZ 203-44, CMZ 203-45, CMZ 203-49, CMZ 203-51, CMZ 203-76, CMZ 203-78, CMZ 203-87, CMZ 203-93 and CMZ 203-95 herein.The synthetic embodiment 19-28 that is provided in respectively of each in these new compounds.

In these particular compound some carried out testing in vitro and found to suppress the transmission of TLR9 signal.See embodiment 29.Therefore Compound C MZ 203-34, CMZ 203-44, CMZ 203-45, CMZ 203-49, CMZ 203-51, CMZ 203-76, CMZ 203-78, CMZ 203-87, CMZ 203-93 and CMZ 203-95 are considered to can be used for method of the present invention.

Wherein

Y does not exist or oxygen atom, sulphur atom, CR9R10 or NR11, wherein R ₉, R ₁₀And R ₁₁Independently be mutually hydrogen atom or alkyl, alkane thiazolinyl or aryl, wherein R ₉, R ₁₀And R ₁₁In any one optional and R ₃Or R ₄Be combined to form and replace or unsubstituted heterocycle;

R ₅, R ₆, R ₇And R ₈Independently be mutually hydrogen atom, halogen atom, or alkyl, alkane thiazolinyl, aryl, heterocyclic radical, nitro, cyano group, carboxyl, ester group, ketone, amino, acylamino-, hydroxyl, alkoxyl, sulfydryl, sulfo-, sulfoxide, sulfone or sulfonamido, wherein R ₅, R ₆, R ₇And R ₈Close on mutually arbitrarily to randomly being combined to form heterocycle or carbocyclic ring.In this respect method can be implemented in external or body according to the present invention.In addition, express described functional TLR cell can but must not be immunocyte.For example, the cell of expressing described functional TLR can be to use the expression vector cells transfected, and wherein said expression vector instructs the described TLR of described cellular expression.In one embodiment, described TLR is TLR9, thereby described method is the method that suppresses through the signal transmission of TLR9.

Except the chemical compound 201-214 that lists in the table 3, below other representativeness and non-limiting quinazoline compound, list in the chemical compound 215-229 of table 4 with reference to formula XIX, can be used for according to the present invention relating to the method that replaces 4-primary amino radical quinoline compound of using in this respect with in all others.Other example table is shown in the following examples.

Table 4. is used for other quinoline components of the inventive method

(b) with effective dose have as defined above that the quinazoline component of structural formula XIX contacts, with lack described quinazoline component under the TLR signal transmission of replying TLR signal agonist compare, suppress the signal transmission that TLR replys TLR signal agonist.The present invention's quinazoline component in this respect can be hydrate or pharmaceutically acceptable salt form.In this respect method can be implemented in external or body according to the present invention.

In one embodiment, described TLR signal agonist is the immunocomplex that comprises nucleic acid as above-mentioned.

(b) with effective dose have as defined above that the quinazoline component of structural formula XIX contacts, with lack described quinazoline component under the immunne response of described antigenicity substance is compared, suppress immunne response to described antigenicity substance.The present invention's quinazoline component in this respect can be hydrate or pharmaceutically acceptable salt form.In this respect method can be implemented in external or body according to the present invention.Described in one embodiment immunne response is that innate immunity is replied.Described in one embodiment immunne response comprises adaptive immune response.

In one embodiment, contact with the antigenicity substance of effective dose, relate to lacking under the quinazoline component immunocyte with expressive function Toll sample receptor: lacking the antigenicity substance of initiatively using effective dose under the quinazoline component, with the step of stimulation to the immunne response of described antigenicity substance to stimulate step to the immunne response of described antigenicity substance.Can use and use described antigenicity substance so that effectively any effective way or the mode of contact are used described antigenicity substance.In the mode of example, can carry out administration by local or systemic injection, suction, per os absorption, topical, mucosa delivery or its combination in any.

Described in one embodiment method relates to and will express the immunocyte of functional Toll sample receptor among the patient

(b) with effective dose have as defined above that the quinazoline component of structural formula XVII contacts, with lack described quinazoline component under the immunne response of described antigenicity substance is compared, suppress the immunne response of described patient to described antigenicity substance.

In one embodiment, contact with the antigenicity substance of effective dose, be passive lacking under the quinazoline component immunocyte with expressive function Toll sample receptor to stimulate step to the immunne response of described antigenicity substance.For example in one embodiment, with the immunocyte of expressive function Toll sample receptor and effective dose have as defined above that the quinazoline component of structural formula XVII contacts, with lack described quinoline component under the immunne response of described antigenicity substance compared inhibition in the step of the immunne response of described antigenicity substance or before, described immunocyte has contacted or the antigenicity substance of contacted effective dose, to stimulate the immunne response to described antigenicity substance.

Described antigenicity substance can be an allergen, as mentioned above.

Described antigenicity substance can be to derive from or be exactly the antigen of infectious microorganism agent, comprises aforesaid antibacterial, virus, fungus or parasitic antigen.

Described antigenicity substance can be a cancer antigen, as mentioned above.

The method of autoimmune disease among the treatment patient is provided in one aspect of the invention.In this respect method relates to the quinazoline component as above-mentioned structural formula XIX of having from effective dose to the patient who suffers from autoimmune disease that use according to the present invention, with the treatment autoimmune disease.Quinazoline component among the present invention in this respect can be hydrate or pharmaceutically acceptable salt form.In one embodiment, described autoimmune disease is selected from systemic lupus erythematosus (sle), rheumatoid arthritis, inflammatory bowel, xerodermosteosis, polymyositis, vasculitis, wegener granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter syndrome, arthritic psoriasis and behcet syndrome.In a specific embodiments, described autoimmune disease is a systemic lupus erythematosus (sle).In a specific embodiments, described autoimmune disease is a rheumatoid arthritis.In one embodiment, described patient is the people.Described method also can be applicable to the animal model of any autoimmune disease listed above.

In one embodiment, described autoimmune disease is the aforesaid disease relevant with immunocomplex.

Described quinazoline component can any appropriate approach use described patient, include but not limited to outside per os and the gastrointestinal tract.Gastrointestinal tract external administration approach is about replacing described in the 4-primary amino radical quinoline those as above.

The effect test

Method of the present invention can be used based on the multiple arbitrarily of TLR/IL-1R signal transduction pathway and may estimate by read-out system.In some embodiments, reading of described method is based on the use natural gene, as an alternative scheme use transfection or by the manually-injected reporter gene construct of alternate manner, they can be made the TLR/IL-1R signal transduction pathway that relates to MyD88, TRAF, p38 and/or ERK and replying.

H?et?al.(1999)EMBO?J18：6973-82。These pathway activation kinases comprise kappa b kinase complex and c-Jun N end kinases.Therefore the reporter gene and the reporter gene construct that specifically can be used for described test for example comprise the reporter gene that can be operationally connected to the responsive promoter of NF-κ B.The example of these promoteres includes but not limited to the promoter of NF-κ B, IL-1 β, IL-6, IL-8, IL-12 p40, IP-10, CD80, CD86 and TNF-α.The reporter gene that can be operationally connected to the responsive promoter of TLR can include but not limited to that enzyme (as luciferase, alkali phosphatase, beta galactosidase, chloramphenicol acetyltransferase (CAT) etc.), bioluminescence mark are (as green fluorescent protein (GFP, as U.S. Patent No. 5,491,084), blue fluorescent protein (BFP, as U.S. Patent No. 6,486,382) etc.), surface expression molecule (as CD25, CD80, CD86) and secretion molecule (as IL-1, IL-6, IL-8, IL-12 p40, TNF-α).In certain embodiments, described report thing is selected from IL-8, TNF-α, NF-κ B-luciferase (NF-κ B-luc;

H et al. (1999) EMBO J 18:6973-82), IL-12 p40-luc (Murphy TL et al. (1995) Mol Cell Biol 15:5258-67) and TNF-luc ( H et al. (1999) EMBO J 18:6973-82).In the test that the dependence enzymatic activity is read, the part of substrate as described test can be provided, detection can relate to other label of measuring chemiluminescence, fluorescence, colour developing, introducing radioactive label, drug resistance or enzymatic activity.For the test that relies on the surface expression molecule, can finish detection with flow cytometry (FACS) analysis or functional test.Secretion molecule available enzyme connection immunoadsorption test (ELISA) or biological test are tested.A lot of these and other suitable read-out system are known in this field and commercial offers arranged.

