CN1845745B - Administration of TLR7 ligands and prodrugs thereof for treatment of infection by hepatitis c virus - Google Patents
Administration of TLR7 ligands and prodrugs thereof for treatment of infection by hepatitis c virus Download PDFInfo
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- CN1845745B CN1845745B CN2004800255325A CN200480025532A CN1845745B CN 1845745 B CN1845745 B CN 1845745B CN 2004800255325 A CN2004800255325 A CN 2004800255325A CN 200480025532 A CN200480025532 A CN 200480025532A CN 1845745 B CN1845745 B CN 1845745B
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- KTJUHWDQDOYXFP-UHFFFAOYSA-N BC1=C(c2ccccc2)N=C(N)NC1=O Chemical compound BC1=C(c2ccccc2)N=C(N)NC1=O KTJUHWDQDOYXFP-UHFFFAOYSA-N 0.000 description 1
- VNSDIJWHOKJBQK-ZFNDLAMFSA-N CC(C)C(C(OC[C@H]([C@H]([C@H]1O)O)OC1N(C(N=C(N)NC1=O)=C1N1CC=C)C1=O)=O)N Chemical compound CC(C)C(C(OC[C@H]([C@H]([C@H]1O)O)OC1N(C(N=C(N)NC1=O)=C1N1CC=C)C1=O)=O)N VNSDIJWHOKJBQK-ZFNDLAMFSA-N 0.000 description 1
- BUIKJNWRANGGHG-JKOSFCEQSA-N CC(C)[C@@H](C(OC[C@H]([C@H]([C@H]1O)O)O[C@H]1N(C(N=C(N)NC1=O)=C1S1)C1=O)=O)N Chemical compound CC(C)[C@@H](C(OC[C@H]([C@H]([C@H]1O)O)O[C@H]1N(C(N=C(N)NC1=O)=C1S1)C1=O)=O)N BUIKJNWRANGGHG-JKOSFCEQSA-N 0.000 description 1
- ZVWUBAVKKBVMQL-UHFFFAOYSA-N CCCCCOC(Nc1nc(C=CCC2)c2c2c1nc[n]2CC(C)C)=O Chemical compound CCCCCOC(Nc1nc(C=CCC2)c2c2c1nc[n]2CC(C)C)=O ZVWUBAVKKBVMQL-UHFFFAOYSA-N 0.000 description 1
- QHBBLPVDZBEZKK-WRKWTSPFSA-N CCC[n]1c(/C(/C)=C(/C=C\C(C)C)\NC)c(CN)nc1 Chemical compound CCC[n]1c(/C(/C)=C(/C=C\C(C)C)\NC)c(CN)nc1 QHBBLPVDZBEZKK-WRKWTSPFSA-N 0.000 description 1
- AVVDRGPJAJNLPU-UHFFFAOYSA-N CCOc(nc(N)nc1-c2ccccc2)c1Br Chemical compound CCOc(nc(N)nc1-c2ccccc2)c1Br AVVDRGPJAJNLPU-UHFFFAOYSA-N 0.000 description 1
- AWBKEIIVIIOGDX-NTSWFWBYSA-N COc(nc(N)nc1N2[C@@H]3O[C@H](CO)CC3)c1SC2=O Chemical compound COc(nc(N)nc1N2[C@@H]3O[C@H](CO)CC3)c1SC2=O AWBKEIIVIIOGDX-NTSWFWBYSA-N 0.000 description 1
- TZYVRXZQAWPIAB-FCLHUMLKSA-N NC(NC1=O)=NC(N2[C@@H]([C@@H]3O)O[C@H](CO)[C@H]3O)=C1SC2=O Chemical compound NC(NC1=O)=NC(N2[C@@H]([C@@H]3O)O[C@H](CO)[C@H]3O)=C1SC2=O TZYVRXZQAWPIAB-FCLHUMLKSA-N 0.000 description 1
- FSUHHAQAUXBQJK-UHFFFAOYSA-N Nc(nc1-c2ccccc2)ncc1Br Chemical compound Nc(nc1-c2ccccc2)ncc1Br FSUHHAQAUXBQJK-UHFFFAOYSA-N 0.000 description 1
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Abstract
This invention relates to methods for treating or preventing hepatitis C virus infections in mammals using Toll-Like Receptor (TLR)7 ligands and prodrugs thereof. More particularly, this invention relates to methods of orally administering a therapeutically effective amount of one or more prodrugs of TLR7 ligands for the treatment or prevention of hepatitis C viral infection. Oral administration of these TLR7 immunomodulating ligands and prodrugs thereof to a mammal provides therapeutically effective amounts and reduced undesirable side effects.
Description
The priority of U.S. Provisional Patent Application 60/518,996 that the application requires that JIUYUE in 2003 submitted on the 5th U.S. Provisional Patent Application was submitted at November 10 in 60/500,339,2003 and the U.S. Provisional Patent Application of submitting on November 10th, 2,003 60/518,997.
1. invention field
The application relates to the method for using Toll sample receptor (TLR) 7 parts and prodrug treatment or prevention of hepatitis C infection in mammal.More particularly, the prodrug that the present invention relates to one or more TLR7 parts of orally give treatment effective dose is used for the treatment of or prevention of hepatitis C infection.These TLR7 immunomodulating parts of orally give mammal and prodrug thereof can provide the treatment effective dose and reduce adverse side effect.
2. background of invention
By finding out the chemical compound of combination and activation Toll sample receptor (TLR), realize the micromolecule immunomodulating.TLR works in mammiferous innate immune response, usually is the first line of defence of opposing such as pathogen such as antibacterial and virus.The content difference of various TLR in different mammalian cell types, content also can change according to the molecular structure in conjunction with TLR and activation signal approach.These signal pathways produce the reply scope relevant with innate immunity.
TLR differentiates PAMP (pathogen correlation molecule model) and by MyD88 dependency interleukin 1 receptor (IL-1R)-TLR signal pathway immune stimulatory cell, causes the activation of transcription factor NF-KB 2.Identified the functional type family member (TLR1-TLR10) of 10 kinds of TLR in the human body.Akira S. etc., Nature Immunol., 2,675-680 (2001).TLR2, TLR4 and TLR5 be key concerning identification polypeptide polysaccharide, lipopolysaccharide and flagellin.Hayashi, F. etc., Nature, 410,1099-1103 (2001).TLR6 discerns the mycoplasma lipoprotein in conjunction with TLR2.Ozinsky, A., etc., Proc.Natl.Acad.Sci USA., 97,13766-13771 (2000).TLR9 detects the DNA of bacteria that contains non-methylated CpG motif, and TLR3 activates the immunocyte with the double-stranded RNA response.Hemmi, H. etc., Nature, 408,740-745 (2000).
Reported chemical compound lot, the pyrimidine, imidazoquinolie that comprise guanosine analogue, replacement are as the TLR7 part.For example, referring to Hemmi etc., Nature Immunol., 3,196-200 (2002) (imiquimod and R-848 (resiquimod)); Jurk etc., Nat.Immunol., 3,499 (2002) (R-848); With Lee etc., Proc.Natl.Acad.Sci USA, 100,6646-6651 (2003) (guanosine analogue loxoribine wherein, 7-sulfur-8-oxo guanosine (isatoribine) and the 7-denitrification guanosine (deazaguanosine) of mixing, imidazoquinolines, the alternative TLR7 that activates of imiquimod and R-848 (resiquimod)).
Before as potential TLR7 part, past 20 years was carried out a lot of research to guanosine analogue and other D-and L-purine nucleotides.For example, referring to Reitz etc., J.Med.Chem., 37,3561-78 (1994); Michael etc., J.Med.Chem., 36,3431-36 (1993) (7-and/or 8-position have substituent immunomodulating guanosine analogue); Be issued to the patent 5,821,236 (disclosing the 6-alkoxyl derivatives of the arabinose furyl glycosyl purine derivative that can be used for oncotherapy) of Krenitsky etc.; Be issued to the United States Patent (USP) 5,041,426 (disclose some and in the BDF1 mice, can effectively treat pyrimido [4,5-d] the pyrimidine nucleoside acids of L1210) of Robins etc.; Revankar etc., J.Med.Chem., 27,1489-96 (1984) (important broad-spectrum antiviral active 3-deazaguanine purine nucleoside and nucleotide) with some DNA of opposing and RNA viruses.
In recent years, many known chemical compounds with immunostimulatory activity are accredited as the TLR7 part in the literature.For example, referring to Heil etc., Eur.J.Immunol., 33 (11), 2987-97 (2003), Lore etc., J.Immunol., 171 (8), 4320-8 (2003), Nagase etc., J.Immunol., 171 (8), 3977-82 (2003), Mohty etc., J.Immunol., 171 (7), 3385-93 (2003), Pinhal-Enfield, Deng, Am.J.Pathol., 163 (2), 711-21 (2003), Doxsee etc., J.Immunol., 171 (3), 1156-63 (2003), Bottcher etc., Neurosci.Lett., 344 (1), 17-20 (2003), Kaisho etc., Curr.Mol.Med., 3 (4), 373-85 (2003), Okada etc., Eur.J.Immunol., 33 (4), 1012-9 (2003), Edwards etc., Eur.J.Immunol., 33 (4), 827-33 (2003), Akira etc., Immunol.Lett., 85 (2), 85-95 (2003), Ito etc., Hum.Immunol., 63 (12), 1120-5 (2002), Rothenfusser etc., Hum.Immunol., 63 (12), 1111-9 (2002), Yamamoto etc., J.Immunol., 169 (12), 6668-72 (2002), Gibson etc., Cell Immunol., 218 (1-2), 74-86 (2002), Horng etc., Nature, 420 (6913), 329-33 (2002), Yamamoto etc., Nature, 420 (6913), 324-9 (2002), Applequist etc., Int.Immunol., 14 (9), 1065-74 (2002), Sato etc., Int.Immunol., 14 (7), 783-91 (2002); Jurk etc., Nat.Immunol., 3 (6), 499 (2002); Hornung etc., J.Immunol., 168 (9), 4531-7 (2002), Hemmi etc., Nat.Immunol., 3 (2), 196-200 (2002); Bruno etc., Eur.J.Immunol., 31 (11), 3403-12 (2001); Jarrossay etc., Eur.J.Immunol., 31 (11), 3388-93 (2001); Miettinen etc., Genes Immun., 2 (6), 349-55 (2001), Chuang etc., Eur.cytokine Netw., 11 (3), 372-8 (2000) and Du etc., Eur.cytokine Netw., 11 (3), 362-71 (2000).
But known these TLR7 parts are in external and immune stimulatory response in animal, and the research that this has just caused these use of a compound is used for several treatments and uses, and comprises antiviral and treatment of cancer.These chemical compounds are characterised in that, are a) guanosine, b) imidazoquinolie and c) analog or the derivant of pyrimidine.Referring to Akira, Current Opinion, 15,5-11 (2003).A kind of (imiquimod) in the discovery imidazoquinolie chemical type can effectively be treated the local genital infection of papillomavirus.Second kind of imidazoquinolie compounds resiquimod can be used for treating HCV, but the not effect of anti-HCV under the tolerance oral dose of this chemical compound.Pockros etc., Gastroenterology, 124 (Suppl 1), A-766 (2003).
Therefore, be applied to treat immune disease and viral infection though the TLR7 part is only limited; For example, referring to the United States Patent (USP) 5,041 that is issued to Robins etc., 426 and 4,880,784 (have remarkable immunocompetence, 3-β-D-nuclear benzofuran sugar yl the thiazole that comprises interior anti-Semliki Forest (Semliki Forest) virus activity of mice spleen cell propagation and body is [4,5-d] pyrimidine also); Be issued to US patent application publication No.US 2003/0199461 and the WO 03/045968 (having also [4,5-d] pyrimidine nucleoside of the acute and chronically infected 3-β of anti-RNA and DNA viruses-D-nuclear benzofuran sugar yl thiazole) of Averett etc.; So far proved that part can not effectively treat or prevention of hepatitis C.
The orally give of known many purine nucleoside analogs has run into difficulty, comprises absorption difference, poor dissolution, or in the gastrointestinal tract because the combination of the effect of acidity or alkali condition or enzyme and/or these phenomenons causes degraded.Therefore, still need the purine nucleosides acid-like substance, it has the oral administration biaavailability of improvement and gives, and is used to regulate immune system.
And, to compare with intravenous route, the oral tolerance of immunomodulating nucleoside is relatively poor.And owing to combine with the immuning tissue of intestinal wall (for example intestinal) in a large number, there is the specificity tolerance barrier to immunoreagent in gastrointestinal tract.Though this is the important biomolecule mechanism that prevents that body from being attacked by intestinal microbial population, behind the orally give immunomodulatory compounds, owing to give the local concentration height of chemical compound in the intestinal, immuning tissue also can be preferentially infected.This just causes adverse side effect, for example, for immunoactivator, observes gastroenteritis and local hemorrhage effect.
Still there is not the method that solves effective oral delivery immunomodulator problem in the document.Evidence shows at present, and behind the oral low dosage, the body concentration that gives such medicine is subjected to the restriction of gastrointestinal toxicity raising.Therefore, still need to have the immunomodulating TLR7 part that improves oral administration biaavailability and reduce GI irritation.
3. summary of the invention
3.1 TLR7 part
The present invention has been contained and has been used for the treatment of or the novel method of prevention of hepatitis C infection, and the pharmaceutical composition that utilizes TLR7 part or its pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer.
In one embodiment, the method of treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, and described method comprises TLR7 part or its pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer or described stereoisomer pharmaceutically acceptable salt or the hydrate that gives patient treatment or prevention effective dose.
In another embodiment, the method of treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, and described method comprises and gives the patient effectively or the analog that is selected from following material of prevention effective dose and the TLR7 part of derivant: imidazoquinolie c a) guanosine b)) adenine and d) pyrimidine.
In another embodiment, the method of treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, described method comprise give patient treatment or prevention effective dose be selected from the following TLR7 part or the pharmaceutically acceptable salt or the hydrate of its pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer or described stereoisomer:
Wherein:
Each R
1Be H, replacement or unsubstituted alkyl, alkenyl or alkynyl, they can be separated by one or more O, S or N hetero atom, or replacement or unsubstituted aryl or heteroaryl;
R
2Be H, OH, SH, halogen, replacement or unsubstituted alkyl, alkenyl or alkynyl, they can be separated by one or more O, S or N hetero atom, or replacement or unsubstituted-O-(alkyl) ,-O-(aryl) ,-O-(heteroaryl) ,-S-(alkyl) ,-S-(aryl) ,-S-(heteroaryl), aryl or heteroaryl;
R
3Be H, OH, SH, replacement or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl ,-O-(alkyl) ,-O-(aryl) ,-O-(heteroaryl) ,-S-(alkyl) ,-S-(aryl) ,-S-(heteroaryl) ,-NH (alkyl) ,-NH (aryl) ,-NH (heteroaryl) ,-NH (R
4) (alkyl) ,-NH (R
4) (aryl) ,-NH (R
4) (heteroaryl), wherein R
4Be to replace or unsubstituted alkyl;
X is O or S;
Y is H, halogen, OH, OR
4, SH, SR
4Or replacement or unsubstituted alkyl or aryl;
Z is H, halogen, OH, OR
4, SH or SR
4
In another embodiment, the method of treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, described method comprises the TLR7 part that is selected from general formula I a, Ib, Ic, Id, Ie, If, Ig and Ih that gives patient treatment or prevention effective dose, wherein R
1Be H or replacement or unsubstituted alkyl, alkenyl or alkynyl; R
2Be H, OH, halogen, replacement or unsubstituted alkyl, alkenyl, alkynyl or-CH
2-O-(alkyl); R
3Be H, OH, SH, replacement or unsubstituted-O-(alkyl) ,-S-(alkyl) or-NH (alkyl); X is O or S; Y is H, halogen, OH, OR
4, SH or SR
4With Z be H, halogen, OH, OR
4, SH or SR
4
In another embodiment, the method of treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, described method comprise give patient treatment or prevention effective dose be selected from following TLR7 part or its pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer:
With
On the one hand, the present invention has been contained in the mammal of needs, preferably the method for treatment or prevention of hepatitis C infection in the people of needs.
In embodiment alternatively, the method of treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, and described method comprises TLR7 part and acceptable accessories, carrier or the vehicle that gives patient treatment or prevention effective dose.
In embodiment alternatively, the method for in the patient of needs treatment or prevention of hepatitis C infection has been contained in the present invention, and that described method comprises is oral, the TLR7 part of mucosal administration, part or transdermal administration patient treatment or prevention effective dose.
In preferred embodiments, the method for treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, and described method comprises that parenteral route gives the TLR7 part of patient treatment or prevention effective dose.
In embodiment independently, the method of treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, described method comprises TLR7 part and other therapeutic agent, preferably other antiviral agent or the immunomodulator that gives patient treatment or prevention effective dose.
The suitable parenteral route that the TLR7 part of the present invention of the treatment that comprises sterile form or pharmaceutically acceptable amount has also been contained in the present invention gives patient's pharmaceutical composition; Comprise the suitable orally give patient's of the treatment or the TLR7 part of the present invention of pharmaceutically acceptable amount pharmaceutical composition, wherein, prepare described compositions, go up contacting of subcutaneous immune anatomical structure and TLR7 part, improve the body absorption of TLR7 part to reduce; Comprise the suitable mucosal administration patient pharmaceutical composition of the treatment or the TLR7 part of the present invention of pharmaceutically acceptable amount, wherein, prepare described compositions, go up contacting of subcutaneous immune anatomical structure and TLR7 part, improve the body absorption of TLR7 part to reduce; With the suitable topical administration patient's who comprises the treatment or the TLR7 part of the present invention of pharmaceutically acceptable amount pharmaceutical composition, wherein, prepare described compositions, go up contacting of subcutaneous immune anatomical structure and TLR7 part to reduce, improve the body absorption of TLR7 part.According to the concrete tissue of being treated, can be before, simultaneously or use other component such as penetration enhancer afterwards with active component of the present invention treatment.In preferred embodiments, every kind of described compositions is the single dose unit form, comprises a certain amount of active component, is enough to treatment or prevention human infection hepatitis C virus.
In specific embodiment, the present invention has been contained and has been comprised the pharmaceutical composition that is selected from following TLR7 part: a) guanosine, b) imidazoquinolie, c) adenine and d) analog and the derivant of pyrimidine.
In another embodiment, the present invention has been contained and has been comprised the pharmaceutical composition that is selected from following TLR7 part: general formula I a, Ib, Ic, Id, Ie, If, Ig and Ih and pharmaceutically acceptable salt thereof, hydrate, metabolite or stereoisomer or described stereoisomer pharmaceutically acceptable salt or hydrate.
3.2 TLR7 part prodrug
The new method of treatment or prevention of hepatitis C infection has also been contained in the present invention, and the new pharmaceutical composition that utilizes TLR7 part prodrug or its pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer.
The method of replying the disease of immunotherapy with the immunoreagent treatment has also been contained in the present invention, described method comprises that orally give needs the patient TLR7 part prodrug of immunotherapy, wherein, TLR7 part prodrug reaches the treatment effective plasma level concentration of TLR7 part in the patient.
In one embodiment, the method of treatment infection with hepatitis C virus in the patient has been contained in the present invention, described method comprises patient TLR7 part prodrug or its pharmaceutically acceptable salt, hydrate or the stereoisomer of orally give needs, wherein, orally give TLR7 part prodrug can reach the treatment effective plasma level concentration of TLR7 part, reduces and the relevant adverse side effect of orally give TLR7 part simultaneously.In preferred embodiments, TLR7 part prodrug is the TLR7 part prodrug of labelling.
In another embodiment, the disease of treatment responsing reaction therapy has also been contained in the present invention, reduce the method for immunoreagent related side effects simultaneously, described method comprises that orally give needs the patient TLR7 part prodrug of immunotherapy, wherein, TLR7 part prodrug can reach the treatment effective plasma level concentration of TLR7 part in the patient.In preferred embodiments, TLR7 part prodrug is the TLR7 part prodrug of labelling.
In another embodiment, orally give TLR7 part prodrug can improve the interior bioavailability of body of TLR7 part.In preferred embodiments, orally give TLR7 part prodrug can reach effective plasma level concentration in the body of TLR7 part, is the 10%-500% of exposure level (exposure) in effective body of obtaining of orally give TLR7 part only.In another preferred embodiment, reaching the effective plasma level concentration of TLR7 part in the TLR7 part prodrug body of orally give labelling, is the 50%-200% of exposure level in effective body of obtaining of orally give TLR7 part only.
In another embodiment, orally give TLR7 part prodrug can reduce side effect.In preferred embodiments, side effect comprises GI irritation, and wherein, that GI irritation comprises is hemorrhage, damage and vomiting.
In another embodiment, compare with orally give TLR7 part only, TLR7 part prodrug can improve oral administration biaavailability at least 25% in the patient, and reduces GI irritation at least 50%.In another embodiment, TLR7 part prodrug improves oral administration biaavailability at least 50%, and reduces GI irritation, makes to compare with oral TLR7 part only, and other toxicity becomes limited among the patient.
In preferred embodiments, TLR7 part prodrug reaches treatment effective plasma level concentration, is the 25%-200% of effective bulk concentration of orally give patient TLR7 part, and the GI irritation minimum.
In one embodiment, the inventive method comprises the prodrug that is selected from following TLR7 part of the patient treatment that needs or prevention effective dose: a) guanosine, b) imidazoquinolie, c) adenine and d) analog and the derivant of pyrimidine.
In another embodiment, the inventive method comprises patient treatment that needs or the prodrug that is selected from following TLR7 part that prevents effective dose: a) guanosine, b) imidazoquinolie, c) adenine and d) analog and the derivant of pyrimidine, wherein, prodrug is the amide after (a) TLR7 part amine substituent group transforms, carbamate or amidine part, (b) ester after TLR7 part alcohol substituent group transforms, carbonic ester, carbamate, ether, imide ester (imidate), acetal, aminal or ketal part, (c) acetal or ketal part after TLR7 part ketone substituent group transforms, (d) the imide ester part after the substituent carbonyl of T LR7 part acylamino-transforms, (e) the deoxidation part after TLR7 part oxo (oxo) substituent group of pyrimidine or guanosine transforms, or (f) amine.