The report construct

In some embodiments, expressive function TLR and the cell that is used for the inventive method have expression vector, and it comprises that coding is used to detect the isolating nucleic acid of the report construct that the TLR signal transmits.Comprising that the expression vector of isolating nucleic acid that coding is used to detect the report construct of TLR signal transmission can comprise the reporter gene that is positioned under promoter response element (enhancer element) control.In some embodiments, described promoter response element is connected with the minimal promoter of response transcription factor, and described transcription factor is thought as the result of TLR signal transmission the applicant and is activated.The example of these minimal promoters includes but not limited to the promoter of following gene: AP-1, NF-κ B, ATF2, IRF3 and IRF7.These minimal promoters contain the corresponding promoter response element to AP-1, NF-κ B, ATF2, IRF3 and IRF7 sensitivity respectively.In other embodiments, comprising that the expression vector of isolating nucleic acid that coding is used to detect the report construct of TLR signal transmission can comprise is positioned at promoter response element control reporter gene down, and described promoter response element is selected from the response element to following sensitivity: the response element (ISRE) of IL-6, IL-8, IL-12 p40 subunit, I type IFN, RANTES, TNF, IP-10, I-TAC and interferon stimulation.Generally there are a plurality of copies in described promoter response element, repeats as series connection.For example in a report construct, the coded sequence of luciferase is positioned under the control of 6 the multiple NF-κ B response elements of series connection in upstream.In another example, being used for ISRE-luciferase reporting construct of the present invention can obtain from Stratagene (catalog number (Cat.No.) 219012), comprises that 5 series connection that are connected to luciferase reporter gene upstream TATA box repeat.As other local further discussion of this paper, described report thing self can be any gene outcome that is suitable for the means known in the art detection.These detection methods for example can comprise: measure spontaneous or stimulated luminescence, enzymatic activity, the expression of shla molecule, the expression of cell surface molecule etc.

Typically read and relate to Toll/IL-1R signal transmitting element commonly used, as MyD88, TRAF and IRAK molecule, although the effect of MyD88 is clear to other TLR family member for TLR3.As showing herein, these are replied and comprise inducing and be positioned at the following gene of specificity promoter such as the control of NF-κ B promoter, increase specific cells factor level, increase specific chemokines level etc.Gene under the control of NF-κ B promoter can be the gene of the natural NF-of comprising κ B promoter, or inserts the gene of NF-κ B promoter in construct.The gene and the construct that comprise NF-κ B promoter include but not limited to IL-8, IL-12 p40, NF-κ B-luc, IL-12 p40-luc and TNF-luc.

The reason that cytokine levels increases can be the cytokine due to the signal of replying the TLR mediation transmits generate increase, stability increases, secretion increases or aforesaid combination in any.Cytokine generally comprises but is not limited to IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-18, IFN-α, IFN-β, IFN-γ, TNF-α, GM-CSF, G-CSF, M-CSF.The Th1 cytokine includes but not limited to IL-2, IFN-γ and IL-12.The Th2 cytokine includes but not limited to IL-4, IL-5 and IL-10.

The reason that the chemotactic factor level increases can be the chemotactic factor due to the signal of replying the TLR mediation transmits generate increase, stability increases, secretion increases or aforesaid combination in any.The especially significant chemotactic factor of the present invention includes but not limited to CCL5 (RANTES), CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC), IL-8 and MCP-1.

To patient's administration

Aspects more of the present invention relate to the patient uses the component of effective dose to obtain the specificity result.Therefore the small molecule component that uses according to the inventive method can be suitable for any way preparation of pharmaceutical applications.

The present invention prescription can pharmaceutically acceptable solution form be used, and salt, buffer agent, antiseptic, compatibility that its routine can contain pharmaceutically acceptable concentration are passed body, adjuvant and other optional therapeutic component.

For therapeutic use, the any-mode that the described chemical compound of effective dose can make suitable target cell absorb described chemical compound is used to the patient." using " pharmaceutical composition of the present invention can finish by any-mode well known by persons skilled in the art.Concrete route of administration includes but not limited to oral cavity, transdermal (as through agent), parenteral injection (subcutaneous, Intradermal, intramuscular, intravenous, intraperitoneal, intrathoracic etc.) or mucosa (in intranasal, the trachea, suction, internal rectum, intravaginal etc.).Injection can be to inject or inculcate continuously form.

For example using according to pharmaceutical composition of the present invention often is to use by intravenous, intramuscular or other parenteral mode, or is administered to epidermis by biological trajectory type (biolistic) " particle gun ".Use that they also can be used by intranasal, suck, surface, oral cavity, or can be used as implant and even rectum or vaginal application.The liquid or solid pharmaceutical dosage forms that is fit to for example be used for the water injecting or suck or saline solution, littlely seal, encochleated, bag by to micro-gold grain, be contained in liposome, spray, aerosol, be implanted to the piller (pellet) in the skin or be dried to the sharp object that is used to scratch skin.Described pharmaceutical composition also comprises granule, powder, tablet, coated tablet, (little) capsule, suppository, syrup, Emulsion, suspension, frost, drop or postpones the preparation of release of active compounds, in these preparations excipient and additive and/or auxiliary agent such as disintegrating agent, binding agent, coating materials, sweller, lubricant, be flavor agent, sweeting agent or cosolvent and as above describe conventional the use.Described pharmaceutical composition is applicable to multiple drug delivery system.Referring to Langer R (1990) Science 249:1527-33, the document is introduced herein as a reference about the summary of existing delivery method.

The compound concentration that is included in the compositions that is used for the inventive method can be that about 1nM is to about 100 μ M.Think that effective dose is that about 10 picomoles/kg is to about 100 micromoles/kg.

Described pharmaceutical composition preferably prepares with dosage unit and uses.Liquid dosage unit is to be used to inject or the pipe or the ampoule of other parenteral administration.Solid dosage unit is tablet, capsule, powder and suppository.Be the treatment patient, according to described compound activity, administering mode, administration purpose (i.e. prevention or treatment), disease character and the order of severity, patient age and body weight, different dosage may be necessary.Can be by form single administration, to carry out the administration of given dose with separate dosage units or several more small dosage units.Be also included within the present invention in particular day, week or month interval repetition and multiple dosing respectively.

Described component can itself (purely) or pharmaceutically acceptable salt form be used.When being used for medicine, described salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salt can be used to prepare corresponding pharmaceutically acceptable salt easily.These salt include but not limited to the salt that is equipped with from following processed with acid: hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-methyl benzenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid.In addition, these salt can be prepared into alkali metal or alkali salt, as described the sodium of hydroxy-acid group, potassium or calcium salt.

Suitable reducing comprises: acetic acid and salt (1-2%w/v); Citric acid and salt (1-3%w/v); Boric acid and salt (0.5-2.5%w/v) and phosphoric acid and salt (0.8-2%w/v).Suitable antiseptic comprises benzalkonium chloride (0.003-0.03%w/v); Methaform (0.3-0.9%w/v); P-Hydroxybenzoate (0.01-0.25%w/v); And thimerosal (0.004-0.02%w/v).

Pharmaceutical composition of the present invention is optional comprises active component and the optional pharmaceutically acceptable body of passing.Term " the pharmaceutically acceptable body of passing " is represented solid or liquid filling agent, the diluent that one or more are compatible or becomes capsule material that they are fit to be applied to people or other vertebrates.Term " is passed body " and is represented natural or synthetic organic or inorganic composition, and described active component makes up with assistance application with it.The composition of described pharmaceutical composition also can with chemical compound blend of the present invention and mutually blend, its blending method makes does not have to hinder in fact the interaction of expected drug curative effect.

The compositions that is suitable for the gastrointestinal tract external administration comprise can with the isoosmotic sterile aqueous preparation of receptor's blood.Acceptable excipient and solvent have water, Ringer ' s solution, phosphate buffer and isotonic sodium chlorrde solution.In addition, aseptic solidified oil is usually also as solvent or suspension media.For this purpose, any brand hardened mineral oil or non-mineral oil can be used, comprise synthesizing singly-or two glyceride.Find that in addition fatty acid such as oleic acid can be used for preparing injection.The body preparation of passing that is suitable for subcutaneous, intramuscular, intraperitoneal, intravenous etc. can be at Remington ' s Pharmaceutical Sciences, MackPublishing Company, and Easton finds among the PA.

Being used for chemical compound of the present invention can send above the form of mixtures of two kinds of these chemical compounds.Except described chemical compound combination, mixture can also comprise one or more adjuvants.

Available multiple route of administration.Certainly, select ad hoc fashion will depend on the dosage that selected specific compound, described patient's age and general health state, disease specific to be treated and curative effect need.In general, method of the present invention can be implemented with any administering mode that medical science allows, and promptly replys and does not cause that any way of unacceptable side effect clinically implements to produce effect level.Preferred administering mode is in above discussion.

Described compositions can provide with unit dosage form easily, and can prepare with the known any method of pharmaceutical field.All methods comprise with described chemical compound with form one or more auxiliary elements pass the bonded step of body.In general, by all even closely with described chemical compound with liquid pass body, the meticulous solid that separates is passed body or two kinds of combinations, if necessary, makes shape of product again, thereby prepare described compositions.

Other delivery system can comprise regularly release, postpone to discharge or lasting release delivery system.These systems can avoid the described chemical compound of repetitive administration, thereby increase described patient and doctor's convenience.The release delivery system of a lot of types can use and be known to those of ordinary skills.They comprise the polymer-matrix system, as poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester), copolymerized oxalate, polycaprolactone, polyesteramide, poe, poly butyric and polyanhydride.The microcapsule that contains the aforementioned polymer of medicine is described in, and for example U.S. Patent No. 5,075, and 109.Delivery system also comprises the non-polymer system, and they are: fat comprises sterin such as cholesterol, cholesteryl ester and fatty acid or neutral fat such as list, two and triglyceride; The hydrogel delivery system; The silicone rubber system; The peptide based system; The wax coating; Use the compressed tablet of conventional binding agent and excipient; The implant of partial fusion etc.Instantiation includes but not limited to: (a) corrosion system, and composition wherein of the present invention is involved with intramatrical form, as is described in U.S. Patent No. 4,452, those in 775,4,675,189 and 5,736,152; (b) diffusion system, wherein active component oozes out from polymer with in check speed, as in U.S. Patent No. 3,854, those that describe in 480,5,133,974 and 5,407,686.In addition, can use the hardware delivery system based on pump, some of them are suitable for implanting.

The present invention will further describe in following examples, and these embodiment do not limit the invention scope that claims are described.

Embodiment

Embodiment 1

The inhibitor of in-vitro screening people TLR9 from the micromolecule storehouse

The candidate's micromolecule that is used for Preliminary screening obtains (PrestwickChemical Library) from 880 the micromolecular commodity of non-patent storehouses, and described micromolecule is selected according to structure diversity with to people's checking bioavailability.Human Embryonic Kidney HEK 293 cells of stable transfection people TLR9 (hTLR9) expression vector (are the EC of ODN 2006 there being 50nM CpG ODN2006 ₅₀Concentration) with from 5 * 10 ^-7To 5 * 10 ^-5Overnight incubation under the selected micromolecule candidate compound of the variable concentrations of M scope.According to cotransfection is tested the TLR9 activity to the inducing of 6 * NF-κ B-luciferase reporting construct in the cell.Measurement result is expressed as lacking the multiple of inducing of baseline uciferase activity that ODN measures for 2006 times, and with individualism EC ₅₀2006 times multiples of inducing to baseline of concentration ODN compare.According to this Preliminary screening, multiple micromolecule is accredited as lead compound, and according to structure and activity classification.Use is selected from other storehouse or the special other micromolecule of preparation for this purpose carries out other screening in a similar manner.Some results of these tests are shown among Fig. 1 and the Biao 5-16.

Described method is more detailed description below.Cell to each independent cell clone (hTLR3-NF κ B-293, hTLR7-NF κ B-293, hTLR8-NF κ B-293 and hTLR9-NF κ B-293) is counted, and is stimulating the previous day with 1.5 * 10 ⁴Cells/well is inoculated in the 96 porocyte culture plates.For adherent, described cell after inoculation at 37 ℃, 5%CO ₂Humid air in overnight incubation.

Screen of the effect of every kind of chemical compound simultaneously at all receptor TLR3, TLR7, TLR8 and TLR9.Every kind of chemical compound test three kinds of different final concentrations (0.5 μ g/ml, 5 μ g/ml and 50 μ g/ml) and each concentration are carried out retest, can use on single porous culture plate up to 15 kinds of test compounds and carry out the stimulation of 16 hour cells.The motherboard of creating 4 * final concentration is added in the cell to prepare different test compounds.

Each independent cell clone on the Tissue Culture Plate adds and repeats contrast.Positive control is by following use (final concentration): 50 μ g/ml poly (I:C) are used for hTLR3-NF κ B-293; 8 μ M resiquimod (R848) are used for hTLR7-NF κ B-293; 30 μ M R848 are used for hTLR8-NF κ B-293; 2.5 μ M CpG-ODN 2006 is used for hTLR9-NF κ B-293.The single culture base is as negative control.Add after contrast and the micromolecule, described cell clone is used EC in addition ₅₀The suitable receptor-specific part of concentration stimulates.

For calculating to EC ₅₀The baseline of the suitable receptor-specific part of concentration is replied, and hole H3 and H6 do not accept the receptor-specific part.(there are cell and EC in every hole for hole H2 and H5 ₅₀The suitable receptor-specific part of concentration) meansigma methods obtains EC divided by the meansigma methods of hole H3 and H6 (every hole has only cell) ₅₀The baseline of the suitable receptor-specific part of concentration is replied (multiple of inducing that is uciferase activity).

After stimulating in 16 hours, remove supernatant, handle cell and before measurement, be stored in-80 ℃ with lysis buffer.

Agonist screens parallel the carrying out of just having described with the front of antagonist.Except not adding EC ₅₀Beyond the receptor-specific part of concentration, described method is similar to described antagonist screening.This results of screening reflection excitability and toxic action.

The pattern evaluation system

Carry out graphic recording for each The selection result.Every kind of chemical compound is determined to describe the digit score of graphic result.Positive compound for as antagonist or agonist or synergist has at least repeated to screen twice.

Antagonist

As the baseline result of above-mentioned each Tissue Culture Plate of measurement (by adding EC ₅₀The receptor-specific part of concentration is measured).By transferring to measure antagonism relatively down with baseline.The antagonist scoring is as follows:

13: the clearly chemical compound of downward modulation is arranged at 0.5 μ g/ml, 5 μ g/ml and 50 μ g/ml;

12: the clearly chemical compound of downward modulation is arranged at 5 μ g/ml and 50 μ g/ml;

11: only the clearly chemical compound of downward modulation is arranged at 50 μ g/ml;

--: in addition 50 μ g/ml also not clearly the downward modulation chemical compound;

Agonist

Be the detection of agonist activity, described cell is handled with aforesaid way, but with not adding EC after the micromolecule processing ₅₀The receptor-specific part of concentration.It is 1 that the baseline of common agonist screening is taken as, because these cells are without EC ₅₀The receptor-specific part of concentration stimulates in addition.When being higher than baseline active, there is agonist activity.The agonist scoring is as follows:

For having the chemical compound of high agonist activity at 0.5 μ g/ml,

-1: relatively induce with baseline to surpass 5 times chemical compound;

-2: relatively induce 2-5 chemical compound doubly with baseline;

-3: relatively induce with baseline to be less than 2 times chemical compound.

For having the chemical compound of high agonist activity at 5 μ g/ml,

0.25: relatively induce with baseline to surpass 5 times chemical compound;

0.5: relatively induce 2-5 chemical compound doubly with baseline;

0.75: relatively induce with baseline to be less than 2 times chemical compound.

For having the chemical compound of high agonist activity at 50 μ g/ml,

1: relatively induce with baseline to surpass 5 times chemical compound;

2: relatively induce 2-5 chemical compound doubly with baseline;

3: relatively induce with baseline to be less than 2 times chemical compound.

Toxicity

In the agonist screening, pass through to analyze activity measurement described micromolecular " toxicity " under the baseline." toxicity " can have different the explanation---the perhaps difference between cell clone, described chemical compound influencing in the described cell clone through the TLR signal transduction pathway, perhaps described chemical compound is to the in fact toxic effect of described cell.