In another embodiment, the inventive method comprises that the patient treatment or the prevention effective dose that need are selected from following TLR7 part prodrug or its pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer or described stereoisomer pharmaceutically acceptable salt or hydrate:
With
Wherein:
Each R
1Be H, replacement or unsubstituted alkyl, alkenyl or alkynyl, they can be separated by one or more O, S or N hetero atom, or replacement or unsubstituted aryl or heteroaryl;
R
2Be H, OH, SH, halogen, replacement or unsubstituted alkyl, alkenyl or alkynyl, they can be separated by one or more O, S or N hetero atom, or replacement or unsubstituted-O-(alkyl) ,-O-(aryl) ,-O-(heteroaryl) ,-S-(alkyl) ,-S-(aryl) ,-S-(heteroaryl), aryl or heteroaryl;
R
3Be H, OH, SH, replacement or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl ,-O-(alkyl) ,-O-(aryl) ,-O-(heteroaryl) ,-S-(alkyl) ,-S-(aryl) ,-S-(heteroaryl) ,-NH (alkyl) ,-NH (aryl) ,-NH (heteroaryl) ,-NH (R
4) (alkyl) ,-NH (R
4) (aryl) or-NH (R
4) (heteroaryl);
R
4Be to replace or unsubstituted alkyl;
R
5Be H independently ,-C (O) (C
1-18Alkyl), raceme, L-or D-amino-C (O) CHNH
2R
9
R
6Be H, OR
10Or N (R
11)
2
R
7Be H or replacement or unsubstituted-C (O) (C independently
1-18Alkyl) or-C (O)
2(C
1-18Alkyl);
R
8Be H ,-OH ,-O-(alkyl) ,-OCO
2(C
1-18Alkyl) ,-OC (O) (C
1-18Alkyl), raceme, L-or D-amino-OC (O) CHNH
2R
1
R
9Be H, replacement or unsubstituted alkyl, C (O) CH (C
1-6Alkyl) NH
2Or-C (O) CH (CH
2-aryl) NH
2
R
10Be H, C independently
1-6Alkyl, C
3-7Alkenyl, C
3-7Alkynyl ,-(CR
12R
13)
t(C
6-C
10Aryl) ,-(CR
12R
13)
t(C
3-C
10Cycloalkyl) ,-(CR
12R
13)
t(C
4-C
10Heterocyclic radical) ,-(CR
12R
13)
T＞1OH ,-(CR
12R
13)
T＞0CO
2C
1-18Alkyl ,-(CR
12R
13)
T＞0N (R
14) CO
2C
1-18Alkyl and SO
2(aryl), wherein, except as otherwise noted, t is the integer of 0-6, abovementioned alkyl, alkenyl, alkynyl, aryl, cycloalkyl and heterocyclic radical randomly are independently selected from following group and replace: halogen, cyano group, nitro, trifluoromethyl, trifluoromethoxy, C
1-C
6Alkyl, C
2-C
6Alkenyl, C
2-C
6Alkynyl, hydroxyl, C
1-C
6Alkoxyl ,-NH
2,-NH-alkyl ,-N (alkyl)
2,-NH-aryl ,-N (alkyl) (aryl) ,-N (aryl)
2,-NHCHO ,-NHC (O) alkyl ,-NHC (O) aryl ,-N (alkyl) C (O) H ,-N (alkyl) C (O) alkyl ,-N (aryl) C (O) H ,-N (aryl) C (O) alkyl ,-NHCO
2Alkyl ,-N (alkyl) CO
2Alkyl ,-NHC (O) NH
2,-N (alkyl) C (O) NH
2,-NHC (O) NH-alkyl ,-NHC (O) N (alkyl)
2,-N (alkyl) C (O) NH-alkyl, N (alkyl) C (O) N (alkyl)
2,-NHSO
2-alkyl ,-N (alkyl) SO
2-alkyl ,-C (O) alkyl ,-C (O) aryl ,-OC (O) alkyl ,-OC (O) aryl ,-CO
2-alkyl ,-CO
2-aryl ,-CO
2H ,-C (O) NH
2,-C (O) NH-alkyl ,-C (O) N (alkyl)
2,-C (O) NH-aryl ,-C (O) N (aryl)
2,-C (O) N (alkyl) (aryl) ,-S (O) alkyl ,-S (O) aryl ,-SO
2Alkyl ,-SO
2Aryl ,-SO
2NH
2,-SO
2The NH-alkyl and-SO
2N (alkyl)
2
R
11Be H, C independently
1-6Alkyl, C
3-C
10Cycloalkyl, or form 5-or 6-unit heterocycle with nitrogen;
R
12And R
13Be H, C independently
1-6Alkyl, C
2-6Alkenyl or C
2-6Alkynyl;
R
14Be H, C
1-6Alkyl or-CH
2-aryl;
X is O or S;
Y is H, halogen, OH, OR
4, SH, SR
4Or replacement or unsubstituted alkyl or aryl; With
Z is H, halogen, OH, OR
4, SH or SR
4
In another embodiment, treatment or prevention of hepatitis C method in the patient of needs have been contained in the present invention, described method comprises the TLR7 part that is selected from general formula I Ia, IIb, IIc, IId, IIe, IIf, IIg and IIh that gives patient treatment or prevention effective dose, wherein R
1Be H or replacement or unsubstituted alkyl, alkenyl or alkynyl; R
2Be H, OH, halogen, replacement or unsubstituted alkyl, alkenyl or alkynyl or-CH
2-O-(alkyl); R
3Be H, OH, SH, replacement or unsubstituted-O-(alkyl) ,-S-(alkyl) or-NH (alkyl); R
5Be independently H ,-C (O) (C
1-18Alkyl), raceme, L-or D-amino-C (O) CHNH
2R
9, R wherein
9It is substituted alkyl not; R
6Be H or OR
10, R wherein
10Be C independently
1-6Alkyl, C
3-7Alkenyl, C
3-7Alkynyl ,-(CR
12R
13)
t(C
6-C
10Aryl) ,-(CR
12R
13)
t(C
4-C
10Heterocyclic radical) and-(CR
12R
13)
T＞0N (R
14) CO
2C
1-18Alkyl, wherein, except as otherwise noted, t is the integer of 0-4, abovementioned alkyl, alkenyl, aryl and heterocyclic radical randomly are independently selected from following group by 1-3 and replace: halogen, cyano group, nitro, trifluoromethyl, trifluoromethoxy, C
1-C
6Alkyl, C
2-C
6Alkenyl, C
2-C
6Alkynyl, hydroxyl, C
1-C
6Alkoxyl ,-CO
2-alkyl ,-CO
2-aryl ,-OC (O) alkyl and-OC (O) aryl, R
12And R
13Be H, C independently
1-6Alkyl or C
2-6Alkenyl; And R
14Be H ,-CH
3Or-CH
2CH
3R
7Be H, replacement or unsubstituted-C (O) (C independently
1-18Alkyl) or-C (O)
2(C
1-18Alkyl); R
8Be H ,-OH ,-O-(alkyl) ,-OCO
2(C
1-18Alkyl), raceme, L-or D-amino-OC (O) CHNH
2R
1X is O or S; Y is H, halogen, OH, OR
4, SH or SR
4With Z be H, halogen, OH, OR
4, SH or SR
4
In specific embodiments, the method of treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, described method comprise give patient treatment or prevention effective dose be selected from the following TLR7 part prodrug or the pharmaceutically acceptable salt or the hydrate of its pharmaceutically acceptable salt, hydrate or stereoisomer or described stereoisomer.
In another embodiment preferred of the present invention, TLR7 part prodrug is the amino-acid ester prodrug of TLR7 part.In another preferred embodiment, the amino-acid ester prodrug of TLR7 part is a valyl ester.
In one embodiment of the invention, R
5Be raceme, L-or D-amino-C (O) CHNH
2R
9In another embodiment, when TLR7 part prodrug is when being selected from the chemical compound of general formula I Ih, R
5Be raceme, L-or D-amino-C (O) CHNH
2R
9
At another alternatively in the embodiment, the method of treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, and described method comprises prodrug and pharmaceutically acceptable excipient (excipient), carrier or the vehicle (vehicle) of the TLR7 part that gives patient treatment or prevention effective dose.
At one independently in the embodiment, the method of treatment or prevention of hepatitis C infection has been contained in the patient of needs in the present invention, described method comprises prodrug and other therapeutic agent, preferably other antiviral or the immunomodulator of the TLR7 part that gives patient treatment or prevention effective dose.
The suitable parenteral route that the TLR7 part prodrug of the present invention of the treatment that comprises sterile form or pharmaceutically acceptable amount has also been contained in the present invention gives patient's pharmaceutical composition; The suitable parenteral route that comprises the TLR7 part prodrug of the present invention of treatment or pharmaceutically acceptable amount gives patient's pharmaceutical composition; The pharmaceutical composition that comprises the suitable mucosal administration patient of the treatment or the TLR7 part prodrug of the present invention of pharmaceutically acceptable amount; Pharmaceutical composition with the suitable topical administration patient who comprises the treatment or the TLR7 part prodrug of the present invention of pharmaceutically acceptable amount.According to the concrete tissue of being treated, can be before, simultaneously or use other component such as penetration enhancer afterwards with active component of the present invention treatment.In preferred embodiments, every kind of described compositions is the single dose unit form, comprises a certain amount of active component, is enough to treatment or prevention human infection hepatitis C virus.
In a specific embodiment, a kind of pharmaceutical composition has been contained in the present invention, and described pharmaceutical composition comprises and is selected from following TLR7 part prodrug: general formula I Ia, IIb, IIc, IId, IIe, IIf, IIg and IIh or its pharmaceutically acceptable salt, hydrate or stereoisomer or described stereoisomer pharmaceutically acceptable salt or hydrate.
In another embodiment, concrete tissue according to treatment, can be before TLR7 part prodrug of the present invention treatment with one or more, simultaneously or use other composition afterwards, include but not limited to penetration enhancer, the molecule of the molecule of targeting infected zone and reduction TLR7 part prodrug toxicity in vivo.
TLR7 part prodrug can be used as immune-system enhancers, and has some immune system characteristic, comprising: adjusting, mitogenesis, increase and/or enhancing, or they are the intermediums with chemical compound of these character.After giving administration, this chemical compound is expected at least a cell mass in the natural killer cell in the host immune system, macrophage, dendritic cell or the lymphocyte is demonstrated effect.Because they have these character, they can be used as anti-infective, include but not limited to antiviral agent and antitumor agent, or are used as the intermediate of antiviral and antitumor agent.They can be used as the active component in the suitable pharmaceutical composition, are used for the treatment of infected host.
In one aspect of the invention, adopt TLR7 part prodrug to treat whole viral diseases, this treatment is to be undertaken by this chemical compound that gives this mammal treatment effective dose.Expect that the viral disease of available TLR7 part prodrug treatment comprises by RNA and the caused acute and chronic infection of DNA viruses.When not limiting the scope of the viral infection that can be treated in any form, TLR7 part prodrug is especially effective in the treatment of diseases that is caused by following viral infection: adenovirus, cytomegalovirus, hepatitis A virus (HAV), hepatitis B virus (HBV), the yellow fever virus and the flaviviridae family that comprises hepatitis C virus (HCV) that comprise yellow fever virus, 1 type and 2 type herpes simplex viruss, herpes zoster, the herpes virus hominis 6, human immunodeficiency virus (HIV), human papillomavirus (HPV), influenza A virus, Influenza B virus, measles, parainfluenza virus, Pestivirus, poliovirus, poxvirus (comprising variola and monkey pox virus), rhinovirus, coronavirus, respiratory syncytial virus (RSV), the multiple virus of the heat that causes bleeding: comprise arenavirus family (LCM, Junin virus, Machup virus, Guanarito virus and Lassa heat), Bunyaviruses (Hanta virus and enzootic hepatitis) and Filovirus family (Ebola and marburg virus); Various viral encephalitiss: comprise west nile virus, LaCrosse virus, Gary Fu Niya encephalitis, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, japanese equine encephalitis virus, Kysanur forest virus; With flat louse-borne virus (tickborne Viruses): as the Chrimean-Congo hemorrhagic fever virus.
In another aspect of this invention, adopt TLR7 part prodrug to treat antibacterial, fungus and protozoal infections in mammal, this treatment is by giving these chemical compounds of this mammal treatment effective dose.Can treat pathogenic microorganism in the four corner by giving TLR7 part prodrug expection, include but not limited to that those have chemical sproof organism to antibiosis.The general ability of a plurality of ingredients of TLR7 part prodrug activated immune system bypass toleration mechanism of finding can reduce antibiotic susceptibility, and therefore to treat the infection that is caused by this class resistant micro-organism in the mammal be a specifically application of the present invention by giving TLR7 part prodrug.
In another aspect of this invention, treat tumor in the mammal by the TLR7 part prodrug that gives mammal treatment effective dose.Expect that medicable tumor or cancer include but not limited to tumor or cancer that those are caused by virus, its effect relates to and suppresses to have infected viral cell transformation is the neoplasm state, suppress virus from being diffused into other normal cell by cell transformed and/or stoping the growth of viral cell transformed.The expection of TLR7 part prodrug can be used for resisting the broad-spectrum tumor, includes but not limited to: cancer, sarcoma and leukemia.This apoplexy due to endogenous wind comprises: breast carcinoma, colon cancer, bladder cancer, pulmonary carcinoma, carcinoma of prostate, gastric cancer and cancer of pancreas and lymphoblast and medullary cell leukemia.
Another aspect of the present invention relates to the mammiferous method of a kind of treatment, and described method comprises the medicine that treats and/or prevents containing of effective dose of TLR7 part of the present invention prodrug.In aspect this, its effect relates to regulates mammiferous immune some part, it specifically is the cytokine activity of regulating Th1 and Th2, include but not limited to: interleukin family, arrive IL-12 as IL-1, with other cytokine, as TNF α and interferon (comprising interferon-ALPHA, interferon beta and interferon gamma) and their downstream effect thing.When the adjusting to Th1 and Th2 cytokine takes place, can expect that this adjusting can comprise: stimulate Th1 and Th2, inhibition Th1 and Th2, stimulation Th1 or Th2 and suppress another or double mode adjusting (when high concentration to the Th1/Th2 level produce a kind of effect (as, the inhibition of generalization), and when low concentration to Th1 or Th2 produce another kind of effect (as, stimulate Th1 or Th2, and suppress another)).
Another aspect of the present invention gives to accept not comprise that with the treatment effective dose mammal of the immunoregulation medicament of The compounds of this invention contains the pharmaceutical composition of TLR7 part prodrug.Of the present invention preferred aspect, the dosage of immunoregulation medicament be reduced to be lower than its conventional effective dose, to reduce side effect.Second preferred aspect, use the routine dose of immunoregulation medicament, but when giving, have the therapeutic effect of improvement with TLR7 part prodrug.
Another aspect of the present invention gives to accept not comprise that with the treatment effective dose mammal of the anti-infectives of The compounds of this invention contains the pharmaceutical composition of TLR7 part prodrug.Of the present invention preferred aspect, the pharmaceutical composition that contains TLR7 part prodrug infects media and gives to suppress its growth or to kill the anti-infectives that infects media with directly acting on the treatment effective dose.
4. Brief Description Of Drawings
Fig. 1 has shown isatoribine and the interferon-ALPHA plasma concentration in mice.
Fig. 2 has shown the variation of virus load in accepting the HCV infected patient of isatoribine.
5. detailed Description Of The Invention
5.1 definition
Being used for following term of the present invention has as giving a definition:
Term " comprises " and " comprising " refers to form open, indefiniteness.
Term " nucleoside " is meant the chemical compound that the pentose by any pentose of the equivalent locations of natural place that is incorporated into heterocyclic particular location or purine (9-position) or pyrimidine (1-position) or analog or modification forms.
Term " purine " is meant nitrogenous bicyclic heterocycle.
Term " D-nucleoside " is meant the nucleoside compound (for example, adenosine) with D-ribose sugar moieties.
Term " L-nucleoside " is meant the nucleoside compound with L-ribose sugar moieties.
Term " immunomodulator " refers to can be by the immune natural or sintetics that stimulates or the inhibition change is normal or unusual.
Term " NOAEL " is meant does not observe the concentration (No Observed AdverseEvent Level) that side effect takes place, it is the toxicity study term, is used for causing not obvious toxic drug dose under the concrete dosage level of selection type, frequency, persistent period condition.
" part " expression can be in conjunction with the low-molecular-weight molecule of biological receptor.Part can be agonist or antagonist, or not effect.
" agonist " is meant by combination, but costimulatory receptor produces the part of the biological effect consistent with the normal biologic activity of receptor.
" antagonist " is meant by combination, causes receptor not produce the part of the normal biologic activity of receptor.
Term " mammal " comprises the animal and human.
Term " prevention " refers to that chemical compound of the present invention or compositions prevent that mammal is diagnosed as disease or has the ability of the disease of ill danger.The further developing of mammalian diseases that pre-Radix Stephaniae Tetrandrae suffers from or have this class disease symptoms also contained in this term.
Term " treatment " is meant:
(i) prevent disease, imbalance or the state of an illness are being tended to this disease, imbalance or the state of an illness take place but N goes out the generation in the mammal that has suffered from;
(ii) suppress disease, imbalance or the state of an illness, promptly stop its development; With
(iii) alleviate disease, imbalance or the state of an illness, even disease, imbalance and/or the state of an illness disappear.
Term " α " and " β " refer in the drawn chemical constitution substituent specific three-dimensional chemical configuration in the chiral carbon atom.
Term " patient " or " object " expression animal (as cattle, horse, sheep, pig, chicken, turkey, Carnis Coturnicis japonicae, cat, Canis familiaris L., mice, rat, rabbit, Cavia porcellus etc.) or mammal comprise hybridization and transgenic animal and mammal.In treatment or prevention HCV infection, term " patient " or " object " are preferably represented monkey or people, are more preferred from the people.In a certain concrete embodiment, the patient or to as if hepatitis c virus infection or be exposed in the hepatitis C virus.In one embodiment, the patient is baby (0-2 year), child (2-17 year), teenager (12-17 year), adult (18 years old and more than) or old man (more than 70 years old) patient.In addition, the patient comprises the patient of immunocompromised host, as HIV positive patient, cancer patient, the patient that stands immunotherapy or chemotherapy.In a certain concrete embodiment, the patient is a healthy individual, does not promptly demonstrate the patient of other viral infection symptom.
Term " treatment effective dose " is meant is enough to treat or prevent virus disease, delay or minimize relevant symptom or healing or alleviation disease or the TLR7 part of the present invention of infection or its cause of disease or the consumption of TLR7 part prodrug of disease that causes with viral infection or virus.Particularly, the treatment effective dose represents to be enough to make the consumption that produces therapeutic effect in the body.Relevant with the use of a certain amount of The compounds of this invention, the symptom or the cause of disease of improving total curative effect, reducing or avoid disease preferably contained in this term, perhaps increases the treatment effect of another kind of therapeutic agent or produce synergistic avirulent consumption with another kind of therapeutic agent.
Term " prevention effective dose " refers to be enough to recurrence or the chemical compound of the present invention of propagation or the consumption of other active component of prevention infection, viral infection.The prevention effective dose can refer to be enough to prevent the recurrence of primary infection or infection or propagation or with the amount that infects diseases associated.Relevant with the use of a certain amount of The compounds of this invention, this term is preferably contained the total prevention of improvement or is increased the prophylactic effect of another kind of preventive or therapeutic agent or produce synergistic avirulent consumption with another kind of preventive or therapeutic agent.
Term " combination " refers to simultaneously or uses in succession more than one preventive and/or therapeutic agent, and makes its effect adduction that produces separately or collaborative.
Term " pharmaceutically acceptable salt " is meant the salt that is comprised inorganic bronsted lowry acids and bases bronsted lowry and the preparation of organic bronsted lowry acids and bases bronsted lowry by pharmaceutically acceptable non-toxicity acid or alkali.If TLR7 part prodrug of the present invention is an alkali, required pharmaceutically acceptable salt can for example, be handled free base with following substances: use mineral acid, example hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid etc. by prepared by any suitable process in the prior art; Or use organic acid, as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, acetone acid, oxalic acid, glycolic, salicylic acid, pyranose thuja acid (pyranosidyl acid), as glucuronic acid or galacturonic acid, 'alpha '-hydroxy acids, as citric acid or tartaric acid; Aminoacid is as aspartic acid or glutamic acid; Aromatic acid is as benzoic acid or cinnamic acid; Sulfonic acid is as p-methyl benzenesulfonic acid or ethyl sulfonic acid etc.If TLR7 part prodrug of the present invention is a kind of acid, required pharmaceutically acceptable salt can pass through prepared by any suitable process, for example, handles free acid with inorganic or organic base, described alkali is for for example, amine (primary, the second month in a season or tertiary amine), alkali metal hydroxide or alkaline earth metal hydroxide etc.The example of acceptable acid addition salts comprises: derived from the organic salt of aminoacid (as glycine and arginine), ammonia, primary amine, secondary amine or tertiary amine and cyclammonium (as piperidines, morpholine and piperazine); With inorganic salt derived from sodium, calcium, potassium, magnesium, manganese, ferrum, copper, zinc, aluminum and lithium.
Term " prodrug " is meant that giving the back can be converted into the another kind of chemical compound that keeps the chemical entities of biologic activity by metabolic response or solvolysis.
Term " TLR7 part prodrug " is meant that giving the back can be converted into by metabolic response or solvolysis and another kind of keep biologic activity and be the chemical compound of the chemical entities of TLR7 part.TLR7 part prodrug itself is the part of TLR7, or can " be sheltered " and can not be effectively as the part of TLR7.
Term " the TLR7 part prodrug of sheltering " is meant that giving the back can be converted into by metabolic response or solvolysis and another kind of keep biologic activity and be any chemical compound of the chemical entities of TLR7 part, and wherein administered compound is the TLR7 part than the chemical compound effectiveness difference that is obtained by metabolic conversion or solvolysis.
Term " pharmaceutically acceptable active metabolite " is meant the pharmaceutically acceptable activated product that the metabolic response by specific compound in the body or its salt obtains.After entering in the body, most drug is the substrate that can change the chemical reaction of its physical property and biological property.These metabolic conversion influence the polarity of TLR7 part usually, change in the medicine body to distribute and excretion pathway.Yet in some cases, drug metabolism should produce therapeutic effect.For example, the transferring anti-cancer medicine of many anti-metabolisms must be converted into its activity form after going into cancerous cell.
Term " alkyl ", as used herein, except as otherwise noted, be meant to have 1-20 carbon atom, preferred 1-10 carbon atom, the most preferably saturated straight chain of 1-4 carbon atom or side chain non-cyclic hydrocarbon.Representational straight chain saturated alkyl comprises methyl, ethyl, n-pro-pyl, normal-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and positive decyl; Saturated branched alkyl comprises isopropyl, uncle's butyl, isobutyl group, the tert-butyl group, isopentyl, the 2-methyl butyl, the 3-methyl butyl, the 2-methyl amyl, the 3-methyl amyl, the 4-methyl amyl, 2-methyl hexyl, 3-methyl hexyl, 4-methyl hexyl, 5-methyl hexyl, 2, the 3-dimethylbutyl, 2,3-dimethyl amyl group, 2,4-dimethyl amyl group, 2,3-dimethyl hexyl, 2,4-dimethyl hexyl, 2,5-dimethyl hexyl, 2,2-dimethyl amyl group, 2,2-dimethyl hexyl, 3,3-dimethyl amyl group, 3,3-dimethyl hexyl, 4,4-dimethyl hexyl, the 2-ethyl pentyl group, the 3-ethyl pentyl group, the 2-ethylhexyl, the 3-ethylhexyl, the 4-ethylhexyl, 2-methyl-2-ethyl pentyl group, 2-methyl-3-ethyl pentyl group, 2-methyl-4-ethyl pentyl group, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethyl amyl group, 3, the 3-diethylhexyl, 2, the 2-diethylhexyl, 3,3-diethylhexyl etc.Alkyl can be not replace or replace.
Term used herein " aryl " except as otherwise noted, is meant the carbocyclic ring aromatic ring with 5-14 annular atoms.The annular atoms of isocyclic aryl all is a carbon atom.Aromatic ring structure comprises the chemical compound with one or more ring structures, and as single, double or tricyclic compound, and benzo-fused isocyclic part is as 5,6,7,8-tetralyl etc.Preferably, aryl is monocycle or dicyclo.Representational aryl comprises phenyl, tolyl, anthryl, fluorenyl, indenyl, azulene base, phenanthryl and naphthyl.Isocyclic aryl can be not replace or replace.
Term " replacement " is meant to have one or more substituent special groups.Term " replacement " is meant there is not substituent special groups." substituted alkyl " or " substituted aryl " is meant by one or more and is selected from following substituent group and replaces: halogen (F, Cl, Br or I), low alkyl group (C
1-6) ,-OH ,-NO
2,-CN ,-CO
2H ,-the O-low alkyl group ,-aryl ,-aryl lower alkyl ,-CO
2CH
3,-CONH
2,-OCH
2CONH
2,-NH
2,-SO
2NH
2, the halogen alkyl (for example ,-CF
3,-CH
2CF
3) ,-O-halogen alkyl (for example ,-OCF
3,-OCHF
2) etc.
Except as otherwise noted, term used herein " optical voidness " or " stereoisomerism is pure " are meant and comprise a kind of chemical compound stereoisomer and the compositions of other stereoisomer of this chemical compound not substantially.For example, the pure chemical compound of stereoisomerism with a chiral centre is gone up substantially and is not contained the relative enantiomer of this chemical compound.Typical stereoisomerism pure compound comprises greater than a kind of stereoisomer of this chemical compound of about 80 weight % with less than other stereoisomer of this chemical compound of about 20 weight %.More preferably greater than a kind of stereoisomer of about 90 these chemical compounds of weight % with less than other stereoisomers of about 10 these chemical compounds of weight %, even more preferably greater than a kind of stereoisomer of about 95 these chemical compounds of weight % with less than other stereoisomers of about 5 these chemical compounds of weight %, most preferably greater than a kind of stereoisomer of about 97 these chemical compounds of weight % with less than other stereoisomers of about 3 these chemical compounds of weight %.Because many The compounds of this invention comprise the polysaccharide of D or L shaped formula, D and L type sugar have also been contained in the present invention.For example, the pure D sugar of stereoisomerism is substantially free of the L type.In embodiment alternatively, use L type TLR7 part then to be substantially free of the D type.Like this, methods described herein and compositions are included in the polymer that uses levulose or prepared by their in the optional embodiment.
The compounds of this invention has tautomerism.Though general formula I and II can not represent all possible tautomeric form, should understand general formula I represented shown in any tautomeric form of chemical compound, the diagram of general formula shown in not being subjected to only is limited in one and has chemical compound.For example, should be understood that not consider whether its alcohol or ketone form have substituent group, the identical chemical compound (shown in following general formula I Ia embodiment) of they representatives.
5.2 identify the TLR7 part
Known TLR7 part includes but not limited to, (1) guanosine analogue, as assorted guanosine of 7-denitrification and related compound, include but not limited to Townsend, J.Heterocyclic Chem, 13,1363 (1976) and Seela, etc., Chem.Ber., 114 (10), the described chemical compound of 3395-3402 (1981); The 7-pi-allyl, 8-oxygen-guanosine (loxoribine) and related compound include but not limited to Reitz, etc., J.Med.Chem, 37, the described chemical compound of 3561-3578 (1994); The 7-methyl, assorted guanosine of 9-denitrification and related compound include but not limited to Girgis etc., J.Med.Chem., 33, the described chemical compound of 2750-2755 (1990); 8-bromo guanosine and other 8-halo purine compound include but not limited to United States Patent (USP) 4,643,992 described chemical compounds; 6-amino-9-benzyl-2-butoxy-9H-purine-8-pure and mild other 2,6,8, the purine that 9-replaces, include but not limited to Hirota etc., J.Med.Chem., 45,5419-5422 (2002), Henry etc., J.Med.Chem., 33,2127-2130 (1990), Michael etc., J.Med.Chem., 36,3431-3436 (1993), Furneaux etc., J.Org.Chem., 64 (22), 8411-8412 (1999), Barrio etc.; J.Org.Chem., 61,6084-6085 (1996), United States Patent (USP) 4,539,205, United States Patent (USP) 5,011,828, United States Patent (USP) 5,041,426, United States Patent (USP) 4,880,784 and the open No.WO 94/07904 described chemical compound of international patent application; (2) imidazoquinolie includes but not limited to 1-(4-amino-2-ethoxyl methyl-imidazo [4,5-c] quinoline-1-yl)-2-methyl-propan-2-ol (imiquimoid), as described in the open WO 94/17043 of international patent application; 1-isobutyl group-1H-imidazo [4,5-c] quinolyl-4 amine (resiquimoid), as open WO 94/17043 of international patent application and U.S. Patent application 10/357,777 (U.S. Patent Application Publication No.US 2003/0195209), 10/357,733 (U.S. Patent Application Publication No.US 2003/0186949), 10/358,017 (U.S. Patent Application Publication No.US2003/0176458), 10/357,995 (U.S. Patent Application Publication No.US 2003/0162806), 10/165,222 (U.S. Patent Application Publication No.US 2003/0100764), 10/011,921 (U.S. Patent Application Publication No.US 2003/0065005) and 10/013,059 (U.S. Patent Application Publication No.US2002/0173655) are described; United States Patent (USP) 5,395,937; The open WO 98/17279 of international patent application; (3) pyrimidine derivatives includes but not limited to 2-amino-6-bromo-5-phenyl-3H-pyrimidin-4-one (bropirimine) and similar substituted pyrimidines, includes but not limited to Wierenga etc., J.Med.Chem, 23,239-240 (1980), Fan etc., J.Heterocyclic Chem., 30,1273 (1993), Skilnick etc., J.Med.Chem., 29,1499-1504 (1986), Fried, Deng, J.Med.Chem., 23,237-239 (1980) and Fujiwara etc., Bioorg.Med.Chem.Lett., the described chemical compound of 10 (12) 1317-1320 (2000).All patents, patent disclosure and publication are included into this paper as a reference.
Except that above-mentioned TLR7 part, also can easily identify other TLR7 part by the known screens choosing method.For example, referring to Hirota etc., J.Med.Chem., 45,5419-5422 (2002); With Akira S. etc., Immunology Letters, 85,85-95 (2003).Use the variant (as described in part 6.1) of one of these known screens choosing methods, can identify that also the analog of adenine and derivant are as the TLR7 part.The open No.EP 1 035 123 of adenine derivative known in the art such as european patent application, EP1 043 021 and EP 0 882 727 are described; United States Patent (USP) 6,376,501; United States Patent (USP) 6,329,381; United States Patent (USP) 6,028,076 and U.S. Patent Application Publication No.US 2003/0162806.
Use those skilled in the art's known method,, can synthesize the TLR7 part of general formula I a-Ih especially according to list of references listed above and patent.
5.3 preparation TLR7 part prodrug
Being prepared as follows of TLR7 part prodrug of the present invention: amide, carbamate or amidine part after preparation (a) TLR7 part amine substituent group transforms, (b) ester, carbonic ester, carbamate, ether, imide ester, acetal or ketal part after TLR7 part alcohol substituent group transforms, (c) acetal or ketal part after TLR7 part amine substituent group transforms, (d) the imide ester part after the carbonyl substituted base of TLR7 part acylamino-transforms, (e) deoxidation products after the oxo substituent group of TLR7 part pyrimidine or guanosine transforms, (f) amine.For example, being prepared as follows of TLR7 part prodrug: by (1) hydroxyl (OH) substituent group of TLR7 part is converted into amino-acid ester, or (2) make the amine substituent group of TLR7 part be converted into amide or carbamate.The method for preparing prodrug is well known in the art, as Burger ' s MedicinalChemistry and Drug Chemistry, and 1,172-178,949-982 (1995) is described.Also can be referring to Bertolini etc., J.Med.Chem., 40,2011-2016 (1997); Shan, etc., J.Pharm.Sci., 86 (7), 765-767; Bagshawe, Drug Dev.Res., 34,220-230 (1995); Bodor, Advances in Drug Res., 13,224-331 (1984); Bundgaard, Design of Prodrugs (Elsevier Press 1985); Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen etc., eds., Harwood Academic Publishers, 1991); Dear etc., J.Chromatogr.B, 748,281-293 (2000); Spraul etc., J.Pharmaceutical ﹠amp; Biomedical Analysis, 10,601-605 (1992); With Prox etc., Xenobiol., 3,103-112 (1992).
Scheme 1-18 has shown the general preparation process of the representative compounds of preparation general formula I I.
Scheme 1-6 has described how to synthesize 5 '-amino-acid ester from guanosine analogue and derivant.
Scheme 1
Townsend，JHC，13，
1976，1363
Seela waits Chem Ber.,
114，10，1981，3395-3402
A) 2,2-dimethoxy propane, acetone, DMSO, MeSO
3H, 0 ℃
b)BOC-NHCHR
1CO
2H，EDC，DMAP，PhMe，0℃-rt
c)anh?HCl，iPrOAc，iPrOH
Scheme 2
Reitz, etc., JMC, 37,1994,3561-3578
A) 2,2-dimethoxy propane, acetone DMSO, MeSO
3H, 0 ℃
b)BOC-NHCHR
1CO
2H，EDC，DMAP，PhMe，0℃-rt
c)anh?HCl，iPrOAc，iPrOH
Scheme 3
Girgis, etc., JMC, 33,1990,2750-2755
A) 2,2-dimethoxy propane, acetone DMSO, MeSO
3H, 0 ℃
b)BOC-NHCHR
1CO
2H，EDC，DMAP，PhMe，0℃-rt
c)anh?HCl，iPrOAc，iPrOH
Scheme 4
8-bromo guanosine [303136-79-0]
Commercially available
A) 2,2-dimethoxy propane, acetone DMSO, MeSO
3H, 0 ℃
b)BOC-NHCHR
1CO
2H，EDC，DMAP，PhMe，0℃-rt
c)anh?HCl，iPrOAc，iPrOH
In typical synthesis path, preferably use acetonide, 2 of the protection general formula I a of elder generation, Ib, Id, Ie or Ih β-D-ribose part ', 3 '-hydroxyl, as 2,6, shown in 10 or 14.Free 5 '-hydroxyl under multiple esterification condition with the aminoacid reaction of N-protected, form 3,7,11 or 15.The nitrogen of amino-acid ester and ribose is unitary 2 ', 3 '-hydroxyl reacts under multiple deprotection condition, preferably reaction simultaneously, and the unhindered amina of amino-acid ester forms salt then, as 4,8, shown in 12 or 16.
Scheme 5
Reitz waits JMC, and 37,1994,3561-3578
A) 2,2-dimethoxy propane, acetone, DMSO, MeSO
3H, 0 ℃
b)BOC-NHCHR
1CO
2H，EDC，DMAP，PhMe，0℃-rt
c)anh?HCl，iPrOAc，iPrOH
Scheme 6
Kini etc., JMC, 34,1991,3006-3010
A) 2,2-dimethoxy propane, acetone, DMSO, MeSO
3H, 0 ℃
b)BOC-NHCHR
1CO
2H，EDC，DMAP，PhMe，0℃-rt
c)anh?HCl，iPrOAc，iPrOH
In the route of synthesis shown in scheme 5 and 6, earlier with 2 of acetonide protection chemical compound 17 and 21 β-D-ribose part ', 3 '-hydroxyl, form 18 and 22 respectively.Free then 5 '-hydroxyl and N-tert-butoxycarbonyl valine carry out esterification, form 19 and 23 respectively.2 of the nitrogen of amino-acid ester and ribose ', 3 '-hydroxyl while deprotection, form the hydrochlorate shown in 20 and 24.
Scheme 7 and 8 has been described how from adenine analog and derivant synthesis of carbamates and carbonic ester.
Scheme 7
a)HOBT，DMF，CH
2Cl
2，0℃
b)TFA，CH
2Cl
2，0℃-rt
In typical synthesis path, the amino with carbonic ester or chloro-formate processing general formula I f forms carbamate.For 27, but the amine deprotection of the terminal protection of amino-acid ester N-forms the salt such as 28.
Scheme 8
Kurimota, etc., Bioorg Med Chem,
12，2004，p?1091-1099
a)C
6H
13OC(O)Cl，(iPr)
2NEt，CH
2Cl
2，MeOH，DMAP，0-35℃
In scheme 8, the hydroxyl with the just own ester esterification of carbonochloridic acid adenine derivative 29 obtains carbonic ester 30.
Scheme 9 and 10 has been described how from the similar thing synthesis of carbamates of imidazoquinolie.
Scheme 9
In typical synthesis path, the amino of general formula I c analog reacts with carbonic ester, pyrocarbonate or chloro-formate under multiple condition, forms carbamate.
Scheme 10
a)[C
5H
11OC(O)]
2O，NEt
3，CHCl
3，40℃
In scheme 10, handle imidazoquinolie 31 with coke acid n-pentyl ester, obtain amyl group carbamate 34.
Scheme 11-12 has described carbamate and the imide ester that how to synthesize general formula I g pyrimidine.
Scheme 11
JMC such as Wierenga, 23, Fan etc., JHC, 30,
1980，239-240 1993，1273-1276
a)[EtO(CO)]
2O，NEt
3，DMF，65℃
In the typical case of carbamate was synthetic, 35 amino reacted with the coke acetoacetic ester under these conditions, formed carbamate 36.
Scheme 12
Wierenga etc., JMC, 23,
1980，239-240
A) PPh of polymer support
3, EtOH, DEAD, THF, rt
In the typical case of imide ester is synthetic, as implied above, 35 amino under Mitsunobu type condition with ethanol synthesis, form ethyoxyl derivant 37.
Scheme 13 has described how to synthesize the similar thing of imidazoquinolie from the similar thing of imidazoquinolie.
Scheme 13
In typical synthesis path, the amino of general formula I c derivant reacts with carbonic ester, pyrocarbonate or chloro-formate under multiple reaction condition, forms carbamate.
Scheme 14 has shown preparation 7-pi-allyl-2-amino-9-β-D-nuclear benzofuran sugar yl-7, the general preparation process of 9-dihydro-purine-8-ketone.
Scheme 14
a)Ac
2O，DMAP，NEt
3，CH
3CN
b)POCl
3，75℃
c)Zn-Cu，AcOH，70℃
d)K
2CO
3，CH
3OH，rt
In typical synthesis path, preferably with acyl group protection 7-pi-allyl-2-amino-9-β-D-nuclear benzofuran sugar yl-7,9-dihydro-1H-purine-6; 2 of β in the 8-diketone 17-D-ribose '; 3 ', 5 '-hydroxyl is shown in 40; 7-pi-allyl-2-amino-9-β-D-nuclear benzofuran sugar yl-7; 9-dihydro-1H-purine-6,8-diketone are converted into C-6 position carbonyl the multiple group that easily is reduced under multiple reaction condition; include but not limited to halogen, shown in 41.After reduction under the similar and different reaction condition, ribose is unitary 2 ', 3 ', 5 '-hydroxyl deprotection forms 43.Can further modify chemical compound 43 if desired.
Scheme 15 has shown 7-pi-allyl-2-amino-6-ethyoxyl-9-β-D-nuclear benzofuran sugar yl-7, the general preparation process of 9-dihydro-purine-8-ketone.
Scheme 15
A) PPh of polymer support
3, EtOH, DEAD, THF, rt
b)K
2CO
3，CH
3OH，rt
In typical synthesis path, 40 are converted into multiple imino group-ether with the carbonyl of C-6 position under multiple condition, include but not limited to ethyl, shown in 44.Then, ribose is unitary 2 ', 3 ', 5 '-hydroxyl deprotection forms 45.Also can further modify if desired chemical compound 45.
Scheme 16 has described how to synthesize ether from adenine analog and derivant.
Scheme 16
Kurimota, etc., Bioorg Med Chem,
12，2004，p?1091-1099
a)Br
2，CH
2Cl
2
b)NaOEt，EtOH
In typical synthesis path, can be at C-8 position halogenation adenine derivative.Then, halogen can carry out displacement reaction with suitable alkoxide, forms derivant as 64.
Scheme 17 has shown alkoxyl-9-β-D-nuclear benzofuran sugar yl-7 that 7-pi-allyl-2-amino-6-replaces, the general preparation process of 9-dihydro-purine-8-ketone.
Scheme 17
A) Et
3SiCl, imidazoles, DMF, rt
B)
The PPh that polymer is supported
3, DEAD, THF, rt
c)HF·NEt
3，CH
3OH，rt
In typical synthesis path, the hydroxyl on the ribose 17 is protected to be silyl ether.Carbonyl on 69 the C-6 position is converted into various imino group-ethers under multiple condition, include but not limited to the ether of 4-methylol-5-methyl-[1,3] dioxo-2-ketone, shown in 70.Then, ribose is unitary 2 ', 3 ', 5 '-hydroxyl deprotection forms 71.
Scheme 18 has shown alkoxyl-9-β-D-nuclear benzofuran sugar yl-7 that 7-pi-allyl-2-amino-6-replaces, the general preparation process of 9-dihydro-purine-8-ketone.
Scheme 18
A) HOCH
2N (CH
3) CO
2Et, the PPh that polymer is supported
3, DEAD, THF, rt
b)HF·NEt
3，CH
3OH，rt
69 C-6 position carbonyl is converted into various imino group-ethers under multiple condition, include but not limited to the ether of N-methyl-N-(methylol) carbamate, shown in 72.Then, ribose is unitary 2 ', 3 ', 5 '-hydroxyl deprotection forms 73.Can further modify chemical compound 73 if desired.
5.4 the treatment of infection with hepatitis C virus and prevention method
The invention provides in the patient of needs the method for treatment or prevention of hepatitis C infection.
The present invention also is provided at and treats and/or prevents in the hepatitis c virus infection process, the combination of the treatment TLR7 part of effective dose or its prodrug or these parts and prodrug is introduced patient's blood flow.
TLR7 part of the present invention or TLR7 part prodrug or its pharmaceutically acceptable salt, hydrate or the stereoisomer value in treatment or prophylaxis of acute or chronic infection will change in the character and the order of severity that infect, the approach that gives active component with determining.Dosage, and the administration frequency under some situation also will be replied according to age, body weight and the individuality of the infection that will treat, individual patient and be changed.The proper dosage scheme can be determined according to these factors at an easy rate by those skilled in the art.
Method of the present invention is particularly suitable for human patients.Particularly, the inventive method and dosage can be used for the immunologic injury patient, include but not limited to: cancer patient, HIV infected patient and suffer from the patient of immune degenerative disease.In addition, these methods can be used for being at present the immunologic injury patient of improvement state.Method of the present invention and dosage also can be used for standing the patient of other antiviral therapy.Prevention method of the present invention specifically can be used for having the patient by risk from viral infection.These patients include but not limited to: health care worker, as, doctor, nurse, asylum nursing staff; The army personnel; The teacher; The child-bearing worker; Travelling or live in the patient of external (the particularly third world) comprises the social helping worker, missionary and diplomatic envoy.At last, these method and compositions comprise to unmanageable patient or to the treatment generation drug resistance patient of (as producing drug resistance to reversing transcripting enzyme inhibitor, protease inhibitor etc.).
Dosage
The toxicity of The compounds of this invention and drug effect can be measured by the standard pharmacology program in cell culture or experimental animal, as measuring LD
50(causing the dosage of 50% population death) and ED
50(50% population is treated effective dosage).The dosage of toxicity and treatment effect is than being therapeutic index, and it can be expressed as LD
50/ ED
50
The data that obtains from cell culture assays and zooscopy can be used to be identified for the dosage of the chemical compound of human body.The dosage of this compounds is preferably comprising almost there is not or do not have toxic ED
50The periphery concentration range in.Can in this scope, change dosage according to used dosage form and route of administration.For any chemical compound that is used for the inventive method, the treatment effective dose can estimation at first from cell culture assays.Can in animal model, prepare dosage to obtain comprising the IC that measures in the test cell line
50Concentration (circulatingplasma concertration) scope in the periphery blood plasma of (that is, reaching the concentration of the test compound of maximum half that suppresses of symptom); Perhaps, the dosage of allotment TLR7 part in animal model with concentration range in the periphery blood plasma that obtains the TLR7 part, makes its concentration required with being fixed the reaction magnitude corresponding one by one.This category information can be used for more accurately determining the dosage useful to human body.Concentration can be passed through in the blood plasma, and for example high performance liquid chromatography is measured.
The solution of the present invention and compositions are before being used for human body, and preferred elder generation carries out in vitro tests to required treatment or prophylactic activity, and then carries out in vivo test.For example, analyzed in vitro can be used to determine whether to give specific therapeutic scheme, and analyzed in vitro comprises the cell in vitro culture assays, wherein will reply the cells contacting part of TLR7 part effect, and the intensity of replying by the suitable technique measurement.Assess the effect of TLR7 part then according to the transforming degree of the usefulness of TLR7 part prodrug and TLR7 part prodrug.The chemical compound that is used for the inventive method can be tested with suitable animal model before giving human trial, and described animal model includes but not limited to: rat, mice, chicken, cattle, monkey, rabbit, hamster etc.Chemical compound can be used for suitable clinical trial then.