Screen every kind of chemical compound to each " toxicity " in described four kinds of cell lines.It is 1 that the baseline of common agonist screening is taken as, because these cells are without EC ₅₀The receptor-specific part of concentration stimulates in addition.The apparent toxicity scoring is as follows:

T3: the clearly chemical compound of downward modulation is arranged at 0.5 μ g/ml, 5 μ g/ml and 50 μ g/ml;

T2: the clearly chemical compound of downward modulation is arranged at 5 μ g/ml and 50 μ g/ml;

T1: only the clearly chemical compound of downward modulation is arranged at 50 μ g/ml;

Embodiment 2

The inhibitor of in-vitro screening people TLR8 from the micromolecule storehouse

The candidate's micromolecule that is used for Preliminary screening identifies in the mode similar to embodiment 1, except HEK293 cytotostatic transfection people TLR8 (hTLR8) expression vector, and at 500nM R848 (R848 is to the EC of TLR8 ₅₀) and 5 * 10 ^-7-5 * 10 ^-5There is overnight incubation down in the selected micromolecule candidate compound of the variable concentrations of M.According to cotransfection is tested the TLR8 activity to the inducing of 6 * NF-κ B-luciferase reporting construct in the cell.Measurement result is expressed as the multiple of inducing to the baseline uciferase activity that lacks measurement under the R848.According to this Preliminary screening, multiple micromolecule is accredited as lead compound, and according to structure and activity classification.Use is selected from other storehouse or the special other micromolecule of preparation for this purpose carries out other screening in a similar manner.Some results of these tests are shown among the 5-16.Scoring is as above reported but is applied to TLR8.

Embodiment 3

The inhibitor of in-vitro screening people TLR7 from the micromolecule storehouse

The candidate's micromolecule that is used for Preliminary screening identifies in the mode similar to embodiment 1, except HEK293 cytotostatic transfection people TLR7 (hTLR7) expression vector, and at 2 μ M R848 (R848 is to the EC of TLR7 ₅₀) and 5 * 10 ^-7-5 * 10 ^-5There is overnight incubation down in the selected micromolecule candidate compound of the variable concentrations of M.According to cotransfection is tested the TLR7 activity to the inducing of 6 * NF-κ B-luciferase reporting construct in the cell.Measurement result is expressed as the multiple of inducing to the baseline uciferase activity that lacks measurement under the R848.According to this Preliminary screening, multiple micromolecule is accredited as lead compound, and according to structure and activity classification.Use is selected from other storehouse or the special other micromolecule of preparation for this purpose carries out other screening in a similar manner.Some results of these tests are shown among the 5-16.Scoring is as above reported but is applied to TLR7.

Embodiment 4

The inhibitor of in-vitro screening people TLR3 from the micromolecule storehouse

The candidate's micromolecule that is used for Preliminary screening identifies in the mode similar to embodiment 1, except HEK293 cytotostatic transfection people TLR3 (hTLR3) expression vector, and at 2.5 μ g/ml poly (I:C) (poly (I:C) is to the EC of TLR3 ₅₀) and 5 * 10 ^-7-5 * 10 ^-5There is overnight incubation down in the selected micromolecule candidate compound of the variable concentrations of M.According to cotransfection is tested the TLR3 activity to the inducing of 6 * NF-κ B-luciferase reporting construct in the cell.Measurement result is expressed as the multiple of inducing to the baseline uciferase activity that lacks measurement under the poly (I:C).According to this Preliminary screening, multiple micromolecule is accredited as lead compound, and according to structure and activity classification.Use is selected from other storehouse or the special other micromolecule of preparation for this purpose carries out other screening in a similar manner.Some results of these tests are shown among the 5-16.Scoring is as above reported but is applied to TLR3.

In the data that in following table 5-17, provide, should be realized that some chemical compound can meet more than a kind of chemical formula at least.

Table 5. formula II examples for compounds and to the inhibitory action of selected TLR

Table 6. formula III examples for compounds and to the inhibitory action of selected TLR

Table 7. formula IV examples for compounds and to the inhibitory action of selected TLR

Table 8. formula V examples for compounds and to the inhibitory action of selected TLR

Table 9. formula VI examples for compounds and to the inhibitory action of selected TLR

Table 10. formula VII examples for compounds and to the inhibitory action of selected TLR

Table 11. formula VIII examples for compounds and to the inhibitory action of selected TLR

Table 12. formula IX examples for compounds and to the inhibitory action of selected TLR

Table 14. formula XI examples for compounds and to the inhibitory action of selected TLR

Table 15. formula XII examples for compounds and to the inhibitory action of selected TLR

Table 16. formula XIII examples for compounds and to the inhibitory action of selected TLR

Table 17. formula XIV examples for compounds and to the inhibitory action of selected TLR

Embodiment 5

The selected micromolecule of screening in the body

Use the source of candidate's micromolecule and PAMP or other the suitable TLR part such as the CpG nucleic acid of experiment mice known quantity.The negative control mice is only accepted the source of independent PAMP or other suitable TLR part such as CpG nucleic acid.Behind the appropriate time, obtain blood sample, and estimate the cytokine serum-concentration with appropriate method such as enzyme linked immunological absorption test (ELISA) from contrast and experiment mice.Substituting or in addition, from two treated animal separating periphery blood monocytic cells (PBMC), and with the expression of appropriate technology such as fluorescence activated cell sorting (FACS) check activation marker thing.Paired mode comparative control and experimental group result.With the negative control animal relatively, the activation marker thing is expressed and is reduced or cytokine concentrations reduces and shows that described micromolecule suppresses the signal transmission TLR mediation, that described TLR part is replied in the experimental group animal.With the negative control animal relatively, the activation marker thing is expressed and is increased or the cytokine concentrations increase shows that described micromolecule promotes the signal transmission TLR mediation, that described TLR part is replied in the experimental group animal.

Embodiment 6

The fixed micromolecule of screening in the body

Use experiment mice candidate micromolecule.The negative control mice is used the independent body of passing.Use micromolecule or use independent pass body after, separate PBMC, stimulate described PBMC to make it to express under the condition of activation marker thing such as CD86 or secretory product such as cytokine (as IFN-α, IL-6, TNF-α) or chemotactic factor (as IP-10), lacking under the micromolecule then with the external CpG nucleic acid that is exposed to of PBMC.With FACS, ELISA or the expression of the quantitative described activation marker thing of other appropriate method or the secretion of described product, and relatively use or do not use the result who obtains under the described micromolecule.With the negative control animal relatively, the activation marker thing is expressed and is reduced or cytokine concentrations reduces and shows that described micromolecule suppresses the signal transmission TLR mediation, that described TLR part is replied in the experimental group animal.With the negative control animal relatively, the activation marker thing is expressed and is increased or the cytokine concentrations increase shows that described micromolecule promotes the signal transmission TLR mediation, that described TLR part is replied in the experimental group animal.

Embodiment 7

4-primary amino radical quinoline is to the antagonism of people TLR9

Luciferase gene with human TLR 9 receptor (hTLR9) and NF-κ B promoters driven is stablized cotransfection HEK293 cell, produces hTLR9-NF-κ B-293 cell line.Counting hTLR9-NF-κ B-293 cell, and with 1.5 * 10 ⁴Cells/well is inoculated on the 96 porocyte culture plates, in 37 ℃/5%CO in test the previous day ₂Cultivate.Test the same day, add the antagonist of 50 μ M, 5.0 μ M or 0.5 μ M respectively.Use described TLR9 agonist CpG-ODN 2006 irritation cells of EC50 dosage then, and in the humidification incubator 37 ℃ cultivated 16 hours.Remove supernatant, handle cell, and before measuring uciferase activity, be stored in-80 ℃ with lysis buffer.Use Promega, the luciferase test macro of USA is measured the luciferase readout according to manufacturer's explanation.The results are summarized in table 18.The antagonist dosage of listing in the table 18 is the minimum effective dose (μ M) of the described TLR9 agonist of blocking-up.Apparent according to result in the table 18, measure through this 3 methods of testing, for all test compounds, the minimum effective dose (μ M) of blocking described TLR9 agonist is 0.5 μ M or 5 μ M.

Table 18.4-quinolin-2-ylamine is to the antagonism of people TLR9

Embodiment 8

Quinazoline is to the antagonism of people TLR9

As described in embodiment 7, count hTLR9-NF-κ B-293 cell, and with 1.5 * 10 ⁴Cells/well is inoculated on the 96 porocyte culture plates in test the previous day, and in 37 ℃/5%CO ₂Cultivate.Test same day, add from the antagonist of the initial various concentration through 1/5 to 1/6 dilution of 7 steps of 50 μ M.Use described TLR9 agonist CpG-ODN 2006 irritation cells of EC50 dosage then, and in the humidification incubator 37 ℃ cultivated 16 hours.Press embodiment 7 and remove supernatant, handle cell with lysis buffer, and before described measurement uciferase activity, be stored in-80 ℃.Obtain ∑ shape antagonism curve, calculate the inhibitor concentration (IC50) that blocking-up 50% agonist is replied.Title bar is that the result of hTLR9 is the IC50 dosage (μ M) that CpG ODN produces signal in the blocking-up hTLR9-NF-κ B-293 cell in the table 19.