TLR7 part of the present invention or its pharmaceutically acceptable salt, solvate, hydrate or stereoisomer, the preventive dose in treatment or prophylaxis of acute or chronic infection or disease or the value of therapeutic dose will be different with the route of administration of the character that infects and the order of severity, active component.Dosage, and possible administration frequency also will be replied and different with the infection that will treat, patient's age, body weight and individuality.Those skilled in the art can select the proper dosage scheme at an easy rate according to the consideration to these factors.In some embodiments, dosage is decided according to the specific compound that uses and patient's the body weight and the state of an illness.And dosage also can be according to various concrete TLR7 part prodrugs and difference; Proper dosage can be according to aforesaid testing in vitro, particularly use the test of the TLR7 part relevant with TLR7 part prodrug, and predict according to zooscopy, thereby make demonstrate when measuring in described system or with reference to content as herein described working concentration be lower than other TLR7 part prodrug but effectively those TLR7 part prodrugs use less dosage.In general, the dosage range of every day is about 0.001-100 mg/kg, preferably about 1-25 mg/kg, more preferably from about 5-15 mg/kg.For treatment hepatitis c virus infection person, in one day, divide the dosage that gives about 0.1 milligram-Yue 15 gram/skies for 1-4 time, preferred 100 mg/day to 12 gram/skies, more preferably 100-8000 mg/day.At chemical compound such as 3-β-D-nuclear benzofuran sugar yl thiazole also in the preferred embodiment of [4,5-d] pyrimidine prodrug, reduction of a fraction gives 200-8000 mg/day 1-4 time in one day.In addition, the daily dose of recommendation can following period of time with unitary agent or with other therapeutic agent combined cycle administration.In one embodiment, daily dose is single agent or five equilibrium dosage.In a relevant embodiment, the daily dose of recommendation can give weekly to give once, weekly twice, weekly give three times, give four times weekly or give weekly five times.
In preferred embodiment, give The compounds of this invention, so that this chemical compound whole body in patient's body distributes.In relevant embodiment, give The compounds of this invention, to produce general action in vivo.
In another embodiment, chemical compound of the present invention is by oral, mucosa (comprising Sublingual, buccal, rectum, nose or vagina) administration, parenteral route (comprising subcutaneous, intramuscular, intravenous injection, intra-arterial or intravenous) administration, transdermal administration or topical.In a certain concrete embodiment, chemical compound of the present invention is by mucosa (comprising Sublingual, buccal, rectum, nose or vagina) administration, parenteral route (comprising subcutaneous, intramuscular, intravenous injection, intra-arterial or intravenous) administration, transdermal administration or topical.In another specific embodiment, chemical compound of the present invention passes through oral administration.In another specific embodiment, chemical compound of the present invention is not by oral administration.
As those of ordinary skill in the art understood, for the available different treatment effective dose of different infection.Similar is, is enough to treat or prevents this class to infect but be not enough to cause or the amount that is enough to reduce the side effect relevant with conventional therapy also is encompassed in above-mentioned dosage range and the administration frequency scheme.
Conjoint therapy
Concrete grammar of the present invention also comprises the therapeutic agent (that is the therapeutic agent of non-The compounds of this invention) that gives other.In the specific embodiment of the present invention, The compounds of this invention can be united use with at least a other therapeutic agent.Described therapeutic agent includes but not limited to: antibiotic, Bendectin, antidepressant and antifungal, antiinflammatory, antiviral agent, anticarcinogen, immunomodulator, beta-interferon, alkylating agent, hormone or cytokine.The preferred embodiment of the present invention contains and gives the HCV activity specific or demonstrate active other therapeutic agent of anti-HCV.
TLR7 part prodrug of the present invention can with antibiotic administering drug combinations or preparation.For example, they can with Macrolide (as, tobramycin (Tobi
_)), cephalosporin is (as, cefalexin (Keflex
_), cefradine (Velosef
_), cefuroxime (Ceftin
_), cefprozil (Cefzil
_), cefaclor (Ceclor
_), cefixime (Suprax
_) or cefadroxil (Duricef
_)), clarithromycin is (as clarithromycin (Biaxin
_)), erythromycin is (as, erythromycin (EMycin
_)), penicillin is (as, penicillin V (V-CillinK
_Or PenVeeK
_)) or quinolione (as, ofloxacin (Floxin
_), ciprofloxacin (Cipro
_) or norfloxacin (Noroxin
_)); aminoglycoside antibiotics (as; apramycin; arbekacin; bambermycin; butirosin; dibekacin; neomycin; undecylenate (salt); netilmicin; paromomycin; ribostamycin; sisomicin and spectinomycin); phenalgin pyridine alcohol (amphenicol) antibiotic (as; azidamfenicol; chloromycetin; florfenicol and thiamphenicol); the ansamycin antibiotic (as; rifamide and rifampicin); carbacephem (as; Loracarbef); carbapenem (as; biapenem and imipenum); cephalosporin (as; cefaclor; cefadroxil; cefamandole; cefatrizine; cefazedone; cefozopran; cefpimizole; cefpiramide and cefpirome); cephamycin-type (as; cefbuperazone; cefmetazole and cefminox); the monobactam class (as; aztreonam; carumonam and tigemonam); oxacephems (as; flomoxef and latamoxef); penicillins (as; nitrogen _ amidine penicillin; amdinocillin pivoxil; the amoxicillin; bacampicillin; benzyl penicillinic acid; benzyl penicillin sodium; epicillin; fenbenicillin; the flucloxacillin; penamecillin; the penethacillin hydriodide; benapen (penicillino-benethamine); penicillin; penicillin V; penicillin V benzathine; abbocillin V; ultrabiotic and benzylpenicillin potassium (phencihicillin potassium); lincosamide (as; clindamycin and lincomycin); amfomycin; bacitracin; capreomycin; polymyxin E; enramycin; enviomycin; Tetracyclines (as; apicycline; chlortetracycline; clomocycline and demeclocycline); 2; 4-diamino-pyridine class (as; brodimoprim); itrofurans (as; levofuraltadone and furazolium chloride); quinolones and its analog (as; cinoxacin; clinafloxacin; flumequine and grepafloxacin); sulfonamides (as; sulfacetamide methoxy pyrazine; benzylsulfamide; noprylsulfamide; phthalylsulfacetamide; sulfachrysoidine and sulfacitine); the sulfone class (as, diathymosulfone; glucosulfone sodium and solapsone); cycloserine; mupirocin and tuberin.
TLR7 part prodrug of the present invention can with antiemetic administering drug combinations or preparation.Suitable antiemetic includes but not limited to: metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine hydrochloride, trimethobenzamide, ondansetron, granisetron, hydroxyzine, the acetylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, Oxipendyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol, thiethylperazine, cephalmin, tropisetron and their mixture.
TLR7 part prodrug of the present invention can with antidepressants administering drug combinations or preparation.Suitable antidepressants include but not limited to: binedaline, caroxazone, citalopram, dimethazan, fencamine, indalpine, the hydrochloric acid indeloxazine, nefopam, nomifensine, oxitriptan, oxypertine, paroxetine, Sertraline, thiazesim, triazolone, 1-(benzoyl)-2-(.alpha.-methylbenzyl)hydrazine, iproclozide, iproniazid, isocarboxazid, nialamide, octamoxin, phenelzine, cotinine, rolicypram, rolipram, maprotiline, metralindole, mianserin, mirtazapine (mirtazepine), adinazolam, amitriptyline, amitriptylinoxide, amoxapine, butriptyline, clomipramine, demexiptiline, desipramine, the dibenzepin, dimetacrine, the degree coloured glaze is flat, doxepin, triflupromazine, imipramine, imipramine N-oxide, iprindole, lofepramine, melitracen, metapramine, nortriptyline, noxiptiline, opipramol, pizotifen, propizepine, protriptyline, quinupramine, tianeptine, trimeprimine, adrafinil, benactyzine, amfebutamone, butacetin, dioxadrol, duloxetine, etoperidone, febarbamate, femoxetine, fenpentadiol, fluoxetine, fluvoxamine, hemoporphyrin, hypericin, acetopherane, the medifoxamine, midalcipran, minaprine, moclobemide, nefazodone, oxaflozane, piberaline, prolintane, 2-(dimethylamino)ethyl, ritanserin, Roxindole, Rubinorm (Ifi)., sulpiride, tandospirone, thozalinone, tofenacin control-om, tranylcypromine, the L-tryptophan, venlafaxine, viloxazine and zimeldine.
TLR7 part of the present invention or TLR7 part prodrug can with antifungal administering drug combinations or preparation.Suitable antifungal includes but not limited to: amphotericin B, itraconazole, ketoconazole, fluconazol, intrathecal, flucytosine, miconazole, butoconazole, clotrimazole, nystatin, terconazole (triaconazole), tioconazole, ciclopirox, econazole, haloprogin, naftifine, terbinafine, undecylenate (salt) and griseofulvin.
TLR7 part of the present invention or TLR7 part prodrug can with antiinflammatory administering drug combinations or preparation.Useful antiinflammatory includes but not limited to: nonsteroidal antiinflammatory drug, as salicylic acid, aspirin, methyl salicylate, diflunisal, salsalate, olsalazine, sulfasalazine, acetaminophen, indomethacin, sulindac, etodolac, mefenamic acid, meclofenamate sodium, tolmetin, ketorolac, diclofenac (dichlofenac), ibuprofen, naproxen, naproxen sodium, fenoprofen, ketoprofen, BTS-18322, oxaprozin, piroxicam, meloxicam, ampiroxicam, Droxicam, pivoxicam, tenoxicam, nabumetone, Phenylbutazone, oxyphenbutazone, phenazone, aminophenazone, azapropazone and nimesulide; Leukotriene antagonist includes but not limited to: zileuton, aurothioglucose, sodium aurothiomalate and auranofin; The steroidal class includes but not limited to: the alclometasone dipropionate, amcinonide, beclomethasone dipropionate, betamethasone, betamethasone benzoate, the betamethasone dipropionate, betamethasone sodium phosphate, celestone-V, the clobetasol dipropionate, clocortolone trimethylace tonitric ester, hydrocortisone, hydrocortisone derivative, desonide, desoximetasone, dexamethasone, flunisolide, flucoxinolide, flurandrenolide, chlorine fluorine pine, medrysone, methylprednisolone, the acetic acid methylprednisolone, Urbason Solubile, mometasone furoate, the paramethasone acetas, prednisolone, the prednisolone acetas, Inflamase, prednisolone tebuatate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate esters and triamcinolone hexacetonide; And other anti-inflammatory agent, include but not limited to: methotrexate, colchicine, allopurinol, probenecid, sulfinpyrazone and benzbromarone.
TLR7 part of the present invention or TLR7 part prodrug can with other antiviral agent administering drug combinations or preparation.Useful antiviral agent includes but not limited to: protease inhibitor, nucleoside reverse transcriptase inhibitor and nucleoside analog.Antiviral agent includes but not limited to: zidovudine, acycloguanosine, ganciclovir, vidarabine, idoxuridine, trifluridine, Levovirin, ribavirin (viramidine) and ribavirin and phosphine formic acid, amantadine, rimantadine, Saquinavir, indinavir, amprenavir, Lopinavir, ritonavir, alpha-interferon, beta-interferon, adefovirdipivoxil, resist to come that furan is fixed, Entecavir, pleconaril.
TLR7 part of the present invention or TLR7 part prodrug can with immunomodulator administering drug combinations or preparation.Immunomodulator includes but not limited to: methotrexate, leflunomide, cyclophosphamide, ciclosporin A, mycophenolate, thunder primycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes (as leflunomide), TXi Baoshouti regulator and cytokine receptor regulator, peptide mimics and antibody (as, the people, the peopleization, chimeric, monoclonal, polyclone, Fvs, ScFvs, Fab or F (ab)
2Fragment or epi-position binding fragment), nucleic acid molecules (as, antisense nucleic acid molecule and triple helical body), micromolecule, organic compound and inorganic compound.The example of TXi Baoshouti regulator includes but not limited to: anti--TXi Baoshouti antibody (as, anti-CD 4 antibodies is (as cM-T412 (Boeringer), IDEC-CE9.1
_(IDEC and SKB), mAB4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD 3 antibodies (as, Nuvion (ProductDesignLabs), OKT3 (Johnson﹠amp; Johnson) or Rituxan (IDEC)), anti--CD5 antibody (as, the immune conjugate that anti--CD5 ricin connects), resist-CD7 antibody (as, CHH-380 (Novartis)), anti--CD8 antibody, anti-CD 40 coordination compound monoclonal antibody (as, IDEC-131 (IDEC)), anti-CD 52 antibody (as, CAMPATH1H (Ilex)), resist-CD2 antibody, resist-CD11a antibody (as, Xanelim (Genentech)) and anti--B7 antibody (as, IDEC-114 (IDEC)) and CTLA4-immunoglobulin.The example of cytokine receptor regulator includes but not limited to: soluble cytokine receptor (as, the TNF α receptor of cell foreign lands or its fragment, cell foreign lands IL-1 beta receptor or its fragment and cell foreign lands IL-6 receptor or its fragment), cytokine or its fragment, (as, interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-α, interferon (IFN)-α, IFN-β, IFN-γ and GM-CSF), the antibacterial agent receptor antibody (as, anti--the IFN receptor antibody, anti--the IL-2 receptor antibody (as, Zenapax (ProteinDesignLabs)), anti--the IL-4 receptor antibody, anti--the IL-6 receptor antibody, anti--IL-10 receptor antibody and anti--IL-12 receptor antibody), anti-cytokine antibody (as, anti--IFN antibody, anti-TNF alpha antibody, anti--IL-1 β antibody, anti--IL-6 antibody, anti--IL-8 antibody (as, ABX-IL-8 (Abgenix)) and anti--IL-12 antibody).
TLR7 part of the present invention or TLR7 part prodrug can with the medicament administering drug combinations or the preparation that suppress viral enzyme.Described medicament includes, but are not limited to, and the inhibitor of HCV protease is as the inhibitor of BILN 2061 and NS5b polymerase, as NM107 and its prodrug NM283 (IdenixPharmaceuticals company, Maryland, USA Cambridge).
TLR7 part prodrug of the present invention can with as Wu at Curr Drug Targets InfectDisord (2003; 3 (3): the medicament of the inhibition HCV polymerase 207-19) or with as people such as BretnerM, at Nucleosides Nucleotides Nucleic Acids (2003; 22 (5-8): the medicament of inhibition 1531) virus spiralization function or with as Zhang X at Drugs (2002; 5 (2): the HCV particular target 154-8) is to the inhibitor administering drug combinations or the preparation of thing.
TLR7 part of the present invention or TLR7 part prodrug can with the medicament administering drug combinations or the preparation that suppress virus replication.
TLR7 part of the present invention or TLR7 part prodrug can with cytokine administering drug combinations or preparation.The example of cytokine includes but not limited to: interleukin II (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin-6 (IL-6), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin-18 (IL-18), from hematoblastic somatomedin (PDGF), erythropoietin (Epo), epithelical cell growth factor (EGF), fibroblast growth factor (FGF), granular leukocyte macrophage stimulus factor (GM-CSF), granulocyte cluster stimulating factor (G-CSF), macrophage cluster stimulating factor, (M-CSF), prolactin antagonist and interferon (IFN) (for example, IFN-α and IFN-γ).
TLR7 part of the present invention or TLR7 part prodrug can with hormons administration or preparation.The example of hormone includes but not limited to: luteinization hormone releasing hormone (LHRH), growth hormone (GH), growth hormone releasing hormone, ACTH, Somat, growth hormone, somatomedin, parathyroid hormone, hypothalamic releasing factor, insulin, glucagon, enkephalin, vassopressin, calcitonin, heparin, low molecular weight heparin, heparinoid, synthetic and natural opium, insulin thyrotropin and endorphins.
TLR7 part of the present invention or TLR7 part prodrug can with beta-interferon administering drug combinations or preparation, beta-interferon includes but not limited to: interferon beta-1a, interferon beta-1b.
TLR7 part of the present invention or TLR7 part prodrug can with alpha-interferon administering drug combinations or preparation, alpha-interferon includes but not limited to: Alfacon-1, Intederon Alpha-2a (roferon), Interferon Alpha-2b, intron, Peg-intron, Pegasys, synonym interferon (ingergen) and albuferon.
TLR7 part of the present invention or TLR7 part prodrug can with absorption enhancer administering drug combinations or preparation, be the absorption enhancer of targeting especially with the lymphsystem, it includes but not limited to: sodium glycocholate; Capric acid sodium salt; N-lauroyl-β-D-Fructus Hordei Germinatus pyranoside; EDTA; Blended micelle; With Muranishi at Crit.Rev.Ther.Drug Carrier Syst, the material of being reported among the 7-1-33, it is for reference that the document is included this paper in full in.Also can use other known absorption promoter.Therefore, the pharmaceutical composition that comprises the prodrug shown in one or more formulas I, the chemical compound shown in the formula II and other chemical compound of the present invention and one or more absorption enhancers has also been contained in the present invention.
TLR7 part of the present invention or TLR7 part prodrug can with alkylating agent administering drug combinations or preparation.The example of alkylating agent includes but not limited to: chlormethine, ethylene imine, methylmelamine, sulfonic acid alkane ester, nitroso ureas, triazenes, chlormethine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, altretamine (hexamethylmelaine), tespamin, busulfan, card chlorine mustard, streptozotocin, dacarbazine and temozolomide.
The compounds of this invention and other therapeutic agent can have adduction, or more preferably have synergism.In preferred embodiment, the compositions that contains The compounds of this invention can give simultaneously with another kind of therapeutic agent, and described another kind of therapeutic agent can be and the identical or different compositions of a compositions part that contains The compounds of this invention.In another embodiment, before or after giving another kind of therapeutic agent, give chemical compound of the present invention.At one independently in the embodiment,, give chemical compound of the present invention to once using another kind of therapeutic agent (particularly antiviral agent) treatment in the past or no longer accepting to use the patient of another kind of therapeutic agent treatment at present.
In one embodiment, method of the present invention comprises the therapeutic agent that gives one or more TLR7 parts of the present invention or TLR7 part prodrug and no longer give other.
Pharmaceutical composition and dosage form
Comprise TLR7 part of the present invention or prodrug, or the pharmaceutical composition of its pharmaceutically acceptable salt, hydrate or stereoisomer and single agent dosage form are also within the scope of the invention.That individual dosage form of the present invention is suitable for is oral, mucosa delivery (comprising Sublingual, buccal, rectum, nose or vagina administration), parenteral route (comprising Sublingual, intramuscular, intravenous injection, intra-arterial or intravenous administration) transdermal administration or topical.Pharmaceutical composition of the present invention and dosage form typically also comprise one or more pharmaceutically acceptable excipient.Sterile preparation also within the scope of the invention.
In an optional embodiment, pharmaceutical composition wherein comprises TLR7 part of the present invention or prodrug, or its pharmaceutically acceptable salt, hydrate or stereoisomer, and at least a other therapeutic agent.The example of other therapeutic agent includes but not limited to the listed material of above-mentioned chapters and sections 5.2.2.
Compositions of the present invention, shape and dosage form type typically change with its application.For example, the dosage form that is used for the acute treatment of a kind of disease or relevant disease can contain than one or more bigger active constituents of chronic treatment same disease institute consumption.Similar is that parenteral formulations can contain one or more active constituents than the peroral dosage form less amount that is used for the treatment of same disease or imbalance.
These and other mode that those skilled in the art can understand the particular dosage form that is covered by among the present invention at an easy rate can be different mutually.Referring to, for example, Remington ' s PharmaceuticalScience, the 18th edition, Mack Publishing, pennsylvania, USA Eston (1990).The example of dosage form includes but not limited to: tablet; Capsule sheet (caplet); Capsule, for example capsule of soft elastic gelatin capsule; Cachet (cachet); Lozenge (troche); Lozenge; Dispersant; Suppository; Ointment; Paste (poutice); Paste; Powder; Dressing; Cream; Plaster; Solution; Patch; Aerosol (as nasal spray or inhalant); Gel; Be fit to patient is carried out the liquid dosage form of oral or mucosa delivery, comprise suspending agent (as, aqueous or non-aqueous liquid suspending agent, oil in water emulsion or Water-In-Oil liquid emulsion), solution and elixir; Be fit to liquid dosage form to patient's parenteral administration; With can be used to reconstruct (reconstitute) be fit to patient's parenteral administration liquid dosage form sterile solid (as, crystallization or amorphous solid).
Typical pharmaceutical composition and dosage form comprise one or more carriers, excipient or diluent.Suitable excipient is known in the pharmaceutical field, and this paper provides the indefiniteness example of suitable excipient.A kind of specific excipient whether is fit to mix pharmaceutical composition or dosage form depends on the known various factors of this technical field, and including but not limited to will be to the approach of patient's administration.For example, the peroral dosage form such as tablet can contain the excipient that is not suitable for parenteral formulations.The fitness of particular excipient is also decided according to the given activity composition in the dosage form.
Anhydrous pharmaceutical composition and the dosage form that comprises active constituent also contained in the present invention, because water can quicken the degraded of some chemical compound.For example, adding entry (water as 5%) is to be generally accepted in the pharmaceutical field, and it can be used as the means of simulation long-term storage and measures such as preparation in character such as pot-life of following period of time or stability.Referring to, as Jens T.Carstensen, Drug Stability:Principles ﹠amp; Practice, the 2nd edition, Marcel Dekker, New York, United States New York, 1995, the 379-80 pages or leaves.Water and heat can quicken the decomposition of some chemical compounds effectively.Therefore, owing to generally can run into moisture and/or humidity when making, handle, pack, store, transporting and using preparation, water is very huge to the influence of preparation.
Anhydrous pharmaceutical composition of the present invention and dosage form can be used the component of anhydrous or low humidity and be prepared at low humidity or low moist condition.
Anhydrous pharmaceutical composition is maintained its no aqueous nature in should and storing in preparation.Therefore, anhydrous composition preferably uses waterproof materials to pack, and they can be included in the suitable prescription test kit.The example of suitable package includes but not limited to: sealed foil, plastics, unit dosage forms container (as vial), blister packing and band packing.
The present invention is also contained and is comprised pharmaceutical composition and the dosage form that one or more reduce the chemical compound of active constituent decomposition rate.This compounds (below be called stabilizing agent) includes but not limited to: antioxidant, and as ascorbic acid, pH buffer agent or salt buffer agent.
As the amount and the type of excipient, the amount of active constituent and specific type can be according to various factorss and different in the dosage form, for example, but be not limited to, because of the approach that gives patient different.But the exemplary dosage form of the present invention that comprises The compounds of this invention or its pharmaceutically acceptable salt or hydrate comprises 0.1 milligram to 1500 milligrams/unit, so that the dosage of about 0.01 to 200 mg/kg every day to be provided.
Peroral dosage form
The pharmaceutical composition of the present invention that is fit to oral administration can be discontinuous dosage form, for example, but is not limited to: tablet (as chewable tablet), capsule sheet, capsule and liquid (as the pulpous state agent of flavoring).This class dosage form contains the active constituent of scheduled volume, available pharmaceutical field technical staff known method preparation.Generally referring to Remington ' s Pharmaceutical Sciences, the 18th edition, Mack Publishing, Pennsylvania, America Easton (1990).
The preparation of typical peroral dosage form of the present invention is by active constituent is fully mixed according to the conventional medicine complex technique with at least a excipient.Excipient can possess various excipient forms according to the preparation form of required administration.For example, the excipient that is suitable for liquid oral or aerosol dosage forms includes but not limited to: water, glycol, oil, alcohol, flavoring agent, antiseptic and coloring agent.The excipient example that is suitable for solid oral dosage form (as powder, tablet, capsule and capsule sheet) includes but not limited to: starch, sugar, microcrystalline Cellulose, diluent, granulating agent, lubricant, binding agent and disintegrating agent.
Because tablet and capsule are easy to administration, they have represented the dosage form of the best in the peroral dosage form, have used solid excipient in the case.If need, the aqueous of tablet available standards or non-aqueous technology are carried out coating.This type of dosage form can be prepared with any method in the pharmacy.In general, the preparation of pharmaceutical composition and dosage form is by active constituent and liquid-carrier, finely divided solid carrier or both evenly being mixed nearly, making product form required shape in case of necessity again.
For example, tablet can prepare by compacting or mold pressing.The tablet of compacting can randomly prepare with active constituent mixed with excipients, free-flowing form (as powder or granule) by suppressing in suitable machine.The preparation of mold pressing tablet can be by the chemical compound of mold pressing in suitable machine with the moistening powdered of inert fluid excipient.
The excipient example that can be used for peroral dosage form of the present invention includes but not limited to: binding agent, filler, disintegrating agent and lubricant.The binding agent that is suitable for pharmaceutical composition and dosage form includes but not limited to: corn starch, potato starch or other starch, gelatin, natural and synthetic glue, cellulose and its derivant (as ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methylcellulose, pregelatinized starch, hydroxypropyl emthylcellulose such as arabic gum, sodium alginate, alginic acid, other alginate, powdered tragacanth, guar gum (as, the 2208th, 2906, No. 2910), microcrystalline Cellulose and its mixture.
Being suitable for the pharmaceutical composition of this paper and the filler in the dosage form includes but not limited to: the cellulose of Pulvis Talci, calcium carbonate (as granule or powder), microcrystalline Cellulose, powdered, dextrin, Kaolin, mannitol, silicic acid, sorbitol, starch, change starch and their mixture in advance.Binding agent or filler typically account for about 50-99 weight % of pharmaceutical composition or dosage form in the pharmaceutical composition of the present invention.
The adequate types of microcrystalline Cellulose includes but not limited to: commodity are called AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation U.S. viscose branch, Avicel sales department, pennsylvania, USA Marcus Hook) and their mixture.Specific binding agent is the mixture of microcrystalline Cellulose and carboxymethyl cellulose, and commodity are called AVICEL RC-581.The excipient or the additive of suitable anhydrous or low humidity comprise AVICEL-PH-103
TMWith Starch 1500LM.
The disintegrate when disintegrating agent that is used for the present composition makes tablet be exposed to water environment.The tablet that contains too many disintegrating agent can disintegrate when storing, can not be with required speed disintegrate or can not disintegrate under required condition and contain the tablet of disintegrating agent very little.Therefore, both not many also not very little capacity disintegrating agent and change the disintegrating agent that active constituent discharges fatefully and can be used to form solid oral dosage form of the present invention.The consumption of disintegrating agent can be different with the type of preparation, and this those of ordinary skill for described field is easy to distinguish.Typical pharmaceutical composition comprises the disintegrating agent of the about 15 weight % of about 0.5-, the disintegrating agent of particularly about 1-5 weight %.
The disintegrating agent that can be used for pharmaceutical composition of the present invention and dosage form includes but not limited to: agar-agar, alginic acid, calcium carbonate, microcrystalline Cellulose, cross-linking sodium carboxymethyl cellulose, polyvinylpolypyrrolidone, polacrillin potassium, Explotab, potato starch or tapioca, pregelatinized Starch, other starch, potter's clay, other algin, other cellulose, colloid and their mixture.
The lubricant that can be used for pharmaceutical composition of the present invention and dosage form includes but not limited to: calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerol, sorbitol, mannitol, Polyethylene Glycol, other glycol, stearic acid, sodium lauryl sulfate, Pulvis Talci, hydrogenated vegetable oil (as Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, sunflower oil, Oleum sesami, olive oil, Semen Maydis oil and Oleum Glycines), zinc stearate, ethyl oleate, ethyl laurate, agar and their mixture.Other lubricant comprises, for example, (AEROSIL 200 for silicate silica gel, make by W.R.Grace Co., Maryland, USA Baltimore), the aerosol glue that condenses of synthetic silica is (by Texas, USA, the Degussa Co of Plano sells .), CAB-O-SIL (pyrogenic silica product, by the Massachusetts, USA state, Bostonian CabotCo. sells) and their mixture.If when needing to use, lubricant share with the consumption that accounts for the about 1 weight % that is lower than pharmaceutical composition or dosage form.
Slow release formulation
Can or adopt the known drug delivery systems of this area ordinary person to give active constituent of the present invention by the controlled release mode.Example includes but not limited to: United States Patent (USP) 3,845,770; 3,916,899; 3,536,809; 3,598,123; With 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556 and 5,733, those described in 566, it is for reference that all these patents are all included this paper in.This class dosage form slow release is put by the following material that uses various ratios or one or more active constituents of controlled release to obtain required release profiles, described material for example is, hydroxypropyl emthylcellulose, other polymeric matrix, gel, permeable membrane, osmosis system, multiple coatings, microparticle, liposome, microsphere or its combination.For can selecting with active constituent of the present invention at an easy rate, uses jointly the known suitable controlled release preparation of those of ordinary skills (comprising as herein described).Therefore the present invention has been contained and has been fit to oral single unit dosage forms, for example, but is not limited to: the tablet, capsule, gel capsule and the capsule sheet that are used for controlled release.
The general objects that all controlled release medicines are had is to promote curative effect of medication by its uncontrolled counter pair.It is desirable to, in Drug therapy, use the controlled release preparation of optimal design to be characterised in that in the shortest time, to make to be used for curing or the minimal drug of disease controlling.The advantage of controlled release preparation comprises the prolongation of pharmaceutically active, the reduction of administration frequency, the increase of patient compliance.In addition, controlled release preparation can be used to influence time or other characteristic that begins to act on, and as the blood level of medicine, and therefore influences concurrent side effect (for example, untoward reaction).
Most of controlled release preparations are designed to begin to discharge a certain amount of medicine (active constituent) with the required therapeutical effect of rapid generation, and the medicine that discharges other amount then gradually and constantly is to keep the level of treatment or preventive effect in the time of an elongated segment.In order to keep this stable blood level in vivo, medicine must discharge from dosage form with the speed that replaces metabolism and excretory medicine.But by the sustained release of various condition stimulating activity components, these conditions include but not limited to: pH, temperature, enzyme, water or other physiological condition or chemical compound.
Parenteral formulations
Parenteral formulations can give patient by all means, includes but not limited to: subcutaneous, intravenous (comprise and injecting), intramuscular and intra-arterial administration.Because the natural defence of patient to pollutant walked around in their administration usually, parenteral formulations is preferably aseptic, or can sterilize before giving patient.The example of parenteral formulations includes but not limited to: standby injection, preparation are dissolved in or are suspended in anhydrous and/or freeze-drying prods (reconfigurable powder), injection suspension and the Emulsion of the pharmaceutically acceptable excipient that supplies injection.
The suitable vehicle that is used for parenteral formulations of the present invention is for known in those skilled in the art.Example includes but not limited to: injection USP water; The aqueous excipient, such as but not limited to, sodium chloride injection, Ringer injection, dextrose injection, dextrose and sodium chloride injection and newborn acidifying Ringer injection; The miscible excipient of water, such as but not limited to: ethanol, Polyethylene Glycol and polypropylene glycol; Non-aqueous excipient, such as but not limited to: Semen Maydis oil, Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, Oleum sesami, ethyl oleate, isopropyl myristate and benzyl benzoate.
The chemical compound that can increase the dissolubility of one or more active components that this paper discloses also can mix parenteral formulations of the present invention.
The transdermal dosage form
The transdermal dosage form comprises " reservoir devices " or " matrix type " patch, and it can be used for skin and wears a period of time so that the active component infiltration of aequum.
Can be used to provide the suitable excipient (as carrier and diluent) of transdermal of the present invention and topical formulations and other material is that technical staff in the pharmaceutical field is known, can decide according to the particular organization that gives pharmaceutical composition or dosage form.For this consideration, typical excipient includes but not limited to: water, acetone, ethanol, ethylene glycol, propylene glycol, fourth-1,3-glycol, isopropyl myristate, isopropyl palmitate, mineral oil and their mixture.
According to particular organization to be processed, before with active component processing of the present invention, in the processing or after handling, can use other chemical compound.For example, can use penetration enhancer to help active component to the tissue infiltration.Suitable penetration enhancer includes but not limited to: acetone; Various alcohol are as ethanol, the pure and mild oxolane of oil base; Alkyl sulfoxide is as dimethyl sulfoxine; Dimethyl acetylamide; Dimethyl formamide; Polyethylene Glycol; Pyrrolidinone compounds is as polyvinylpyrrolidone: kollidon (Kollidon) level (pyrrole dimension ketone, polyvidone); Urea and various water-soluble or water-insoluble sugar ester are as Tween 80 (polysorbate80) and sorbester p18 (sorbose monostearate).
Also the pH of the tissue that gives of the pH of scalable pharmaceutical composition or dosage form or pharmaceutical composition or dosage form improves sending of one or more active components.Similar is, the polarity of scalable solvent carrier, its ionic strength or tension force improve and sends.Thereby also can be added in pharmaceutical composition or the dosage form to improve with the hydrophilic that advantageously changes one or more active components or lipotropy such as stearic chemical compound sends.Thus, stearate can be used as the lipid carrier of preparation, as emulsifying agent or surfactant and can be used as and send promoter or penetration enhancer.Different salt, hydrate or the solvate of active component can be used to further regulate the character of resulting composition.
The topical dosage form
Topical formulations of the present invention includes but not limited to: cream, washing liquid, unguentum, gel, solution, Emulsion, suspending agent or be other dosage form known in those skilled in the art.Referring to, as Remington ' s Pharmaceutical Sciences, the 18th edition, Mack Publishing, pennsylvania, USA Easton (1990); With Introduction to Pharmaceutical Dosage Forms, the 4th edition, Lea ﹠amp; Febiger, U.S. Philadelphia (1985).
The suitable excipient that can be used to provide transdermal and topical formulations that the present invention is contained (for example, carrier and diluent) and other material are known for the pharmaceutical field technical staff, and the particular organization that gives according to pharmaceutical composition or dosage form decides.For this consideration, typical excipient includes but not limited to: water, acetone, ethanol, ethylene glycol, propylene glycol, fourth-1,3-glycol, isopropyl myristate, isopropyl palmitate, mineral oil and other mixture.
According to particular organization to be processed, can or handle the back and use other component before with active component processing of the present invention, in the processing.For example, can use penetration enhancer to help active component to the tissue infiltration.Suitable penetration enhancer includes but not limited to: acetone; Various alcohol are as ethanol, oleyl alcohol and oxolane; Alkyl sulfoxide is as dimethyl sulfoxine; Dimethyl acetylamide; Dimethyl formamide; Polyethylene Glycol; Pyrrolidinone compounds is as polyvinylpyrrolidone: kollidon (Kollidon) level (pyrrole dimension ketone, polyvidone); Urea and various water-soluble or water-insoluble sugar ester are as Tween 80 (polysorbate80) and sorbester p18 (sorbose monostearate).
The mucosa delivery dosage form
Mucosa dosage form of the present invention includes but not limited to: known other form of medicament for the eyes solution, spray and aerosol or those skilled in the art of the present technique.Referring to, as Remington ' s PharmaceuticalSciences, the 18th edition, Mack Publishing, pennsylvania, USA Easton (1990); With Introduction to Pharmaceutical Dosage Forms, the 4th edition, Lea ﹠amp; Febiger, U.S. Philadelphia (1985).The dosage form that is fit to be used for treating mucosal tissue in the oral cavity can be mixed with washing liquid of oral cavity or buccal cavity gel.In one embodiment, aerosol comprises carrier.In another embodiment, aerosol does not contain carrier.
TLR7 part of the present invention or TLR7 part prodrug also can be by sucking directly to pulmonary administration.For inhalation, TLR7 part of the present invention or TLR7 part prodrug can carry out conventional sending to pulmonary by many different equipment.For example, metered dose inhaler (" MDI ") uses the canister that contains suitable low boiling propellant directly chemical compound to be delivered to pulmonary, described propellant be as, dichlorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane, carbon dioxide or other suitable gas.MDI equipment can be buied from many suppliers, as 3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, Schering Plough and Vectura.
Perhaps, can use Diskus (DPI) equipment to pulmonary administration TLR7 part (referring to, as Raleigh etc., Proc.Amer.Assoc.Cancer Research Annual Meeting 1999,40,397, includes in for reference at this in full).DPI equipment typically uses such as gas blow-through mechanism and produce dry powder smog in container, is sucked by patient then.DPI equipment also is known in this field, can buy from many suppliers, comprise, as, Fisons, Glaxo-Wellcome, Inhale TherapeuticSystems, ML Laboratories, Qdose and Vectura.The variant of popularizing is multiple dose DPI (" MDDPI ") system, and it can send more than one therapeutic dose.MDDPI equipment can available from, as AstraZeneca, GlaxoWellcome, IVAX, Schering Plough, SkyePharma and Vectura.For example, gelatine capsule and the cartridge case that is used for inhaler or insufflator can be mixed with the mixture of powders that contains chemical compound and be used for the suitable powder substrate (as lactose or starch) of these systems.
The another kind of equipment that is used for The compounds of this invention is delivered to pulmonary is liquid spray equipment, as, the equipment that Aradigm Corporation is produced.Liquid spray equipment uses minimum nozzle to make liquid pharmaceutical formulation aerosolized, can directly suck pulmonary then.
In preferred embodiment, sprayer unit is used to TLR7 part of the present invention or TLR7 part prodrug are delivered to pulmonary.Aerosol apparatus is by using, and for example ultrasonic energy forms the fine particle that is easy to suck, and from liquid pharmaceutical formulation, make aerosol (referring to, as Verschoyle etc., BritishJ.Cancer, 1999,80, Suppl 2,96, it is for reference to include this paper at this).The example of aerosol apparatus comprise Sheffield/Systemic Pulmonary Delivery Ltd. (referring to, Armer etc., U.S. Patent No. 5,954,047; Van der Linden etc., U.S. Patent No. 5,950,619; Van der Linden etc., U.S. Patent No. 5,970,974, it is for reference to include this paper at this); The equipment that Aventis and BatellePulmonary Therapeutics provide.
In particularly preferred embodiments, Electrofluid Mechanics (" EHD ") aerosol apparatus is used to pulmonary delivery TLR7 part of the present invention or TLR7 part prodrug.The EHD aerosol apparatus use electric energy make aerosol liquids drug solution or suspension aerosolized (referring to, as Noakes etc., U.S. Patent No. 4,765,539; Coffee, U.S. Patent No. 4,962,885; Coffee, PCT application, WO94/12285; Coffee, PCT application, WO94/14543; Coffee, PCT application, WO95/26234; Coffee, PCT application, WO95/26235; Coffee, the PCT application, WO95/32807, it is for reference to include this paper at this).When using the EHD aerosol apparatus to pulmonary drug delivery, the electrochemical properties of TLR7 part and TLR7 part prodrug formulation may be to need optimized important parameter, and this class optimization can be carried out routinely by those skilled in the art.The comparable existing pulmonary delivery technology of EHD aerosol apparatus is more effectively to pulmonary drug delivery.Other intrapulmonary delivery send the method for TLR7 part of the present invention and TLR7 part prodrug known for those skilled in the art, and also within the scope of the invention.
Be fit to generally include TLR7 part or TLR7 part prodrug and pharmaceutically acceptable carrier with the liquid pharmaceutical formulation of aerosol apparatus and liquid spray equipment and the use of EHD aerosol apparatus.Preferably, pharmaceutically acceptable carrier is the liquid such as alcohol, water, Polyethylene Glycol or perfluorocarbon.Optional is to add the aerosol character that another kind of material changes the preceding drug solns or the suspension of TLR7 part or TLR7 part.Preferably, this material is the liquid such as alcohol, glycol, polyhydric alcohol or fatty acid.Other method that preparation is suitable for the liquid medicine solution of aerosol apparatus or suspension be known in those skilled in the art (referring to, as Biesalski, U.S. Patent No. 5,112,598; Biesalski, 5,556,611, it is for reference to include this paper at this).TLR7 part or TLR7 part prodrug also can be mixed with rectal compositions or vaginal compositions, as suppository or delay enema, for example contain the conventional suppository bases such as cacao bean ester or other glyceride.
Except aforesaid preparation, TLR7 part or TLR7 part prodrug also can be mixed with storage storehouse (depot) preparation.The preparation of this class long term can be implanted by (for example subcutaneous or intramuscular) and give or give by intramuscular injection.Thus, chemical compound can be with suitable polymers material or hydrophobic material (for example, the Emulsion in acceptable oil) or ion exchange resin preparation, perhaps as sl. sol. derivant, for example as sl. sol. salt.
Perhaps, can use other drug delivery system.Liposome and Emulsion are the known delivery vectors that can be used to send TLR7 part of the present invention and TLR7 part prodrug.Can use some organic solvent, though have bigger toxicity usually such as dimethyl sulfoxine.TLR7 part or TLR7 part prodrug also can be sent in controlled release system.Can use pump agent (Sefton, CRC Crit.RefBiomed Eng, 1987,14,201 in one embodiment; Buchwald etc., Surgery, 1980,88,507; Saudek etc., N.Engl.J.Med., 1989,321,574).(referring to Medical Applications of Controlled Release, Langer and Wise compile, CRC Pres., Boca Raton, Fla. (1974) can to use polymeric material in another embodiment; Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball edit, Wiley, N.Y. (1984); Ranger and Peppas, J.Macromol.Sci.Rev.Macromol.Chem., 1983,23,61; Also can be referring to Levy etc., Science, 1985,228,190; During etc., Ann.Neurol., 1989,25,351; Howard etc., 1989, J.Neurosurg.71,105).In another embodiment, controlled release system can be positioned at The compounds of this invention the targeting thing around, as pulmonary, so only need part whole-body dose (referring to, as, Goodson, in Medical Applications ofControlled Release, the same, the 2nd volume, 115 pages (1984)).Other controlled release system also can use (referring to, as Langer, Science, 1990,249,1527).
What can be used to provide the suitable excipient (as carrier and diluent) of mucosa delivery dosage form of the present invention and other material is that the technical staff is known in the pharmaceutical field, can decide according to specific part or method that compositions or dosage form give.Consider for this point, typical excipient includes but not limited to: water, ethanol, ethylene glycol, propylene glycol, fourth-1,3-glycol, isopropyl myristate, isopropyl palmitate, mineral oil and their mixture, described excipient are nontoxic and pharmaceutically acceptable.The example of this class annexing ingredient is well-known in the art.Referring to, as, Remington ' sPharmaceutical Sciences, the 18th edition, Mack Publishing, pennsylvania, USA Easton (1990).
Also the pH of the tissue of the pH of scalable pharmaceutical composition or dosage form or drug administration compositions or dosage form improves sending of one or more active components.Similar is, can regulate the polarity of solvent carrier, the ionic strength or the tension force of solvent carrier is sent with improvement.Chemical compound such as stearate also can be added to hydrophilic or the lipotropy advantageously to change one or more active components in pharmaceutical composition or the dosage form, sends thereby improve.Thus, stearate can be used as the liquid-carrier of preparation, as emulsifying agent or surfactant, and as sending promoter or penetration enhancer.Different salt, hydrate or the solvate of active component can be used to further regulate the character of resulting composition.
Test kit
The invention provides the drug packages or the test kit that comprise one or more containers, described container comprises the TLR7 part prodrug that is used for the treatment of or prevents hepatitis c virus infection.In other embodiments, the invention provides and comprise and contain the drug packages or the test kit of one or more containers that is used for the treatment of or prevents the TLR7 part prodrug of hepatitis c virus infection, with contain the one or more containers of other therapeutic agent, described other therapeutic agent includes but not limited to the listed medicine of those above-mentioned chapters and sections 5.2.2, the medicament of antiviral agent, interferon, inhibition viral enzyme or suppress the medicament of virus replication particularly, preferably other therapeutic agent is the HCV specificity or demonstrates anti-HCV activity.
The present invention also provides drug packages or the test kit that comprises one or more containers, wherein comprises the composition of one or more pharmaceutical compositions of the present invention.With this class container randomly related be one by the medicine of government organs regulations or biological product production, use or the description of selling desired form, this description shows human body is given manufacturing, the use of described medicine or biological product or sell to have obtained government organs' permission.
Analyze
Measure the activity of TLR7 part of the present invention, TLR part prodrug, compositions and dosage form in the external or body by multiple means known in the art.For example, referring to, used method among following method and the whole embodiment.
The active method of known multiple evaluation TLR7, as described in following publication, all publications are included into this paper as a reference: Hirota etc., J.Med.Chem., 45,5419-5422 (2002); With Akira S. etc., Immunology Letters, 85,85-95 (2003).For example, a kind of system that can be used for analyzing the TLR7 part is, is transfected in the suitable cell line by those skilled in the art's known method clone's TLR7 gene and with it, makes and expresses TLR7 and be coupled to NFkB-luciferase indication plasmid.In this cell system, contact TLR7 part produces the fluorescence signal that can measure in the cell culture.For example, referring to Lee etc., Proc.Nat.Acad.USA, 100,6646-6651 (2003); Hemmi etc., Nat.Immunol., 3,196-200 (2002); With Jurk etc., Nat.Immunol., 3,499 (2002) (wherein, Lee etc., Hemmi etc. and Jurk etc., full content is included into this paper as a reference).
Another example of in vitro method is the TLR7 part prodrug that makes human peripheral blood mononuclear cell (PBMC) contact candidate, continues predetermined space (for example, 2-24 hour), measures immunocompetence then.Immunocompetence can comprise the synthetic of the inducing cell factor, and this can be determined by the cytokine albumen that the elisa assay method is measured in the culture supernatant, for example 1 type interferon (interferon-ALPHA, interferon beta) or 2 type interferon (interferon gamma).Perhaps, can hatch back collection PBMC with candidate's TLR7 part prodrug, extracting PBMC RNA, and by extracting RNA being carried out the level of inducing that the immune system gene is determined in the analysis of RNA enzyme protection.General inductive gene comprises 2 ' 5 '-OAS or interferon gamma, but can measure the various kinds of cell factor.For example, referring to Hirota etc., J.Med.Chem., 45,5419-5422 (2002).