In addition, the TLR9 part antagonism among the monitoring human peripheral blood mononuclear cell (PBMC).Obtain from the yellowish chromatograph of healthy male and women's peripheral blood (buffy coat) prepared product from Dusseldorf blood bank of university (Blood Bank ofthe University of Dusseldorf) (Germany), and from these through Ficoll-Hypaque (Sigma) centrifugal purification PBMC.The PBMC of purification is resuspended in and adds the hot deactivation people of 5% (v/v) AB serum (BioWhittaker is Belgium) or in RPMI 1640 culture medium of 10% (v/v) heat-inactivated fetal bovine serum (FCS), 1.5ml L-glutaminate, 100U/ml penicillin and 100 μ g/ml streptomycins (all from Sigma).Concentration 3 * 10 ⁶/ ml to 5 * 10 ⁶The fresh PBMC of/ml adds in the 96 hole circle base plates (150 μ l/ hole).Add the initial antagonist of various concentration behind the cell bed board respectively through 1/5 to 1/6 dilution of 7 steps from 50 μ M.Use described TLR9 agonist CpG-ODN 2006 irritation cells then, and in the humidification incubator 37 ℃ cultivated 16 hours.Collect culture supernatant,, freeze at-20 ℃ during until needs if do not use immediately.With commodity ELISA test kit (IL-6, Diaclone, USA) interleukin 6 (IL-6) in the quantitative assessment supernatant.Obtain ∑ shape antagonism curve and calculate IC50.Title bar is that the result of IL-6 is the IC50 dosage (μ M) of the IL-6 production that CpG ODN produces in the blocking-up PBMC cell in the table 19.

ND: free of data

Embodiment 9

Compare with the quinoline of structural similarity, the toxicity in vivo of quinazoline compound reduces

Inject CMZ 203-43 (1.0 or 4.0mg), the CMZ 203-34 (1.0 or 4.0mg) of (bolus injection) 0.2ml volume or CMZ 203-49 (1.0 or 4.0mg) fast to female BALB/c mouse (n=3/ group) intraperitoneal.Compound C MZ203-43 has structural formula

Other mice is accepted 10% dimethyl sulfoxide (DMSO) in the same manner.(the 1st day) animal of weighing up to the 5th day every day before inject.The 5th day heart puncture blood collecting and analyzing blood are learned and biochemical parameter.Monitor the animal M ﹠ M every day.The result is shown in Fig. 2 and Fig. 3.

Fig. 2 A represents not body weight change (with respect to the body weight before the injection for the first time) ± meansigma methods standard error on the same group.Fig. 2 B represents not average weight ± meansigma methods standard error on the same group.The mice body weight of accepting 1.0mg CMZ 203-43 significantly reduces, and the mice of accepting two kinds of dosage quinazolines is not like this.All accept the mice of 4.0mg CMZ 203-43 death in the 4th day.4.0mg the mice postmortem of CMZ 203-43 shows intestinal obstruction.

Fig. 3 represents leukocyte (WBC) differentiation with on the same group total leukocyte mean percentage ± meansigma methods standard error not.The mice of handling with 1.0mg CMZ 203-43 has a large amount of neutrophil(e) cells, and the mice of accepting two kinds of dosage quinazolines does not have.Because all mices are dead before the 5th day, so the data of the mice that 4.0mg CMZ 203-43 of no use handles.

Embodiment 10

Suppress in 4-primary amino radical quinoline and quinazoline the body IP-10

Peritoneal injection 40 μ g quinoline 203-43, quinazoline 203-34,203-49 or 203-51, or inject the contrast of 100 μ l sterile phosphate buffer (PBS), (n=5) carries out pretreatment to BALB/c mouse.Injected back 1 hour, to mouse subcutaneous injection 100 μ g CpG ODN 2006.Injection CpG ODN 2006 got blood to animal in back 3 hours, and with IP-10 level in the specific enzyme-linked immunoadsorption test of IP-10 (ELISA) measurement blood plasma.The result is shown in Fig. 4.Significantly reduce with serum I P-10 concentration in 203-43 (comparing p=0.00013) or the pretreated mice of 203-34 (comparing p=0.00044) with the PBS contrast with the PBS contrast.

Embodiment 11

Suppress in 4-primary amino radical quinoline and quinazoline the body TNF-α

Also TNF-α is analyzed for the BLAB/c mice group of handling as embodiment 10.Animal was got blood in 1 hour behind the injection CpG ODN2006, and measure TNF-alpha levels in the blood plasma with TNF-alpha specific ELISA.The result is shown in Fig. 5.Significantly reduce with serum TNF-α concentration in 203-43 (comparing p=0.0023) or the pretreated mice of 203-34 (comparing p=0.0012) with the PBS contrast with the PBS contrast.And have the trend that serum TNF-α concentration reduces with the mice that 203-49 handles.

Embodiment 12

Synthetic CMZ 203-84

4-fluorine benzophenone (4-flurobenzophenone) (16g, 0.08mol) and N methyl piperazine (14g, (12.7g stirs in water 0.12mol) and refluxed 20 hours mixture 0.14mol) containing sodium carbonate.The cooling back extracts mixture with ethyl acetate (200mL).Water (100mL) washs organic facies and then (3 * 50mL) extract with 10% hydrochloric acid solution.Acid extraction liquid with ethyl acetate (100mL) washing merges adds 40% sodium hydroxide solution then and makes it to become alkali.The solid aminobenzophenone is extracted in the ethyl acetate (200mL), and with salt water washing extract.The vacuum evaporation ethyl acetate obtains white solid product mutually, productive rate 7.0g, 33%.

Embodiment 13

Synthetic CMZ 203-85

(15g 0.056mol) stirs and reflux with the mixture of Powdered sodium hydroxide (15g) in ethanol (150mL) described benzophenone.Stop to heat and adding zinc powder (15g) several times with the speed that keeps mixture to reflux.Add the post-heating mixture and make it to reflux 1 hour.Reaction mixture is also filtered and is dezincified.Add (500mL) in the entry with ethanol (20mL) washing zinc and with merging filtrate.After filtration, washing with water also, drying is separated to the white crystals product.Obtain described carbinol 12.1g, productive rate 80%.

Gained carbinol (1.13g, 4.18 * 10 ^-3Mol) nmp solution (10mL) stirs under blanket of nitrogen and along with adding sodium hydride dispersion (0.336g, 60%, 8.36 * 10 ^-3Mol).Stirring this mixture at 65 ℃ stops until the hydrogen generation.Add hydrochloric acid 2-(dimethylamino) diethylaluminum monochloride (0.602g, 4.18 * 10 then ^-3And continue to stir these mixture 1 hour mol), at 65 ℃.To add the sodium hydride dispersion and hydrochloric acid 2-(dimethylamino) diethylaluminum monochloride of twice equal proportion in one hour at interval again.After last the adding, stir the mixture and heated cooling then two hours.Mixture is poured in 10% sodium hydroxide solution (200mL), and (2 * 100mL) extract this suspension with dichloromethane.With 10% sodium hydroxide solution (100mL) washing combining extraction liquid, coupling vacuum stripping then obtains that some NMP pollute and the crude product of a small amount of starting material.On silica gel chromatography, use the dichloromethane eluting impurity that contains 20% methanol, thus purified product.Then with the dichloromethane eluted product that contains 20% methanol that contains 1% diethylamide.Obtain the CMZ 203-85 of 0.540g light brown oily, productive rate 37%.

Embodiment 14

Synthetic CMZ 203-88

Described benzophenone (4.65g, 0.0173mol) and oxammonium hydrochloride. (1.89g 0.027mol) is incorporated in the ethanol (25mL).Adding 40% sodium hydroxide solution in this mixture (8.7g, 0.087mol).Stirring and refluxing gained mixture shows that until TLC (silica gel contains the dichloromethane of 10% methanol) reaction finishes (about 30 minutes).After the cooling, add sodium bicarbonate (20g) aqueous solution (300mL), and the isolated by filtration precipitated solid.Wash with water and drying after, obtain 4.85g (95%) CMZ 203-88 white solid.