It is means known in the art that the RNA enzyme protection is analyzed (RPA), wherein, by earlier will to analyze the special radio-labeled RNA sequence hybridization of RNA to the RNA analyte with quantitatively, enzymic digestion then, selectivity degraded single stranded RNA.Then, under the condition of the double-stranded RNA that can decompose hybridization, protection, sample is carried out gel electrophoresis.Then, gel is carried out autoradiography, disclose the position and the intensity of electrophoresis band.They can be undertaken quantitatively by means known in the art.If protect segmental difference to be enough to make them to separate, can measure simultaneously multiple analysis RNA by gel electrophoresis.The comparison of analysis RNA that will expression is identical in cell and contrast RNA class level is as interior mark, even but make the RNA total amount change the also level of check and analysis RNA class.The process that this RNA enzyme protection is analyzed is as follows:
According to manufacturer's indication, use the RNA of " RNAeasy " test kit (Qiagen) purification from PMBC.Gang form can be from PharMingen (BD Biosciences); The commercially available useful template of a cover from this supplier comprises the material that can analyze TNF-a, IL12p35, IP10, IL-1a, IL-1b, IL-6, interferon gamma and contrast RNA class L32 and GAPDH.This template cover comprises DNA, and it is applicable to synthetic every kind of listed gene suitable R NA probe.
The PharMingen in vitro transcription test kit buffered probe that provides in the test kit is provided.Reactant comprises the RNA enzyme inhibitor; Transcribe buffer; 50ng template cover; Each 0.1375mM rGTP, rCTP, rATP; 0.003mM rUTP; 10mM DTT, 0.010mCi[α 32P] 20 unit t7 rna polymerases of UTP and 20 microlitres.Reactant mixture was hatched under 37 ℃ 1 hour, add the DNA enzyme that 2 units do not contain the RNA enzyme then, hatch 30 minutes cessation reactions again under 37 ℃.Synthetic rna probe 5.2mM EDTA in above-mentioned the hatching, the saturated phenol of 25 μ l Tris, 25 μ L chloroforms and 4 μ g yeast tRNA extract once, and then with 50 μ L chloroform extraction.The ice ethanol that adds 50 μ L 4M ammonium acetates and 250 μ L 100% makes the RNA precipitation, hatch 30 minutes under-80 ℃ after, described preparation high speed centrifugation 30 minutes.In 100% ethanol, wash centrifuge tube, remove ethanol after, the resuspending probe is used for the analysis of RNA enzyme protection.
The probe substance of above-mentioned preparation and the RNA that extracts from PBMC are used in the analysis of RNA enzyme protection.PBMC RNA is washed in 100% ethanol, undertaken quantitatively by trap under the 260nm.Carry out RPA according to the scheme in the PharMingen RiboQuant test kit.8 μ L PBMC RNA samples are mixed in the thin-walled test tube with 2 μ L probe sets, mix well, the short time, centrifugal back covered with mineral oil.Then, test tube is placed 90 ℃ of PCR modules, be cooled to 56 ℃, hatched 16 hours.Sample is cooled to 37 ℃ then, adds RNA enzyme A and RNA enzyme T1.Mixture was hatched under 30 ℃ 45 minutes, with E.C. 3.4.21.64 and yeast tRNA cessation reaction.With phenol-chloroform extraction RNA, with ammonium acetate-ethanol it is precipitated from phenol-chloroform then.Use the washing with alcohol tubule, and in buffer, carry out resuspending, be used to carry out electrophoresis.According to the biology field known method, use the gel electrophoresis analysis sample.
Can adopt many analytical methods to measure the antiviral activity of The compounds of this invention among the present invention, for example cell culture, animal model and human trial.Test described herein can be used for measuring virus growth in time, to determine the growth characteristics of virus in the presence of The compounds of this invention.
In another embodiment, give the animal subject that susceptible viral infects with virus and The compounds of this invention.Can be with incidence of infection, seriousness, persistent period, virus load, mortality rate etc. during with the study subject that only gives virus (no The compounds of this invention) observed sickness rate, seriousness, persistent period, virus load, mortality rate make comparisons.The The compounds of this invention antiviral activity shows as reductions such as the incidence of infection in the presence of The compounds of this invention, seriousness, persistent period, virus load, mortality rate.In a specific embodiment, give animal target simultaneously with virus and The compounds of this invention.In another specific embodiment, before giving The compounds of this invention, give animal target with virus.In another embodiment, before giving virus, give animal target with The compounds of this invention.
In another embodiment, the mensuration of viral growth rate can followingly be carried out: exist or do not exist under the The compounds of this invention condition, metainfective a plurality of time points from human body or animal target take out biofluid/clinical sample (as, the cleaning piece of nose exhalation thing, throat, expectorant, Bronchio-teeth groove lavation thing, urine, saliva, blood or serum) and detect the level of virus.In specific embodiment, the analysis of viral growth rate can followingly be carried out: the virus in sample is through after the cell culture growth, in growth back on the growth medium or after growing in the experimenter, use the existence of virus in any methods analyst sample well-known in the art, described method be such as but not limited to, immunoassay is (as ELISA; About the discussion of ELISA can referring to, volume such as Ausubel for example, 1994, Current Protocolsin Molecular Biology, I volume, John Wiley ﹠amp; Sons, Inc., New York, 11.2.1), immunofluorescence staining or immune spot-ing analysis, utilize immunologic opsonin identification want the antibody of analyzed virus or detect virus-specific nucleic acid (as, analyze by Southern blot or RT-PCR etc.).
In specific embodiment, the mensuration of virus titer is following carries out: obtain biofluid/clinical sample from the object of the cell that infects or infection, the a series of dilutions that prepare this sample, and can single plaque (plagues) the viral dilution degree down infection to the cell monolayer (as archeocyte, transformation cell lines, tissue of patient sample etc.) of viral susceptible.Calculate the plaque number then, virus titer is expressed as speckle and forms the units per ml sample.
In a specific embodiment, the viral growth rate in the object can be assessed by the titre of the virus in the anti-object of antibody.The antibody serum titre can be measured by any method known in this area, such as but not limited to, in the blood serum sample amount of antibody or antibody fragment can by as, ELISA carries out quantitatively.
In addition, the activity in vivo of TLR7 part or TLR7 part prodrug can followingly be measured: directly give experimental animal with this chemical compound, the antiviral activity of collection of biological liquid (as cleaning piece, expectorant, Bronchio-teeth groove lavation thing, urine, saliva, blood or the serum of nose exhalation thing, throat) and test liquid.
At the sample of wanting analyzed virus levels is that sample can comprise or not comprise complete cell in the embodiment of biofluid/clinical sample (as cleaning piece, expectorant, Bronchio-teeth groove lavation thing, urine, saliva, blood or the serum of nose exhalation thing, throat).Sample from the object that contains intact cell can directly be handled, and the separator that does not contain intact cell can be at first through or need not permissive cell system (as primary cell, transformation cell lines, tissue of patient sample etc.) or growth medium (as, LB meat soup/agar, YT meat soup/agar, blood agar etc.) on cultivate.Can by centrifugal (as, under the room temperature, 300 * g, 5 minutes), then under identical condition with PBS (the no Ca of pH 7.4
++And Mg
++) washing makes cell suspension become clarification.Cell mass (cell pellet) but resuspending in small size PBS for analyzing.The constitutional clinical isolates that contains intact cell can mix with PBS, and at room temperature with 300 * g centrifugal 5 minutes.Remove mucus with sterilization dropper point from interface, and cell mass can be washed once more with PBS under the same conditions.Then the cell mass resuspending is supplied to analyze in the PBS of small size.
In another embodiment, the people that chemical compound of the present invention is infected by the virus.Be infected by the virus and give the people of The compounds of this invention susceptible rate, the order of severity, persistent period, viral load, mortality rate etc. can with the anthroposcopy that is subjected to viral infection but does not give The compounds of this invention or give placebo to susceptible rate, the order of severity, persistent period, viral load, mortality rate etc. compare.The antiviral activity of The compounds of this invention can be by The compounds of this invention existence the reduction of the susceptible rate that infects, the order of severity, persistent period, viral load, mortality rate is shown.Any method well-known in the art all can be used to be determined at the antiviral activity in the object (for example, those aforesaid objects).
In addition, the activity in vivo of TLR7 part or TLR7 part prodrug can followingly be measured: directly give animal or human's body object this chemical compound, collection of biological liquid/clinical sample (as, the cleaning piece of nose exhalation thing, throat, expectorant, Bronchio-teeth groove lavation thing, urine, saliva, blood or serum), and the antiviral activity in test organisms liquid/clinical sample (as, add chemical compound of the present invention in the cell in the presence of virus in cultivating).
Above narrated the relevant and key character of the present invention.It will be apparent to one skilled in the art that and to carry out many changes and embodiment.Therefore, appended claims be should understand and all this change and embodiments contained.
6. embodiment
The following examples only are in order to set forth, rather than limitation of the scope of the invention.
6.1 the TLR7 part detects
Known three class TLR7 parts: guanosine class, imidazoquinolines and miazines (seeing chapters and sections 5.2).As mentioned above, the known screens choosing method has been identified other TRL7 parts.For example, the screening step below adopting identifies that adenine analog and derivant are the TLR part, sees Table 1 and 2.
(San Diego California) obtains stable HEK293-hTLR7 cell line, carries out transfection with selected pNiFty2-Luc, NF-kB inductivity luciferase indication plasmid (Invivogen) and (dual) stable transfection from Invivogen.By folding luciferase induction experiment, with no medicine to compare in the same old way, functional assays gained dual (hTLR7/pNiFty2-Luc) cell line is to the response of loxoribine and isatoribine.Select C23 system to be because itself and these (and other) gratifying response of TLR7 agonist and sensitivity feature.The Biological Principles relevant with the NF-kB activation with the TLR7 coupling is widely accepted (summary, referring to Akira S. etc., Immunol.Lett., 85,85-95 (2003)), therefore, HEK293-TLR-NF-kB inductivity reporting system is as code test, no matter be usually used in analyzing TLR (7) agonist, be temporary transient or stable system model.For example, referring to Hemmi H. etc., Nat.Immunol., 3,196-200 (2002); Jurk M. etc., Nat.Immunol., 3,499 (2002); With Lee J. etc., Proc.Natl.Acad.Sci.USA, 100,6646-51 (2003).
For typical C 23 tests, with 6 * 10
4Individual cells/well with cell inoculation in 96 well culture plates, after 4-24 hour with the compound treatment of various concentration.Contact after 2-48 hour,, carry out the test of Lampyridea luciferase with luciferase analytical reagent (Promega), as described in the manufacturer with passive dissolution buffer agent (Promega) dissolved cell monolayer.Luciferase activity is expressed as and does not have the induce multiple of medicine to compare in the same old way relatively.Think that surpassing inducing of background twice is real TLR7 agonist, show that thus significance,statistical increases.
Table 1: the TLR7 that deposits isatoribine activation people in the HEK293 test
Table 2: adenine derivative activates people's TLR7 in the HEK293 test
In table 1, isatoribine is joined in the C23 cell contact 48 hours, collecting cell is analyzed luciferase activity then.Each time point is analyzed three times.Shown in data representation and the induce multiple of no medicine to compare in the same old way, and bracket in standard deviation.
In table 2, adenine derivative 29 is joined in the C23 cell contact 24 hours, collecting cell is analyzed luciferase activity then.Each time point is analyzed three times.Shown in data representation and the induce multiple of no medicine to compare in the same old way, and bracket in standard deviation.
6.2 test TLR7 part is as the anti-HCV agent
The HCV virus load reduces
The isatoribine trial drug that is included in 1 mg/ml physiological saline solution solution form in the 50mL test tube is provided.Venoclysis gives the human body isatoribine, once a day, continues 7 days, and dosage is 200,400,600 or 800 milligrams/dosage.All dosage constant speed gasing injection in 60 fens clock times gives, and removing 800 milligrams of dosage is to give in 80 minutes time.The flow velocity of every kind of dosage is as follows: 200 milligrams of dosage are 3.33 ml/min; 400 milligrams of dosage are 6.67 ml/min; 500 milligrams of dosage are 8.33 ml/min; 600 milligrams and 800 milligrams of dosage are 10.0 ml/min.
Each dosage group is recruited 4-12 position patient (200 milligrams, 400 milligrams, 600 milligrams and 800 milligrams/dosage) venoclysis every day once, lasting 7 days.Before the administration, every patient gets blood to estimate its HCV virogene type.
For these day (* 7 days) dosage groups, before beginning venoclysis every day isatoribine in 2-7 days, measure blood plasma HCV RNA baseline (meansigma methods that 2 pretreatment of the previous day or pretreatment and first day are measured).Referring to Fig. 2.The branched DNA method is measured virus load (Versant
TMV3.0bDNAassay, Bayer Diagnostics).For blood plasma HCV RNA, estimate to change from the maximum of pretreatment baseline with the log transformed value.
Blood plasma HCV RNA reduces during isatoribine is handled, and bigger variation generally occurs among the patient who accepts higher daily dose (Fig. 2).12 8 of accepting in 800 milligrams of QD * 7 of isatoribine day show that plasma viral loads reduce greater than 0.5log10 unit, and the mean change among 12 patients be-0.76log10 unit, and scope is-2.85 to arrive+0.21log10 unit.The reduction of this virus load has significance,statistical (p=0.008) in 800 milligrams of QD dosage groups.The reduction of plasma viral load recovers usually during stopping to handle.
Viral organism analysis based on the HCV replicon
Shown that the HCV replicon is extremely sensitive to the inhibitory action of interferon-' alpha ' and interferon-.Therefore, the HCV replicon becomes very useful system, is used for measuring the amount of the human PBMC's who comes the excitement of free TLR7 agonist supernatant biologic activity interferon.Carry out quantitative analysis, be integrated into the activity of the luciferase indicator of HCV replicon based on mensuration.By using this system, measure the interferon of handling PBMC from the TLR7 agonist, and estimate it with luciferase indication replicon and suppress active.
The human PBMC who separates from healthy donors is placed many cells culture hole (5 * 10
6Cells/well).Do not containing test-compound, 37 ℃, contain 5%CO
2Wet environment down cultivated PBMC 24 hours so that condition of culture is stable, then with the TLR7 part or do not contain medicine to join in the multiple hole of containing from the PBMC of identical donor in the same old way.Can change the TLR7 ligand concentration to be fit to special test, then with the PBMC culture 37 ℃, contain 5%CO
2Wet environment in hatched 8 hours.In 8 hours time points (or be 24 hours time point), will handle and the cell culture supernatant of control wells be taken a sample the generation of elisa assay interferon-' alpha ' from the TLR7 part for loxoribine and sampling thereof.To be diluted in the RPMI culture fluid with 1: 10,1: 100 and 1: 1000 in the same old way supernatant from compound treatment cell and no medicine, and be applied to contain luciferase and indicate in hepatocellular 96 well culture plates of Huh7 of replicon.37 ℃ of following cultured cells are 48 hours in incubator for tissue culture.
After hatching, wash 96 orifice plates, with passive dissolution buffer agent (Promega) dissolving with 2 * PBS.Jolted culture plate under the room temperature 20 minutes, and by injection standard luciferase analytical reagent (Promega) was joined in every hole, Lmax photometer (Molecular Devices) is gone up reading.Original relative light unit is converted into respect to no medicine in the same old way inhibition percent, to determine observed inhibition level in the replicon test.The estimated value that mensuration inhibition HCV replicon duplicates required interferon Cmax is the supernatant of the exciting cell of PBMC of dilution in 1: 10, and it drops in the trial stretch of a series of dilutions.Tried the TLR7 part for all, when dilution in 1: 10, observe 100% of luciferase indication replicon system is suppressed.
Data representation shown in the table 3-8, incubation time shown in the chemical compound of PBMC cells contacting initial concentration continues and as the specified dilution in first hurdle, the suppression ratio of HCV replicon system (" PBMC contacts chemical compound ").To collect cell from PBMC, do not contact chemical compound and the supernatant that shown in first hurdle, dilutes with comparing (" blank supernatant ").The PBMC cell separation is from single blood donors.
Table 3: the antivirus action of isatoribine in the bioanalysis of external HCV replicon
Numbering 1
Incubation time: 8 hours
Initial concentration: 100 μ M
Blood supply numbering: FL72035
| 1∶1000 | 0 | 94 |
Numbering 2
Incubation time: 8 hours
Initial concentration: 100 μ M
Blood supply numbering: FL75287
Numbering 3
Incubation time: 24 hours
Initial concentration: 100 μ M
Blood supply numbering: FL75864
Table 4: the antivirus action of loxoribine in the bioanalysis of external HCV replicon
Numbering 1
Incubation time: 24 hours
Initial concentration: 100 μ M
Blood supply numbering: FL75864
Table 5: the antivirus action of imiquimod in the bioanalysis of external HCV replicon
Numbering 1
Incubation time: 8 hours
Initial concentration: 3.2 μ M
Blood supply numbering: FL75287
Numbering 2
Incubation time: 8 hours
Initial concentration: 3.2 μ M
Blood supply numbering: FL75287
Table 6: the antivirus action of resiquimod in the bioanalysis of external HCV replicon
Numbering 1
Incubation time: 8 hours
Initial concentration: 10 μ M
Blood supply numbering: FL75287
Numbering 2
Incubation time: 8 hours
Initial concentration: 10 μ M
Blood supply numbering: FL75287
Table 7: the antivirus action of bropirimine in the bioanalysis of external HCV replicon
Numbering 1
Incubation time: 8 hours
Initial concentration: 100 μ M
Blood supply numbering: FL72035
Numbering 2
Incubation time: 8 hours
Initial concentration: 100 μ M
Blood supply numbering: FL72036
Table 8: the antivirus action of adenine derivative in the bioanalysis of external HCV replicon
Numbering 1
Incubation time: 8 hours
Initial concentration: 0.1 μ M
Blood supply numbering: FL76418
Compound form: a tfa salt
Numbering 2
Incubation time: 8 hours
Initial concentration: 0.1 μ M
Blood supply numbering: FL76418
Compound form: tfa salt
6.3TLR7 the preparation of part prodrug
According to the synthetic The compounds of this invention of method described in the such scheme 1-18.Except as otherwise noted, all temperature are degree centigrade that all umbers and percent are based on weight.Reagent is all available from commercialization supplier, for example Aldrich Chemical Company or Lancaster Synthesis Ltd., unless otherwise indicated, the not purified direct use of described reagent.Oxolane (THF) and N, dinethylformamide (DMF) be available from Aldrich, and they are in the airtight bottle of safety and can directly use.Unless otherwise indicated, following solvent and reagent distill under drying nitrogen.Distillation THF and Et in Na-benzophenone ketyl (Na-benzophenone ketyl)
2O; At CaH
2Middle distillation CH
2Cl
2, diisopropylamine, pyridine and Et
3N; At P
2O
5, be CaH then
2Middle distillation MeCN; From Mg, distill MeOH; From CaH
2Middle distillation PhMe, EtOAc and i-PrOAc; Under dry argon gas by simple air-distillation purification TFAA.
Under argon malleation and room temperature, (remove nonspecific pointing out), in anhydrous solvent, carry out following reaction, and reaction flask is fixed with rubber septum so that introduce substrate and reagent by syringe.Oven drying and/or heat drying glass drying oven.Reaction is analyzed with TLC, judges the termination of reaction by the consumption of parent material.The thin layer chromatography (TLC) of analyzing usefulness is the silica gel 60F at the aluminum backing
2540.2 carry out on the millimeter plate (EM Science),, heat with commercially available alcohol system phosphomolybdic acid then with UV light (254nm) developing.Preparation thin layer chromatography (TLC) is at aluminum backing silica gel 60F
2541.0mm carry out on the plate (EM Science), with UV light (254nm) developing.HPLC carries out in Waters Micromass ZQ system, and this system comprises 2525 type binary gradient pumps and Alltech model 800ELSD detector and Waters model 996 light () diode matrix detectors.
Unless otherwise indicated, general be performed such post processing: with reaction dissolvent or extractant reaction volume is doubled, then with the appointment aqueous solution washing of 25 volume % of extraction volume.Use anhydrous Na
2SO
4And/or Mg
2SO
4Desciccate solution, filter then and on Rotary Evaporators solvent evaporated under reduced pressure, and indicate " vacuum removal solvent ".Use 230-400 order silica gel or 50-200 order neutral alumina to carry out column chromatography under positive pressure.Under specified pressure of embodiment or ambient pressure, carry out hydrogenolysis.
1The H-NMR spectrum is on Varian Mercury-VX400 instrument, under the 400MHz operation, writes down, and
13The C-NMR spectrum then writes down under the 75MHz operation.The NMR spectrum is from CDCl
3Obtaining in the solution (ppm of unit), with the reference material (7.27ppm and 77.00ppm) of chloroform as reference, is CD in the time of suitably
3OD (3.4 and 4.8ppm and 49.3ppm), DMSO-d
6, or interior target tetramethyl monosilane (0.00ppm).Other NMR solvent can use as required.If during record peak multiplicity, use following abbreviation: s (unimodal), d (bimodal), t (triplet), q (quartet), m (multiplet), br (peak of widening), dd (paired bimodal), dt (paired triplet).Coupling constant is recorded as hertz (Hz).
On Thermo Nicolet Avatar 370FT-IR, write down infrared (IR) spectrum with pure oil or solid form, when the report gained as a result the time with wave number (cm
-1) record.Mass spectrum is recorded as (+)-ESThermoFinnegan LCQ LC/MS, is undertaken by the analytical chemistry portion of Anadys medicine limited company.Elementary analysis is by Atlantic Microlab, and (Norcross, GA) or San Diego, the NuMega of CA carries out Inc..The mensuration of fusing point (mp) is to carry out in the capillary device of opening, and is not calibrated.
Used many common chemical abbreviations: THF (oxolane) in described route of synthesis and the experimentation, DMF (N, dinethylformamide), EtOAc (ethyl acetate), DMSO (dimethyl sulfoxine), DMAP (4-dimethylamino naphthyridine), DBU (1,8-two azo rings [5.4.0] 11-7-alkene), DCM (4-(dicyano methylene)-2-methyl-6-(4-dimethylamino-styryl)-4H-pyrans), MCPBA (3-chloroperoxybenzoic acid), EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), HATU (O-(7-azepine benzo triazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphoric acid ester), HOBT (I-hydroxybenzotriazole hydrate), TFAA (trifluoroacetic anhydride), pyBOP (benzotriazole-1-base oxygen base) tripyrrole alkyl _ hexafluorophosphoric acid ester), DIEA (diisopropylethylamine) or the like.
Embodiment 1:7-pi-allyl-2-amino-9-β-D-nuclear benzofuran sugar yl-7,9-dihydro-purine-8-ketone (43)
Step 1: preparation 7-pi-allyl-2-amino-9-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-7,9-dihydro-1H-purine-6,8-diketone (40)
In anhydrous acetonitrile (25 milliliters), stir 7-pi-allyl-2-amino-9-β-D-nuclear benzofuran sugar yl-7,9-dihydro-1H-purine-6,8-diketone 17 (1.00 grams, 2.95 mMs, according to Reitz etc., JMC, 37,3561-3578 (1994) preparation), DMAP (0.036 milligram, 0.29 mM) and NEt
3The heterogeneous mixture of (2.05 milliliters, 14.74 mMs).Acetic anhydride (0.862 milliliter, 9.13 mMs) is slowly joined in the suspension, under the room temperature this reaction mixture is stirred 16h.Solvent removed in vacuo is dissolved in dichloromethane (DCM) with residue.Use saturated sodium bicarbonate aqueous solution (NaHCO then
3), salt water washing organic facies, use anhydrous magnesium sulfate (MgSO then
4) drying.The vacuum concentration solvent, dry in high vacuum under the room temperature.Obtain 1.33 gram faint yellow solids 40 (97%):
1H NMR (400MHz, CDCl
3) δ 6.12 (t, J=6.0Hz, 1H), 6.01 (d, J=3.6Hz, 1H), 5.89 (m, 1H), 5.82 (t, J=6.0Hz, 1H), 5.39 (br s, 2H), 5.21 (m, 2H), 4.58 (br s, 2H), 4.51 (m, 1H), 4.32 (m, 2H), 2.16 (s, 3H), 2.15 (s, 3H), 2.10 (s, 3H); MS (+)-ES[M+H]
+466.2m/z.
Step 2: preparation 7-pi-allyl-2-amino-6-chloro-9-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-7,9-dihydro-purine-8-ketone (41)
Chemical compound 40 (0.65 gram, 1.39 mMs) is dissolved in the phosphorus oxychloride (10 milliliters), is heated to 75 ℃ of lasting 16h.This reactant mixture of vacuum concentration, with dissolving crude product in DCM.Then, use NaHCO
3The solution washing mixture, salt washing, dry (MgSO
4) and filter.Vacuum concentrated filtrate.Adopt the hexane solution of the ethyl acetate of 10-50% gradient to carry out purified by flash chromatography.Remove and desolvate, obtain 280 milligrams (41%) required product 41:
1H NMR (400MHz, CDCl
3) δ 6.04 (d, J=4.0Hz, 1H), 6.03 (t, J=5.6Hz, 1H), 5.87 (m, 1H), 5.86 (t, J=5.6Hz, 1H), 5.18 (m, 4H), 4.59 (d, J=8.0Hz, 2H), 4.45 (d, J=7.6Hz, 1H), 4.31 (m, 2H), 2.10 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H); MS (+)-ES[M+H]
+484.2m/z.
Step 3: preparation 7-pi-allyl-2-amino-9-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-7,9-dihydro-purine-8-ketone (42)
Chemical compound 41 (0.27 gram, 0.56 mM) is dissolved in acetic acid, and adding Zn-Cu is right in this solution.With this mixture heated to 70 ℃, continue 18h.The elimination particle, vacuum concentrated filtrate.Adopt the hexane solution of 10%-100% gradient ethyl acetate to carry out purified by flash chromatography.Remove and desolvate, obtain 150 milligrams of beige solids (60%) 42:
1H NMR (400MHz, CDCl
3) δ 6.05 (t, J=4.0Hz, 1H), 6.03 (d, J=4.0Hz, 1H), 5.87 (t, J=6.0Hz, 1H), 5.83 (m, 1H), 5.48 (br s, 2H), 5.33 (s, 1H), 5.29 (d, J=5.6Hz, 1H), 4.49 (d, J=3.2Hz, 1H), 4.46 (d, J=3.2Hz, 1H), 4.41 (d, J=5.6Hz, 2H), 4.27 (m, 2H), 2.12 (s, 3H), 2.10 (s, 3H), 2.05 (s, 3H); MS (+)-ES[M+H]
+450.0m/z.
Step 4: preparation 7-pi-allyl-2-amino-9-β-D-nuclear benzofuran sugar yl-7,9-dihydro-purine-8-ketone (43)
In 42 (0.13 gram, 0.29 mM) methanol (4 milliliters) solution, add solid K
2CO
3(0.024 gram, 0.17 mM) stirs this reactant liquor 18h under the room temperature.In this muddiness liquid, add AmberliteCG-50 (0.5 gram), be stirred to neutrality, filter.Concentrated filtrate obtains beige solid, washing, high vacuum is down dry, obtain certain productive rate 93.5 milligrams of beige solids pure 43:
1H NMR (400MHz, d
6-DMSO) δ 7.88 (s, 1H), 6.33 (br s, 2H), 5.85 (m, 1H), 5.66 (d, J=6.0Hz, 1H), 5.30 (d, J=5.6Hz, 1H), 5.20 (s, 1H), 5.16 (d, J=8.4Hz, 1H), 5.01 (d, J=4.8Hz, 1H), 4.89 (q, J=5.6Hz, 1H), 4.75 (br s, 1H), 4.35 (d, J=5.2Hz, 2H), 4.10 (t, J=8.4Hz, 1), 3.80 (q, J=3.6Hz, 1H), 3.57 (m, 1H), 3.44 (m, 1H) .MS (+)-ES[M+H]
+324.1m/z.
Embodiment 2:7-pi-allyl-2-amino-6-ethyoxyl-9-β-D-nuclear benzofuran sugar yl-7,9-dihydro-purine-8-ketone (45)
Step 1: preparation 7-pi-allyl-2-amino-6-ethyoxyl-9-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-7,9-dihydro-purine-8-ketone (44)
In anhydrous THF (15 milliliters) solution of 40 (0.30 gram, 0.64 mMs), add triphenyl phasphine (0.89 gram, 1.93 mMs) and EtOH (0.11 milliliter, 1.93 mMs) that polymer is supported under the room temperature.In the mixed liquor of this stirring, add diethylazodicarboxylate (diethyl azodicarboxylate) (0.12 milliliter, 0.77 mM), continue to stir 18h.The polymer holder that elimination consumes, solvent removed in vacuo.Then, adopt the hexane solution of 10-50% gradient ethyl acetate to carry out the purified by flash chromatography residue.Remove and desolvate, obtain the required product 6 of 85 milligrams of (26%) clarified oil forms:
1H NMR (400MHz, CDCl
3) δ 6.07 (d, J=4.0Hz, 1H), 6.06 (d, J=4.0Hz, 1H), 6.01 (d, J=3.6Hz, 1H), 5.96 (t, J=6.0Hz, 1H), 5.87 (m, 1H), 5.14 (d, J=2.2Hz, 1H), 5.15 (m, 1H), 4.80 (br s, 2H), 4.46 (m, 4H), 4.37 (q, J=7.2Hz, 2H), 4.29 (m, 2H), 2.09 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 1.35 (t, J=7.6Hz, 3H); MS (+)-ES[M+H]
+494.1m/z.
Step 2: preparation 7-pi-allyl-2-amino-6-ethyoxyl-9-β-D-nuclear benzofuran sugar yl-7,9-dihydro-purine-8-ketone (45)
In methanol (4 milliliters) solution of 44 (0.084 gram, 0.17 mMs), add solid K
2CO
3(0.014 gram, 0.17 mM) stirs this reactant liquor 1h under the room temperature.In this muddiness liquid, add Amberlite CG-50 (0.5 gram), be stirred to neutrality, filter.Concentrated filtrate adopts the DCM solution of 100%DCM-10% methanol to carry out purified by flash chromatography.Remove and desolvate, obtain 7 (99%) of 62 milligrams of clarified oil forms:
1H NMR (400MHz, CDCl
3) δ 5.97 (d, J=8.0Hz, 1H), 5.93 (m, 1H), 5.25 (d, J=32.4, Hz, 1H), 5.21 (s, 1H), 5.02 (t, J=8.0Hz, 1H), 4.62 (br s, 2H), 4.47 (d, J=5.6Hz, 2H), and 4.25-4.45 (m, 3H), 4.21 (q, J=6.8Hz, 2H), 3.77 (ABq, Δ υ
AB=0.14, J
AB=12.4Hz, 2H), 1.37 (t, J=6.8Hz, 3H), 1.27 (t, 7.6,2H); MS (+)-ES[M+H]
+368.0m/z.
Embodiment 3:5-bromo-4-ethyoxyl-6-phenyl-pyrimidine-2-base amine (37)
Step 1: preparation 5-bromo-4-ethyoxyl-6-phenyl-pyrimidine-2-base amine (37)
Being similar to the mode of step 2 among the embodiment 2, from the title compound of 2-amino-5-bromo-6-phenyl-3H-pyrimidin-4-one 35 preparation white solid forms (Wierenga, etc., JMC, 23,239-240 (1980)), productive rate 13%:
1H NMR (400MHz, CDCl
3) δ 7.61 (m, 2H0,7.42 (m, 3H), 5.15 (br s, 2H), 4.23 (q, J=7.2Hz, 2H), 1.44 (t, 6.8Hz, 3H); MS (+)-ES[M]
+294.1[M+2]
+296.0m/z. elementary analysis C
12H
12BrN
3O: value of calculation: C, 49.00; H, 4.11; N, 14.29; Measured value: C, 48.94; H, 4.18; N, 14.01.
Embodiment 4:4-(2-amino-5-bromo-6-phenyl-pyrimidine-4-yloxymethyl)-5-methyl-[1,3] Dloxole-2-ketone (dioxol-2-one) (46)
Step 1: preparation 4-(2-amino-5-bromo-6-phenyl-pyrimidine-4-yloxymethyl)-5-methyl-[1,3] Dloxole-2-ketone (46)
To be similar to the mode of step 2 among the embodiment 2, from the title compound of 2-amino-5-bromo-6-phenyl-3H-pyrimidin-4-one 35 preparation white solid forms, productive rate 4%:
1H NMR (400MHz, CDCl
3) δ 7.62 (m, 2H), 7.45 (m, 3H), 5.18 (s, 2H), 5.07 (s, 2H), 2.26 (s, H); MS (+)-ES[M]
+378.2[M+2]
+380.1m/z. elementary analysis C
15H
12BrN
3O
4: value of calculation: C, 47.64; H, 3.20; N, 11.11; Measured value: C, 46.98; H, 3.23; N, 10.70.
Embodiment 5:5-bromo-4-phenyl-pyrimidine-2-base amine (48)
Step 1: preparation 4-phenyl-pyrimidine-2-base amine (47)
Under-78 ℃, in the anhydrous THF (100 milliliters) of bromobenzene (4.43 milliliters, 42.06 mMs) solution, add BuLi (394 milliliters, 63.08 mMs), this mixed liquor is stirred 2h down at-78 ℃.In 15 minutes time, in this mixed liquor, add hot toluene (80 milliliters) solution of 2-aminopyrimidine (2.0 grams, 21.03 mMs).With this mixed-liquor return 16h, be cooled to room temperature, use NaHCO
3The careful cancellation of aqueous solution.Filter mixed liquor, vacuum concentrated filtrate.Then residue is dissolved among the DCM, uses NaHCO
3Aqueous solution, salt water washing, dry (MgSO
4).Remove and desolvate, obtain 350 milligrams of faint yellow solids 47 (10%):
1H NMR (400MHz, CDCl
3) δ 8.32 (and d, J=4.8Hz, 1H), 7.97 (m, 2H), 7.45 (m, 3H), 7.02 (J=4.8Hz, 1H) .5.27 (br s, 2H); MS (+)-ES[M+H]
+172.2m/z.
Step 2: preparation 5-bromo-4-phenyl-pyrimidine-2-base amine (48)
Chemical compound 47 (0.30 gram, 1.75 mMs) is dissolved in the glacial acetic acid (15 milliliters), is heated to 45 ℃, slowly add Br
2(0.09 milliliter, 1.75 mMs).Then, the gained mixed liquor is at room temperature stirred 3h.Solvent removed in vacuo obtains solid residue.Then, residue is transferred on the filter funnel,, washed with water again with the DCM washing.Then, dry residual solids 16h under the high vacuum obtains 197 milligrams of faint yellow solids 13 (45%):
1H NMR (400MHz, d
6-DMSO) δ 8.40 (s, 1H), 7.61 (m, 2H), 7.45 (m, 3H), 6.96 (s, 2H); MS (+)-ES[M]
+250.0[M+2]
+252.0m/z. elementary analysis C
10H
8BrN
3: value of calculation: C, 48.02; H, 3.22; Br, 31.95; N, 16.80; Measured value: C, 47.91; H, 3.28; Br, 32.15; N, 16.80.
Embodiment 6:(5-bromo-6-oxygen-4-phenyl-1,6-dihydro-pyrimidine-2-base)-urethanes (36)
Step 1: preparation (5-bromo-6-oxygen-4-phenyl-1,6-dihydro-pyrimidine-2-base)-urethanes (36)
In DMF (8 milliliters) solution of 35 (0.25 gram, 0.94 mMs), add NEt
3(0.14 milliliter, 0.99 mM) and pyrocarbonic acid diethyl ester (0.27 milliliter, 1.89 mMs).This reaction mixture is remained on 65 ℃, continue 20h.Except that desolvating and handling residue with DCM.Filter the gained mixed liquor to remove residual initial substance 35, filtrate is used NaHCO
3Aqueous solution is washed, salt washing, dry (MgSO
4).Concentrated filtrate, HPLC purification (Thomson ODS-A 100A 5 μ 150 * 21.2mm post; Flow velocity=30 ml/min; The CH that contains 0.05%TFA
3CN (A) contains the water (B) of 0.05%TFA; Make-up pump flow velocity=0.9 ml/min; Make-up pump mobile phase; The MeOH that contains 0.05%TFA adopts following gradient system: t=0; 15%A, 85%B; T=3.0 minute; 15%A, 85%B; T=9.5 minute; 70%A, 30%B; T=10.0 minute; 100%A, 0%B; T=12.0 minute; 100%A, 0%B; T=12.5 minute; 15%A, 85%B; T=15.0 minute; 15%A 85%B.), obtains 54 milligrams of clarified oils 36 (17%):
1H NMR (400MHz, CDCl
3) δ 7.66 (m, 1H), 7.44 (m, 3H), 4.26 (q, J=7.6Hz, 2H), and 1.32t, J=6.8Hz, 3H); MS (+)-ES[M]
+338.1[M+2]
+340.0m/z. elementary analysis C
13H
12BrN
3O
3: value of calculation: C, 46.17; H, 3.58; N, 12.43; Measured value: C, 46.43; H, 3.74; N, 11.95.
Embodiment 7:(5-bromo-6-oxygen-4-phenyl-1,6-dihydro-pyrimidine-2-base)-amyl carbamate (49)
Step 1: preparation (5-bromo-6-oxygen-4-phenyl-1,6-dihydro-pyrimidine-2-base)-amyl carbamate (49)
With the mode that is similar to step 1 among the embodiment 6 from 35 and coke acid diamyl ester prepare the title compound of clarified oil, HPLC purification (Thomson ODS-A 100A 5 μ 150 * 21.2mm post; Flow velocity=30 ml/min; The CH that contains 0.05%TFA
3CN (A) contains the water (B) of 0.05%TFA; Make-up pump flow velocity=0.9 ml/min; Make-up pump mobile phase; Contain the MeOH of 0.05%TFA, gradient system is as follows: t=0; 35%A, 65%B; T=3.0 minute; 35%A, 65%B; T=10 minute; 100%A, 0%B; T=12.0 minute; 100%A, 0%B; T=12.5 minute; 35%A, 65%B; T=15.0 minute; 35%A, 65%B.) the back productive rate is 9%:
1H NMR (400MHz, CDCl
3) δ 7.69 (br s, 1H), 7.67 (m, 2H), 7.43 (d, J=2.0Hz, 3H), 4.17 (t, J=7.2Hz, 2H), 1.64 (t, J=6.8Hz, 2H), 1.34 (m, 4H), 0.92 (t, J=6.4Hz, 3H); MS (+)-ES[M]
+380.1[M+2]
+382.1m/z.
Embodiment 8:(1-isobutyl group-1H-imidazo [4,5-c] quinolyl-4)-amyl carbamate (34)
Step 1: preparation (1-isobutyl group-1H-imidazo [4,5-c] quinolyl-4)-amyl carbamate (34)
CHCl at 1-isobutyl group-1H-imidazo [4,5-c] quinolyl-4 amine 31 (0.15 gram, 0.62 mM is according to the described method preparation of WO94/17043)
3In (5 milliliters) suspension, add NEt
3(0.09 milliliter, 0.65 mM) and coke acid diamyl ester (0.231 gram, 0.94 mM).Under 40 ℃ this mixed liquor is stirred 60h.Reaction mixture NaHCO
3Solution washing, the salt washing is also used MgSO
4Dry.Concentrated filtrate, the purified by flash chromatography of the hexane solution of employing 10%-70% gradient ethyl acetate obtains 50.5m gram white solid 34 (23%):
1H NMR (400MHz, CDCl
3) δ 8.31 (brs, 1H), 8.15 (t, J=8.0Hz, 2H), 7.85 (t, J=7.2Hz, 1H), 7.77 (t, J=8.0Hz, 1H), 4.43 (d, J=7.6Hz, 2H), 4.36 (t, J=7.2Hz, 2H), 2.31 (m, 1H), 1.75 (t, J=6.8Hz, 2H), 1.36 (m, 4H), 1.06 (d, J=6.4Hz, 6H), 0.89 (t, J=6.8Hz, 2H); MS (+)-ES[M+H]
+355.3m/z.
Embodiment 9:(1-isobutyl group-1H-imidazo [4,5-c] quinolyl-4)-urethanes (50)
Step 1: preparation (1-isobutyl group-1H-imidazo [4,5-c] quinolyl-4)-urethanes (50) to be being similar to the mode of step 1 among the embodiment 8, from 31 and pyrocarbonic acid diethyl ester prepare the title compound of white solid, productive rate 67%:
1H NMR (400MHz, CDCl
3) δ 9.32 (br s, 2H), 8.2 (d, J=8.0Hz, 2H) 8.12 (d, J=8.0Hz, 1H), 7.83 (t, J=7.2Hz, 1H), 7.74 (t, J=8.0Hz, 1H) 4.43 (m, 4H), 2.35 (m, 1H), 1.39 (t, J=7.2Hz, 3H), 1.08 (d, J=6.4Hz, 6H); MS (+)-ES[M+H]
+313.2m/z.
Embodiment 10: carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester ethyl ester (51)
Step 1: preparation carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester ethyl ester (51)
With 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-alcohol, 29 (11.75 milligrams, 0.027 mM, according to Kurimota, etc., Bioorg.Med.Chem., 12, the described method preparation of 1091-109 (2004)) are suspended in CH
2Cl
2In (0.6 milliliter), and be cooled to 0 ℃.Then with DIEA (11.96 microlitres, 0.068mmol) and ethyl chloroformate (3.86 milligrams, 0.036 mM adds with the dichloromethane solution of 10 volume %) join in the suspension.Under 0 ℃ reaction mixture was stirred 10 minutes, be heated to room temperature then and kept 15 minutes.There is initial substance in the TLC demonstration of reaction mixture.Reaction mixture is heated to 35 ℃, adds DMAP (catalytic amount), methanol (60 μ L part) adds ethyl chloroformate (3.86 milligrams, 0.036 mM is with the dichloromethane solution adding of 10 volume %) up to reacting completely again to dissolve 29.Adopt the hexane solution of 0-100% gradient ethyl acetate to carry out the thick mixture of purified by flash chromatography.Collect required peak, vacuum concentration obtains 8.5 milligrams of (80%) white solid chemical compounds 51:
1H NMR (400MHz, CDCl
3) δ 7.45 (d, J=Hz, 2H), 7.27 (m, 3H), 4.98 (s, 2H), 4.46 (m, 4H), 3.74 (m, 2H), 3.42 (s, 2H), 1.46 (t, 3H); MS[M+H]+m/z 388.3.
Embodiment 11-20 is according to embodiment 10 described methods, and from 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-alcohol, 29 with the preparation of suitable chloro-formate.
Embodiment 11: carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester propyl ester (52)
The white solid of productive rate 86%:
1H NMR (400MHz, CDCl
3) δ 7.45 (d, J=6.4Hz, 2H), 7.27 (m, 3H), 4.98 (s, 2H), 4.46 (m, 4H), 3.74 (t, J=5.2Hz, 2H), 3.42 (s, 3H), 1.46 (t, J=7.6Hz, 3H); MS[M+H]
+M/z 402.2.
Embodiment 12: carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester isobutyl ester (53)
The white solid of productive rate 92%:
1H NMR (400MHz, CDCl
3) δ 7.45 (d, J=6.4Hz2H), 7.27 (m, 3H), 4.99 (s, 2H), 4.45 (m, 2H), 4.17 (d, J=7.4Hz, 2H), 3.73 (t, J=4.8Hz, 2H), 3.4 (s, 3H), 2.15 (m, 1H), 1.06 (d, J=6.8Hz, 6H); MS[M+H]+m/z 416.3.
Embodiment 13: carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester pentyl ester (54)
The white solid of productive rate 92%:
1H NMR (400MHz, CDCl
3) δ 7.45 (d, J=6.4Hz, 2H), 7.27 (m, 3H), 4.99 (s, 2H), 4.45 (t, J=4.8Hz, 2H), 4.39 (t, J=7.2Hz, 2H), 3.73 (t, J=5.2Hz, 2H), 3.4 (s, 3H), 2.15 (m, 1H), 1.82 (m, 2H), 1.40 (m, 4H), 0.93 (t, J=6.8Hz, 3H); MS[M+H]+m/z 430.2.
Embodiment 14: allyl carbonate 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester (55)
The white solid of productive rate 93%:
1H NMR (400MHz, CDCl
3) δ 7.46 (d, J=6.0Hz, 2H), 7.25 (m, 3H), 6.0 (m, 1H), 5.5 (m, 1H), 5.35 (m, 1H), 4.99 (s, 2H), 4.89 (d, J=2.4Hz, 2H), 4.46 (t, J=4.8Hz, 2H), 3.74 (d, J=5.2Hz, 2H), 3.42 (s, 3H); MS[M+H]+m/z 400.2.
Embodiment 15: carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester 4-chloro-butyl ester (56)
The white solid of productive rate 98%: 1H
1H NMR (400MHz, CDCl
3) δ 7.44 (d, J=6.0Hz, 2H), 7.25 (m, 3H), 4.98 (s, 2H), 4.45 (m, 4H), 3.63 (t, J=5.2Hz, 2H), 3.42 (s, 3H), 1.99 (m, 4H); MS[M+H]+m/z 450.2.
Embodiment 16: carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester butyl ester (57)
The white solid of productive rate 100%:
1H NMR (400MHz, CDCl
3) δ 7.45 (d, J=6.0Hz, 2H), 7.27 (m, 3H), 4.99 (s, 2H), 4.46 (m, 2H), 4.41 (t, J=4.4Hz, 2H), 3.73 (t, J=7.2Hz, 2H), 3.42 (s, 3H), 1.79 (m, 2H), 1.48 (m, 2H), 0.96 (t, J=7.6Hz, 3H); MS[M+H]+m/z 416.2.