Embodiment 15

Synthetic CMZ 203-88-1

Described oxime (1.0g, 3.39 * 10 ^-3Mol) alcoholic solution (20mL) exists 10% carbon to carry stirring and refluxing under the palladium (200mg).In this mixture, dropwise add 90% formic acid (0.35g, 6.77 * 10 ^-3Mol).After adding formic acid fully, heating reflux reaction mixture 30 minutes.Cooling mixture and isolated by filtration catalyst, filtrate is stripping under vacuum.The rapid curing of oil residues quantitatively obtains CMZ 203-88-1.

Embodiment 16

Synthetic CMZ 203-89

Stir described carbinol (1.4g, 5.0 * 10 ^-3Mol) slowly add thionyl chloride (0.6g, 5.0 * 10 in the time of DMF (10mL) and dichloromethane (20mL) solution ^-3Mol).After the stirring at room 30 minutes, dropwise add N, N-dimethyl-ethylenediamine (0.88g, 1.0 * 10 ^-3Mol) in turbid solution.Stirring at room mixture 30 minutes is poured in 5% sodium hydroxide solution (400mL) then.(2 * 100mL) extract this mixture, and combining extraction liquid washs with 5% sodium hydroxide (200mL) with dichloromethane.Extract filters after dried over mgso, and stripping obtains grease under vacuum.Thereby with the dichloromethane that contains 20% methanol through silica gel chromatography eluting impurity purified product.Then with the dichloromethane eluted product that contains 20% methanol that contains 1% diethylamine.Obtain light brown oily CMZ 203-89, productive rate 0.588g, 33.4%.

Embodiment 17

Synthetic CMZ 203-91

Stir described carbinol (0.6g, 2.03 * 10 ^-3Mol) slowly add thionyl chloride (0.242g, 2.03 * 10 in the time of DMF (5mL) solution ^-3Mol).After the stirring at room 30 minutes, dropwise add N methyl piperazine (0.400g, 4.0 * 10 ^-3Mol) in solution.Stirring at room mixture 30 minutes is poured in 5% sodium hydroxide solution (200mL) then.(2 * 100mL) extract this suspension, combining extraction liquid water (100mL) washing with dichloromethane.Extract stripping under vacuum obtains grease.Thereby with the dichloromethane that contains 20% methanol through silica gel chromatography eluting impurity purified product.Then with the dichloromethane eluted product that contains 20% methanol that contains 1% diethylamine.Obtain oily CMZ203-91, through placement and crystallization, productive rate 0.58g, 77%.

Embodiment 18

Novel diarylmethanes chemical compound of the present invention is to the antagonism of people TLR9

As counting hTLR9-NF-κ B-293 cell as described in the embodiment 7 and with 1.5 * 10 ⁴Cells/well is inoculated on the 96 porocyte culture plates in test the previous day, and is incubated at 37 ℃/5%CO ₂Test same day, add from the antagonist of the initial various concentration through 1/5 to 1/6 dilution of 7 steps of 50 μ M.Use TLR9 agonist CpG-ODN 2006 irritation cells of EC50 dosage then, and in the humidification incubator 37 ℃ cultivated 16 hours.Press embodiment 7 and remove supernatant, handle cell and before described measurement uciferase activity, be stored in-80 ℃ with lysis buffer.Obtain ∑ shape antagonism curve and calculate the inhibitor concentration (IC50) that blocking-up 50% agonist is replied.Title bar is that the result of hTLR9 is the IC50 dosage (μ M) that CpG ODN produces signal in the blocking-up hTLR9-NF-κ B-293 cell in the table 20.

The novel diarylmethanes chemical compound of table 20. the present invention is to the antagonism of people TLR9

Embodiment 19

Synthetic CMZ203-34

Step 1

Contain sodium carbonate (12.7g, the 4-fluorobenzaldehyde in water 0.12mol) (80mL) (10g, 0.08mol) and N methyl piperazine (14g, 0.14mol) mixture stirs also and refluxed 21 hours.After the cooling mixture is poured in the separatory funnel of moisture (200mL), the oily product is extracted into dichloromethane (in 3 * 50mL).Combining extraction liquid washes with water, then stripping under vacuum.Isolating oily product becomes the sepia solid through cooling curing.Solid product is pulverized in hexane and is separated after filtration.Obtain almost solid N-(4-formoxyl phenyl)-the N '-methyl piperazine of white of 14.9g (91%) after the drying.

Step 2

Anthranilamide (9.94g, 0.073mol) and N-(4-formoxyl phenyl)-N '-methyl piperazine (14.9g 0.073mol) is incorporated among the NMP (100mL).Stir this mixture and insulation until dissolving fully.In warm solution, add p-methyl benzenesulfonic acid monohydrate (2.0g), then 100 ℃ of heated solutions 30 minutes.Add acetic acid (50mL), continue to stir and heating blends 30 minutes at 100 ℃.During about 2 minutes, add chloranil (17.9g, 0.073mol).Heated dark solution 10 minutes at 100 ℃.Solution cool to room temperature and water (500mL) and t-butyl methyl ether (TBME, 500mL) dilution.Stir the mixture and add solid sodium carbonate and alkalize.Stir 15 minutes after-filtration precipitation separation products.Crude product is resuspended in the warm water, stirs 5 minutes, filters then and collects.After water, TBME wash successively, air drying gained solid.Exsiccant quinazoline recrystallization in n-butyl alcohol obtains colourless needle-like product 11.1g (50%).

Step 3

Described quinazoline (2.4g, 7.5 * 10 ^-3Mol) under agitation add several times in the phosphorus oxychloride (10mL).Described mixture is warmed to 80 ℃ and obtains red solution, and solid therefrom begins precipitation.Mixture stirred 15 minutes at 80 ℃, and heating makes of short duration backflow then.Cooling back gained slurry under agitation slowly adds in cold 10% sodium carbonate liquor (650mL).Stir after 15 minutes, the solid chloro-quinazoline is extracted into chloroform (in 2 * 150mL).Dried over mgso is used in combining extraction liquid salt water washing then.Filtering solution is removed desiccant, and filtrate stripping under vacuum obtains yellow solid.(NMP, 20mL) solid is through the dissolving of heating to add N-Methyl pyrrolidone.Add N, and the N-dimethyl-ethylenediamine (1.32g, 0.015mol), then at 100 ℃ of solution 30 minutes of heating.Cooling back water (200mL) and strong aqua ammonia (50mL) dilution nmp solution.Precipitated product is extracted into dichloromethane (in 2 * 100mL).Combining extraction liquid through dried over mgso, filters, and stripping obtains shallow brown oil then.Make eluant with the dichloromethane that contains 10% methanol and continue purification through silica gel chromatography.Separate like this and obtain 600mg (1.54 * 10 ^-3Mol) the pure quinazoline of oily.Product is dissolved in t-butyl methyl ether, and (TBME 50mL), and under agitation adds toluenesulfonic acid monohydrate (584mg, 3.1 * 10 that are dissolved among the TBME (50mL) ^-3Mol).Isolated by filtration obtains the two toluene fulfonates of solid, obtains the 1.0g yellow solid product with TBME washing and drying.

Embodiment 20

Synthetic CMZ 203-44

N, (3.5g, 0.04mol) (2.4g 0.01mol) is incorporated in the n-butyl alcohol (50mL) and backflow the N-methyl ethylenediamine with 2-phenyl-4-chloro-quinazoline.Once you begin reflux, TLC (silica gel contains the dichloromethane of 10% methanol) shows that reaction finishes.Cooling solution and stripping.Residue is dissolved in t-butyl methyl ether (TBME) and washes solution with water.Through dried over mgso after-filtration and stripper solution, obtain 1.37g (0.0047mol, 47%) oily crude product.Product is dissolved in the ethanol (25mL) and uses concentrated sulphuric acid (0.46g, 0.0047mol) solution-treated that is dissolved in the ethanol (5mL).Begin to separate out crystalline sulfuric acid salt in several seconds, room temperature 30 minutes is after isolated by filtration.With washing with alcohol gained salt and dry.Productive rate is 1.77g, (from crude product 96%).

Embodiment 21

Synthetic CMZ 203-45

60% sodium hydride (0.80g, 0.02mol) slurry in NMP (50mL) stirs in blanket of nitrogen, slowly adds N through syringe simultaneously, the N-methylethanolamine (1.96g, 0.022mol, 2.2mL).After adding described amine, 30 ℃ of agitating solutions 10 minutes, once add subsequently 2-phenyl-4-chloro-quinazoline (2.4g, 0.01mol).Continuation was stirred 30 minutes at 40 ℃, and TLC (silica gel contains the dichloromethane of 10% methanol) shows that reaction finishes subsequently.Cooling solution is also poured in the water (250mL).Product is extracted into dichloromethane (in 3 * 50mL).Dried over mgso after-filtration and stripper solution obtain the oily crude product.Product is dissolved in the ethanol (20mL), and with the concentrated sulphuric acid (0.85g, 0.0087mol) solution-treated that are dissolved in the ethanol (10mL).Crystalline sulfuric acid salt is slowly separated out, and 60 minutes after-filtration of room temperature separate.With washing with alcohol gained salt and dry.Productive rate is 1.37g, (is 40% based on sulphuric acid).

Embodiment 22

Synthetic CMZ 203-49

(3.2g 0.01mol) under agitation adds in the phosphorus oxychloride (10mL) described quinazoline several times.Described mixture refluxed 30 minutes.Cool off orange slurry and add TBME (100mL).Stir 10 minutes after-filtration and separate described chloro-quinazoline, and add N, in the nmp solution (50mL) of N-dimethylaminoethanol sodium salt.By being prepared as follows N, the nmp solution of N-dimethylaminoethanol sodium salt.60% sodium hydride (2.1g, 0.05mol) slurry in NMP (50mL) stirs in blanket of nitrogen, slowly adds N through syringe simultaneously, the N-dimethylethanolamine (4.9g, 0.055mol).After adding described amine, 30 ℃ of agitating solutions 10 minutes.This mixture stirred 30 minutes at 100 ℃.Cooling back solution adds in the dichloromethane (150mL), and water (3 * 150mL) extract this solution.Stripping dichloromethane solution, residue are dissolved in the ethanol (50mL).In this solution, add the concentrated sulphuric acid be dissolved in the ethanol (5mL) (0.8g, 0.008mol).The solid salt that isolated by filtration is separated out, and successively with ethanol and TBME washing, drying.Productive rate is 1.3g, 23%.

Embodiment 23

Synthetic CMZ 203-51

Step 1

Under agitation (6.8g 0.05mol) (adds p-methyl benzenesulfonic acid monohydrate (1.0g) with two phenyl formaldehyde among the 9.1g, DMF solution (100mL) 0.05mol) to anthranilamide.Along with dihydroquinazoline precipitation yellow solution becomes thick paste rapidly.Heating serosity to 100 ℃ makes most solids dissolvings.(12.3g 0.05mol), forms dark solution repeatedly to add chloranil in this hot mixt during in 2 minutes.From then on product begins crystallization in the solution.Heating blends obtains dark solution to boiling.The described quinazoline in cooling back forms colourless acicular crystal.The isolated by filtration product thoroughly washs dry then with DMF and acetone.Productive rate is 11.9g, 80%.

Step 2

The phosphorus oxychloride (12mL) and the described pair of phenylquinazoline (2.98g, 0.01mol) stirring and refluxing 1 hour together.Excessive phosphorus oxychloride is removed in distillation, and the oily yellow residue is removed the trace phosphorus oxychloride with vacuum suction device.In hexane, pulverize the gained yellow solid and topple over and remove hexane.In yellow solid, add n-butyl alcohol (50mL) and N, N-dimethyl-ethylenediamine (6mL).This solution of stirring and refluxing 30 minutes.The cooling butanol solution is also poured in the separatory funnel that contains TBME (200mL) and 10% hydrochloric acid (200mL).The thermal agitation funnel is standing demix then.The separation water layer also is collected in the flask, scrapes flask with glass rod, and product begins to separate out with light yellow solid immediately.30 minutes after-filtration separated products of room temperature are also used a small amount of cold water washing.Dry back obtains 3.64g (82%) light yellow solid product.

Embodiment 24

Synthetic CMZ 203-76

Step 1

2,4-two chloro-6, (5.2g, 0.02mol) and N, (1.76g, dichloromethane 0.02mol) (65mL) solution was stirring at room 2 hours for the N-dimethyl-ethylenediamine for the 7-dimethoxyquinazoline.Add another part N in the serosity that forms, (1.76g 0.02mol) forms clear solution to the N-dimethyl-ethylenediamine.This solution of stirring at room spends the night.Solution poured in 10% sodium carbonate liquor (200mL) product is separated out with solid.Isolated by filtration solid, water and washed with dichloromethane successively then.This solid recrystallization in the 2-propanol obtains the purified product of 2.6g (42%) white crystalline solid.

Step 2

Described chloro-quinazoline (930mg, 0.003mol) and the mixture of N methyl piperazine (5mL) 100 ℃ the heating 3 hours.Cooling solution and coupling vacuum stripping.Add n-butyl alcohol (10mL) and once more the described solution of coupling vacuum stripping to remove remaining N methyl piperazine.Residue is dissolved in ethanol (20mL), adds the sulphuric acid (0.59g, 0.006mol) solution that are dissolved in the ethanol (5mL).Mixture refluxed 1 minute, then cooling.The isolated by filtration product is used ethanol and TBME washed product successively, and is dry then.Productive rate is 0.85g, (50%).

Embodiment 25

Synthetic CMZ203-78

(1.55g, 0.005mol) (1.6g, 0.01mol) mixture in n-butyl alcohol (5mL) was 100 ℃ of heating 2 hours with the N-phenylpiperazine for described chloro-quinazoline.With n-butyl alcohol (15mL) dilution gained serosity, mixture heated is to seething with excitement to obtain clear solution.Separate out solid and isolated by filtration after the cooling.Stir solids 15 minutes is to remove small amount of N-phenylpiperazine in warm water.The isolated by filtration product with warm water washing and dry, obtains 1.0g (46%) white solid product.

Embodiment 26

Synthetic CMZ 203-87

4-chloro-quinazoline (2.74g, 8.0 * 10 ^-3Mol) and N-aminoethyl morpholine (2.10g, 1.6 * 10 ^-2Mol) mixture in ethanol (25mL) refluxed 2 hours.Reaction mixture is also poured in the water (200mL) that contains ammonia (50mL 28%), and precipitated product is extracted into dichloromethane (in 3 * 100mL).Dried over mgso is used in combining extraction liquid water (200mL) washing then.Remove by filter the described solution of desiccant final vacuum stripping, obtain the sepia solid crude product.This solid is dissolved in warm dichloromethane (10mL), and solution dilutes with warm hexane (20mL).After the cooling, product is separated out with the sepia solid crystal.Solid is also dry with the hexane wash that contains 30% dichloromethane after the isolated by filtration.Productive rate is 1.2g, (35%).

Embodiment 27

Synthetic CMZ 203-93

Step 1

(13.6g, 0.10mol) (10.7g adds p-methyl benzenesulfonic acid monohydrate (2.0g) in NMP 0.10mol) (100mL) and acetic acid (50mL) solution with the 3-pyridine carboxaldehyde to anthranilamide.50 ℃ of agitating solutions 30 minutes, and at this moment point by funnel during 1 minute, add several times chloranil (24.6g, 0.10mol).Stirring dark solution and cool to room temperature makes described quinazoline form crystallization.The isolated by filtration solid, and use acetone/NMP (1: 1) and washing with acetone successively.Dry back crude product recrystallization in n-butyl alcohol (200mL) and NMP (90mL).Separate the sepia filament crystal that obtains described quinazoline, productive rate 10.6g, 47.5%.

Step 2

(4.46g's described quinazoline of a part 0.02mol) refluxed 1 hour in phosphorus oxychloride (20mL).Carefully solution is poured in cold 20% sodium carbonate liquor (500mL) after the cooling.Isolated by filtration solid chloro-quinazoline washes with water and is dissolved in the dichloromethane (400mL).Solution obtains described chloro-quinazoline light yellow solid, productive rate 3.7g, 76.5% with dried over mgso, filtration and stripping.(3.7g, 0.015mol) (3.99g's described 4-chloro-quinazoline altogether 0.031mol) refluxed 1 hour with the N-2-aminoethyl morpholine in ethanol.The cooling back is stripper solution at once, in the residue water-soluble (400mL).Add sodium carbonate and make solution becomes alkali, product is extracted into dichloromethane (in 2 * 100mL).Dried over mgso is used in combining extraction liquid water (50mL) washing then.Filter back stripping extract, obtain the sepia solid product.Recrystallization solid in ethyl acetate (25mL) and hexane (50mL).Be separated to the almost solid product 203-93 of white, productive rate 2.86g, 56.0%.

Embodiment 28

Synthetic CMZ 203-95

Step 1

(13.6g, 0.10mol) (10.7g adds p-methyl benzenesulfonic acid monohydrate (2.0g) in methanol 0.10mol) (250mL) and acetic acid (25mL) solution with the 2-pyridine carboxaldehyde to anthranilamide.50 ℃ of agitating solutions 30 minutes, and at this moment point by funnel during 1 minute, add several times chloranil (24.6g, 0.10mol).With NMP (25mL) washing funnel.Mixture heated is to refluxing 5 minutes.Stirring dark solution and cool to room temperature makes described quinazoline form crystallization.The isolated by filtration solid is also used washing with acetone.Twice in n-butyl alcohol recrystallization of crude product of dry back.A small amount of under these conditions tetrachloro hydroquinone meeting cocrystallization, this can remove by stirring in warm 5% sodium carbonate liquor.Described quinazoline separates as the sepia solid, productive rate 9.5g, 43%.

Step 2

(4.46g's described quinazoline of a part 0.02mol) refluxed 30 minutes in phosphorus oxychloride (20mL).Carefully solution is poured in the cold water (500mL) after the cooling.Add 40% sodium hydroxide solution neutralization solution, thereby described chloro-quinazoline is separated out with white solid.Isolated by filtration solid chloro-quinazoline washes with water and is dissolved in the dichloromethane (300mL).Solution obtains described chloro-quinazoline light yellow solid, productive rate 2.75g, 57% with dried over mgso, filtration and stripping.(2.75g, 0.0114mol) (2.97g's described 4-chloro-quinazoline altogether 0.0228mol) refluxed 30 minutes with the N-2-aminoethyl morpholine in ethanol.The cooling back is poured solution in 10% sodium hydroxide solution (200mL) at once, and product is extracted into dichloromethane (in 3 * 100mL).Combining extraction liquid is used dried over mgso then with 10% sodium hydroxide solution (150mL) washing.Filter back stripping extract and obtain the sepia solid product.Recrystallization solid in n-butyl alcohol (10mL) and hexane (20mL).Be separated to the almost solid product 203-95 of white, productive rate 1.32g, 34.5%.

Equivalent

The description of more than writing is enough to make those skilled in the art to implement the present invention.The scope of the embodiment that provides is provided, because being intended that as the single of one aspect of the invention of these embodiment illustrates, the embodiment of other function equivalence is included in the scope of the present invention.Except those provided herein and described, from the explanation of front, multiple improvement of the present invention it will be apparent to those skilled in the art that, and drops in the scope of appended claims.Advantage of the present invention and purpose are not to be comprised by each embodiment of the present invention.

The full content of all lists of references, patent and the patent disclosure of quoting among the application is all introduced herein as a reference.

Sequence table

＜110〉Coley Pharm GmbH

Coley Pharm Group Inc.

＜120〉micromolecule TOLL sample receptor (TLR) antagonist

<130>C1041.70036WO00

<150>US?60/556,007

<151>2004-03-23

<150>US?60/480,588

<151>2003-06-20

<160>4

<170>PatentIn?version?3.1

<210>1

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>1

tcgtcgtttt?gtcgttttgt?cgtt 24

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>2

gggggacgat?cgtcgggggg 20

<210>3

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>3

tcgtcgtttt?cggcgcgcgc?cg 22

<210>4

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<220>

＜221〉modified base

<222>(2)..(2)

<223>m5c

<220>

＜221〉modified base

<222>(5)..(5)

<223>m5c

<220>

＜221〉modified base

<222>(13)..(13)

<223>m5c

<220>

＜221〉modified base

<222>(21)..(21)

<223>m5c

<400>4

tcgtcgtttt?gtcgttttgt?cgtt 24

Claims

1. the quinazoline compound and pharmaceutically acceptable hydrate and the salt that have structural formula XVIII,

Formula XVIII

Wherein

X does not exist or aryl or heterocyclic radical, if X is a phenyl, and NR ₁R ₂It is heterocyclic part;

R ₁And R ₂Independently be mutually hydrogen atom or alkyl, wherein R ₁And R ₂Randomly be combined to form piperazine ring;

Y is oxygen atom or NR ₁₁, R wherein ₁₁Be hydrogen atom or alkyl, wherein R ₁₁Optional and R ₃Or R ₄Be combined to form and replace or unsubstituted morpholine ring;

There is not or contains the alkyl of 1 to 10 carbon in L;

R ₃And R ₄Independently be mutually hydrogen atom or alkyl, wherein R ₃And R ₄Randomly be combined to form the morpholine ring; And

R ₅, R ₆, R ₇And R ₈Independently be mutually hydrogen atom or alkoxyl.

2. the quinazoline compound of claim 1, wherein

R ₆And R ₇It independently is mutually alkoxyl.

3. the quinazoline compound of claim 1, wherein

R ₆And R ₇It independently is mutually methoxyl group.

4. the quinazoline compound of claim 1 and pharmaceutically acceptable hydrate and salt, wherein

X does not exist or aryl; With

L does not exist or C ₂-C ₆Alkyl.

5. the quinazoline compound of claim 4 and pharmaceutically acceptable hydrate and salt, wherein

X does not exist or aryl;

NR ₁R ₂Be to replace or unsubstituted piperazine ring;

Y is NH; With

L is C ₂-C ₆Alkyl.

6. the quinazoline compound of claim 5 and pharmaceutically acceptable hydrate and salt, wherein

X is an aryl;

NR ₁R ₂Be to replace or unsubstituted piperazine ring;

Y is NH;

L is C ₂-C ₆Alkyl;

R ₃And R ₄Independently be mutually methyl or ethyl, or R ₃And R ₄Randomly be combined to form the morpholine ring; And

R ₅, R ₆, R ₇And R ₈Independently be mutually hydrogen atom or alkoxyl.

7. the quinazoline compound of claim 6 and pharmaceutically acceptable hydrate and salt, wherein

X is a phenyl;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄It all is methyl; And

R ₅, R ₆, R ₇And R ₈In each be hydrogen atom.

8. the quinazoline compound of claim 6 and pharmaceutically acceptable hydrate and salt, wherein

X is a phenyl;

NR ₁R ₂It is N methyl piperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄Be combined to form the morpholine ring; And

R ₅, R ₆, R ₇And R ₈In each be hydrogen atom.

9. the quinazoline compound of claim 5 and pharmaceutically acceptable hydrate and salt, wherein

X does not exist;

NR ₁R ₂It is the N-phenylpiperazine;

Y is NH;

L is-CH ₂CH ₂-;

R ₃And R ₄It all is methyl;

R ₆And R ₇It all is methoxyl group; And

R ₅And R ₈It all is hydrogen atom.

10. the chemical compound that has structural formula XVIII,

Formula XVIII

Wherein

X is an aryl;

NR ₁R ₂It is N-methyl-N '-piperazine;

Y is NH;

L is C ₂H ₄

R ₃And R ₄Form the morpholine ring;

R ₅And R ₈It is respectively hydrogen atom; With

R ₆And R ₇Be respectively alkoxyl independently,

And the acceptable salt of pharmacy.

11. the chemical compound of claim 10, wherein R ₆And R ₇Be respectively methoxyl group, and the acceptable salt of pharmacy.

12. the chemical compound of claim 10, wherein R ₆And R ₇It is respectively methoxyl group.

13. the chemical compound of claim 10 is used for the purposes of inhibition through the medicine of Toll sample receptor 9 (TLR9) signal transmission in preparation.

14. the chemical compound of claim 11 is used for the purposes of inhibition through the medicine of Toll sample receptor 9 (TLR9) signal transmission in preparation.

15. the chemical compound of claim 12 is used for the purposes of inhibition through the medicine of Toll sample receptor 9 (TLR9) signal transmission in preparation.

16. the chemical compound of claim 10 is used for the treatment of purposes in the medicine of autoimmune disease among the patient in preparation.

17. the purposes of claim 16, wherein said patient is the people.

18. the chemical compound of claim 11 is used for the treatment of purposes in the medicine of autoimmune disease among the patient in preparation.

19. the purposes of claim 18, wherein said patient is the people.

20. the chemical compound of claim 12 is used for the treatment of purposes in the medicine of autoimmune disease among the patient in preparation.

21. the purposes of claim 20, wherein said patient is the people.