Embodiment 17: carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester phenyl ester (58)
The white solid of productive rate 100%:
1H NMR (400MHz, CDCl
3) δ 7.52 (d, J=6.8Hz, 2H), 7.41 (m, 2H), 7.30 (m, 3H), 7.25 (m, 3H), 4.99 (s, 2H), 4.47 (t, J=4.8Hz, 2H), 3.75 (t, J=4.8Hz, 2H), 3.43 (s, 3H); MS[M+H]+m/z 436.2.
Embodiment 18: carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester 2,2-dimethyl-propyl ester (59)
The white solid of productive rate 100%:
1H NMR (400MHz, CDCl
3) δ 7.43 (d, 2H), 7.25 (m, 3H), 4.99 (s, 2H), 4.47 (t, 2H), 4.08 (s, 2H), 3.75 (t, 2H), 3.42 (s, 3H), 1.07 (s, 9H); MS[M+H]+m/z 430.2.
Embodiment 19: carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base ester heptyl ester (60)
The white solid of productive rate 100%:
1H NMR (400MHz, CDCl
3) δ 7.45 (d, J=6.0Hz, 2H), 7.27 (m, 3H), 4.99 (s, 2H), 4.46 (t, J=5.2Hz, 2H), 4.39 (t, J=7.2Hz, 2H), 3.74 (t, J=5.2Hz, 2H), 3.42 (s, 3H), 1.8 (m, 2H), 1.4 (m, 2H), 1.3 (m, and 6H) 0.87 (t, J=7.2Hz, 3H); MS[M+H]+m/z 458.3.
Embodiment 20: carbonic acid 6-amino-9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-8-base own ester of ester (30)
The white solid of productive rate 74%:
1H NMR (400MHz, CDCl
3) δ 7.45 (d, J=5.6Hz, 2H), 7.27 (m, 3H), 4.98 (s, 2H), 4.45 (t, J=4.8Hz, 2H), 4.41 (t, J=6.8Hz, 2H), 3.73 (t, J=4.4Hz, 2H), 3.42 (s, 3H), 1.81 (m, 2H), 1.34 (m, 2H), 1.31 (m, 2H), 1.26 (m, 2H), 0.89 (t, J=2Hz, 3H); MS[M+H]+m/z444.4.
Embodiment 22 and 23 is from 9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-6-base amine, 62, by 9-benzyl-8-bromo-2-(2-methoxyl group-ethyoxyl)-9H-purine-6-base amine, 63 and Sodium ethylate or Feldalat NM preparation, above-mentioned raw materials is separately according to Kurimota etc., Bioorg.Med.Chem., 12, the described method of 1091-1099 (2004) makes.
Embodiment 21:9-benzyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-6-base amine (62)
The brown solid of productive rate 100%:
1H NMR (400MHz, CDCl
3) δ 7.59 (s, 1H), 7.27 (m, 5H), 5.83 (s, 2H), 5.26 (s, 2H), 4.49 (t, J=4.8Hz, 2H), 3.75 (t, J=5.2Hz, 2H), 3.43 (s, 3H); MS[M+H]
+M/z 300.2.
Embodiment 22:9-benzyl-8-ethyoxyl-2-(2-methoxyl group-ethyoxyl)-9H-purine-6-base amine (64)
The brown solid of productive rate 91%:
1H NMR (400MHz, d
6-DMSO) δ 7.24 (m, 2H), 7.24 (m, 3H), 5.00 (s, 2H), 4.42 (m, 2H), 4.27 (t, J=4.8Hz, 2H), 3.58 (t, J=4.8Hz, 2H), 3.26 (s, 3H), 1.32 (t, J=7.2Hz, 3H); MS[M+H]
+M/z344.1.
Embodiment 23:9-benzyl-8-methoxyl group-2-(2-methoxyl group-ethyoxyl)-9H-purine-6-base amine (65)
The brown solid of productive rate 91%:
1H NMR (400MHz, d
6-DMSO) δ 7.28 (m, 2H), 7.22 (m, 3H), 6.86 (s, 2H), 5.01 (s, 2H), 4.26 (t, J=4.4Hz, 2H), 4.02 (s, 3H), 3.58 (t, J=4.8Hz, 2H), 3.25 (s, 3H); MS[M+H]
+M/z 330.2.
Embodiment 24:7-pi-allyl-2-amino-9-(5 '-O-L-figured silk fabrics acyl group-β-D-nuclear benzofuran sugar yl)-7,9-dihydro-1H-purine-6,8-diketone (68)
Step 1: preparation 7-pi-allyl-2-amino-9-(2 ', 3 '-O-isopropylidene-β-D-nuclear benzofuran sugar yl)-7,9-dihydro-1H-purine-6,8-diketone (66)
Chemical compound 17 (0.17 gram, 0.49 mM) is dissolved among the DMF (4.0 milliliters), and acetone (3.0 milliliters) is joined in this solution.In mixed liquor, add 2,2-dimethoxy propane (0.18 milliliter, 1.47 mMs) and MeSO
3H (0.02 milliliter, 0.05mmol).Under the room temperature with this reaction mixture and saturated NaHCO
3Aqueous solution stirs 20h.Use CH then
2Cl
2Aqueous phase extracted (4 *).With the organic facies drying (MgSO that merges
4), filtering, vacuum concentration obtains 130m gram white solid 66, productive rate 70%:
1H NMR (400MHz, CD
3OD) δ 5.97 (d, J=2.4Hz, 1H), 5.93 (m, 1H), 5.34 (dd, J=4.4,2.0Hz, 1H), 5.15 (m, 1H), 5.12 (dd, J=7.6,1.2Hz, 1H), 4.98 (m, 1H), 4.52 (d, J=5.6,2H), 4.16 (m, 1H), 3.71 (m, 2H), 1.56 (s, 3H), 1.36 (s, 3H); MS (+)-ES[M+H]
+380.0m/z.
Step 2: preparation 7-pi-allyl-2-amino-9-(2 ', 3 '-O-isopropylidene-5 '-N tert-butoxycarbonyl-L-figured silk fabrics acyl group)-β-D-nuclear benzofuran sugar yl)-7,9-dihydro-1H-purine-6,8-diketone (67)
The THF (8.0 milliliters) of (0.13 gram, 0.34 mMs), BOC-valine (0.08 gram, 0.36 mM), EDC (0.07 gram, 0.38 mM), DMAP (0.05 gram, 0.38 mM) and pyridine (0.8 milliliter) the mixed liquor N at room temperature with 66
2Stir 16h in the environment.Solvent removed in vacuo, and residue is dissolved among the EtOAc.The saturated NaHCO of organic facies
3Solution washing, salt washing, dry (MgSO
4) and filter.Vacuum concentrated filtrate, the CH of employing 2%-5% gradient MeOH
2Cl
2Solution carries out purified by flash chromatography, obtains 180 milligrams of faint yellow solids 67 (91%):
1H NMR (400MHz, CDCl
3) δ 6.07 (s, 1H), 5.80 (m, 1H), 5.56 (br s, 2H), 5.41 (d, J=5.6Hz, 1H), 5.15 (m, 3H), 4.97 (br s, 1H), 4.53 (br s, 2H), 4.46 (m, 1H), 4.32 (m, 2H), 4.20 (m, 1H), 2.11 (m, 1H), 1.56 (s, 3H), 1.44 (s, 9H), 1.36 (s, 3H), 0.93 (d, J=6.8Hz, 3H), 0.84 (d, J=6.8Hz, 3H); MS (+)-ES[M]
+578.9m/z.
Step 3:7-pi-allyl-2-amino-9-(5 '-O-L-figured silk fabrics acyl group-β-D-nuclear benzofuran sugar yl)-7,9-dihydro-1H-purine-6,8-diketone (68)
In MeOH (10 milliliters) solution of 67 (0.18 gram, 0.311 mMs), N
2Environment adds AcCl (0.86 milliliter, 12.07 mMs) down.With behind this reaction mixture stirring 18h, use saturated NaHCO carefully under the room temperature
3The aqueous solution neutralization.In this mixed liquor, add silica gel, vacuum concentration.Adopt the CH of 10%-20% gradient MeOH
2Cl
2Carry out the purified by flash chromatography residue, obtain 80m gram white solid 68 (59%):
1H NMR (400MHz, d
6-DMSO) δ 6.72 (br s, 2H), 5.84 (m, 1H), 5.59 (d, J=4.8Hz, 1H), 5.43 (d, J=5.6Hz, 1H), 5.15 (brs, 1H), 5.07 (d, J=12Hz, 1H), 5.0 (d, J=18.8Hz, 1H), 4.77 (q, J=4.8Hz, 1H), 4.36 (m, 3H), 4.26 (t, J=4.4Hz, 1H), 4.20 (m, 1H), 3.93 (m, 1H), 3.57 (br s, 1H), 2.01 (m, 1H), 0.86 (d, J=4.4Hz, 3H), 0.85 (d, J=5.2Hz, 3H); MS (+)-ES[M+H]
+439.1m/z.
Embodiment 25:7-pi-allyl-2-amino-9-β-D-nuclear benzofuran sugar yl-6-(5-methyl-2-oxygen-[1,3] Dloxole-4-base (dioxolyl) methoxyl group)-7,9-dihydro-purine-8-ketone (71)
Step 1: preparation 7-pi-allyl-2-amino-9-(2 ', 3 ', 5 '-three-O-triethylsilyl-β-D-nuclear benzofuran sugar yl)-7,9-dihydro-1H-purine-6,8-diketone (69)
(0.93 restrains, and in DMF 13.64mol) (13 milliliters) solution, dropwise adds chloro triethyl-silicane (0.92 milliliter, 5.46 mMs), stirs 2.5h under the room temperature for (0.46 gram, 1.36 mMs) and imidazoles 17.Use saturated NaHCO
3Aqueous solution processing reaction mixed liquor, separating obtained biphase.With diethyl ether (2 *) washing water.Merge organic facies and wash dry (MgSO with water
4), filter the back and concentrate.Adopt the CH of 2%-10% gradient MeOH
2Cl
2Solution carries out purified by flash chromatography, obtains 69 (89%) of 830 milligrams of light yellow oils:
1H NMR (400MHz, CDCl
3) δ 5.92 (m, 1H), 5.85 (d, J=6.8Hz, 1H), 5.3 (m, 1H), 5.15-5.23 (m, 2H), 5.09 (br s, 2H), 4.54 (d, J=4.4Hz, 2H), 4.33 (m, 1H), 3.98 (m, 1H), 3.67-3.80 (m, 2H), 0.85-1.02 (m, 26H), 0.48-0.71 (m, 19H): MS (+)-ES[M]
+682.6m/z.
Step 2: preparation 7-pi-allyl-2-amino-9-(2 ', 3 ', 5 '-three-O-triethylsilyl-β-D-nuclear benzofuran sugar yl)-6-(5-methyl-2-oxygen-[1,3] Dloxole-4-ylmethoxy)-7,9-dihydro-purine-8-ketone (70)
To be similar to the mode of embodiment 2 steps 1, from chemical compound 69 and 4-methylol-5-methyl-[1,3] Dloxole-2-ketone is (according to Alepegiani, Syn.Comm., 22 (9), the described method preparation of 1277-82 (1992)) preparation chemical compound 70, productive rate 5%, HPLC purification (Thomson ODS-A 100A 5u50 * 21.2mm post; Flow velocity=30 ml/min; The CH that contains 0.05%TFA
3CN (A) contains the water (B) of 0.05%TFA; Make-up pump flow velocity=1.0 ml/min; Make-up pump mobile phase; Contain the MeOH of 0.05%TFA, gradient system is as follows: t=0; 50%A, 50%B; T=2.0 minute; 50%A, 50%B; T=5.0 minute; 100%A, 0%B; T=9.5 minute; 100%A, 0%B; T=10.0 minute; 50%A, 50%B; T=13.0 minute; 50%A, 50%B).Obtain white solid: MS (+)-ES[M behind the purification]
+794.1m/z.
Step 3: preparation 7-pi-allyl-2-amino-9-β-D-nuclear benzofuran sugar yl-6-(5-methyl-2-oxygen-[1,3] Dloxole-4-ylmethoxy)-7,9-dihydro-purine-8-ketone (71)
In the MeOH (1.5 milliliters) of 70 (9.0 milligrams, 0.012 mM) solution, add 3HFNEt
3(0.01 milliliter, 0.07 mM), and at room temperature stir 16h.Solvent removed in vacuo is also used the purified by flash chromatography residue.The required product CH of 2%-5% gradient MeOH
2Cl
2Eluant solution obtains 3.26m gram white solid 71 (64%):
1H NMR (400MHz, CDCl
3) 5.96 (d, J=7.6Hz, 1H), 5.85 (m, 1H), 5.17 (m, 4H), 4.96 (t, J=7.2Hz, 1H), 4.47 (d, J=6.0Hz, 2H), 4.25 (J=5.2Hz, 1H), 4.24 (s, 1H), 3.81 (ABq, Δ υ AB=0.17, JAB=11.6Hz, 2H), 2.22 (s, 3H): MS (+)-ES[M+H]
+452.4m/z.
Embodiment 26:(7-pi-allyl-2-amino-9-β-D-nuclear benzofuran sugar yl-8-oxygen-8,9-dihydro-7H-purine-6-yloxymethyl)-methyl-urethanes (73)
Step 1: preparation (7-pi-allyl-2-amino-9-(2 ' 3 ' 5 '-three-O-triethylsilyl-β-D-nuclear benzofuran sugar yl)-8-oxygen-8,9-dihydro-7H-purine-6-yloxymethyl)-methyl-urethanes (72)
Being similar to the mode of embodiment 2 steps 1, from chemical compound 69 and N-methyl-N-(methylol) urethanes (Kelper, JOC, 52,1987, p.453-455) preparation chemical compound 72, productive rate 4%, white solid behind the HPLC purification:
1H NMR (400MHz, CDCl
3) δ 5.94 (m, 1H), 5.82 (d, J=6.4Hz, 1H), 5.53 (br s, 1H), and 5.23-5.29 (m, 2H), 5.15 (t, J=9.6Hz, 1H), 4.57 (d, J=5.2Hz, 2H), 4.35 (br s, 1H), 4.21 (m, 2H), 3.96 (br s, 1H), 3.73 (m, 2H), 3.06 (s, 3H), 1.30 (m, 4H), 0.87-1.01 (m, 24H), 0.57-0.68 (m, 19H): MS (+)-ES[M+H]
+797.7m/z.
Step 2: preparation (7-pi-allyl-2-amino-9-β-D-nuclear benzofuran sugar yl-8-oxygen-8,9-dihydro-7H-purine-6-yloxymethyl)-methyl-urethanes (73)
To be similar to the mode of step 3 among the embodiment 25, preparation chemical compound 73, productive rate 64%, HPLC purification (Thomson ODS-A 100A 5 μ 50 * 21.2mm post; Flow velocity=30 ml/min; The CH that contains 0.05%TFA
3CN (A) contains the water (B) of 0.05%TFA; Make-up pump flow velocity=1.0 ml/min; Make-up pump mobile phase; Contain the MeOH of 0.05%TFA, gradient system is as follows: t=0; 50%A, 50%B; T=2.0 minute; 50%A, 50%B; T=5.0 minute; 100%A, 0%B; T=9.5 minute; 100%A, 0%B; T=10.0 minute; 50%A, 50%B; T=13.0 minute; 50%A, 50%B) back is a white solid.
1H?NMR(400MHz，CDCl
3)δ5.92(m，1H)，5.92(d，J＝7.6Hz，1H)，5.51(m，2H)，5.22(d，J＝15.6Hz，1H)，5.16(d，J＝9.2Hz，1H)，4.92(t，J＝7.2Hz，1H)，4.57(d，J＝6.0，2H)，4.40(d，J＝6.0，1H)，4.21(m，3H)，3.79(ABq，ΔυAB＝0.178，JAB＝14.0Hz，2H)，3.06(s，3H)，1.31(t，J＝7.2Hz，3H)：MS(+)-ES[M]
+455.4m/z.
27: two hydrochloric acid 5-of embodiment amino-3-(5 '-O-L-figured silk fabrics acyl group-β-D-nuclear benzofuran sugar yl) thiazole is [4,5-d] pyrimidine-2 also, 7-diketone (24)
Step 1: preparation 5-amino-3-(2 ', 3 '-O-isopropylidene-β-D-nuclear benzofuran sugar yl) thiazole is [4,5-d] pyrimidine-2 also, 7-diketone (22)
In the 250mL Morton kieldahl flask, at 21 (5.37 grams, 17.0 mM, according to United States Patent (USP) 5,041, the preparation of method described in 426 (embodiment 2), its content is included into this paper as a reference) the non-homogeneous mixed liquor of acetone (40 milliliters) in, add 2 in succession under the room temperature, 2-DMP (6.26 milliliters, 50.9 mMs), DMSO (6.6 milliliters) and MeSO
3H (220 microlitres, 3.39 mMs).The vigorous stirring reaction mixture is along with the consumption of the diketone homogenizing and golden yellow that becomes.TLC analyzes (SiO
2, 10%MeOH-CHCl
3) show that 6 hours afterreactions are complete.Use the undissolved solid of Whatman 1 type filter paper gravity filtration of band groove.Then, filtrate is poured in the frozen water of 10 volumes (～400 milliliters), white solid is directly precipitated.Short time adds the NaHCO that is dissolved in the water (10 milliliters) after stirring
3(285 milligrams, 3.39 mMs), with in and MeSO
3H.In the Morton reactor, continued vigorous stirring 15 minutes, mixed liquor is filtered by the thick agglomerating funnel of peeling off.Solid matter washs with frozen water (100 milliliters), air drying, and 65 ℃ of high vacuums are further dry down then, obtain 280-81 ℃ of 5.36 gram (88%) white solid ketal 22:mp;
1H (DMSO-d
6) 1.28 (s, 3H), 1.47 (s, 3H), 3.43-3.55 (m, 2H), 3.95-3.99 (m, 1H), 4.77-4.80 (m, 1H), 4.88-4.91 (m, 1H), 5.24-5.26 (m, 1H), 5.99 (s, 1H), 6.97 (br s, 2H), 11.25 (s, 1H).
Step 2: preparation 5-amino-3-(2 ', 3 '-O-isopropylidene-5 '-N-tert-butoxycarbonyl-L-figured silk fabrics acyl group)-β-D-nuclear benzofuran sugar yl)-thiazole [4,5-d] pyrimidine-2 also, 7-diketone (23)
Under 0 ℃, in THF (9 milliliters) solution of N-butoxy carbonyl-(L)-valine (671 milligrams, 2.81 mMs), add EDC (588 milligrams, 3.07 mMs).0 ℃ was stirred gained intimate mixing liquid down after 45 minutes, and it is non-homogeneous that mixed liquor becomes, and added the solid acetonide 2 (1.00 grams, 2.81 mMs) of a part from above-mentioned steps 1.Add solid DMAP (522 milligrams, 4.27 mMs) then.Make reaction mixture return to room temperature, restir 5 hours, 25 ℃ are rotated evaporation and concentration down, obtain yellow slurry.Residue is dissolved among the EtOAc (50 milliliters), distributes, use saturated NaHCO then with 1N HCl (10 milliliters)
3Aqueous solution (10 milliliters) neutralizing acid.Acid water is further used EtOAc (2 * 50 milliliters) extraction, distributes with alkaline water then.With the organic facies Na that merges
2SO
4Drying is by short filler SiO
2Filter, concentrate, obtain the amino-acid ester 23 of 1.480 gram form of foam (96%) Boc-protections: 158 ℃ of fusing points (mp) (dec);
1H (CDCl
3) 0.86 (d, J=7.0,3H), 0.95 (d, J=7.0,3H), 1.35 (s, 3H), 1.44 (s, 9H), 1.56 (s, 3H), 1.75 (brs, 1H), 2.08-2.19 (m, 1H), 4.20-4.24 (m, 2H), 4.30-4.37 (m, 1H), 4.56 (dd, J=11.0,5.9,1H), 4.96 (dd, J=6.2,3.7,1H), 5.11 (br d, J=8.8,1H), 5.29 (br d, J=6.6,1H), 5.88 (br s, 2H), 6.23 (s, 1H).
Step 3: prepare also [4,5-d] pyrimidine-2 of two hydrochloric acid 5-amino-3-(5 '-O-L-figured silk fabrics acyl group-β-D-nuclear benzofuran sugar yl) thiazole, 7-diketone (24)
HCl gas is by dense H
2SO
4Behind the bubbler, (by the glass separator tube) imports and contains in the 250mL three neck Morton flasks of Glacial acetic acid isopropyl ester (80 milliliters) under 0 ℃, up to obtaining saturated solution.In this solution, add isopropyl acetate (30 milliliters) solution of Boc-amino-acid ester (5.53 grams, 9.95 mMs) from above-mentioned steps 2, form the white solid precipitation in 5 minutes.Add 10% (volume/volume) IPA (11 milliliters).This reaction mixture is heated to room temperature, restir 12 hours.Dilute heterogeneous reaction mixture with dry toluene (100 milliliters).With sintering (scintered) glass funnel of intermediate pore size at N
2Following filtration obtains cream-coloured amorphous solid.Abrasive solid in anhydrous THF is filtered, and 65 ℃ of vacuum dryings obtain 3.677 gram white solid (81%) 166-68 ℃ of 24:mp of title compound (dec);
1H (DMSO-d
6) 0.90 (d, J=7.0,3H), 0.94 (d, J=7.0,3H), 2.14-2.18 (m, 1H), 3.83-3.85 (m, 1H), 3.96-4.00 (m, 1H), 4.23-4.28 (m, 2H), 4.42 (dd, J=11.7,3.4,1H), 4.75 (dd, J=10.3,5.5,1H), 5.81 (d, J=4.4,1H), 6.46 (br s, 3H), 7.23 (br s, 2H), 8.47 (s, 3H), 11.5 (br s, 1H). elementary analysis C
15H
21N
5O
7S2HCl: value of calculation: C, 36.89; H, 4.75; Cl, 14.52; N, 14.34; S, 6.57; Measured value: C, 37.03:H, 4.74; Cl, 14.26; N, 14.24; S, 6.42.
Embodiment 28:5-acetyl-amino-3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl) thiazole is [4,5-d] pyrimidines-2,7 (6H)-diketone (74) also
Step 1: preparation 5-acetyl-amino-3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl) thiazole is [4,5-d] pyrimidines-2,7 (6H)-diketone (74) also
Anhydrous 21 (8.0 grams, 39.5 mMs) are dissolved in the anhydrous pyridine (65 milliliters).Add DMAP (3.1 grams, 25.3 mMs) and acetic anhydride (19.1mL 202.4 mMs) in succession.After reacting 2 hours under the room temperature, use saturated NaHCO
3(100 milliliters) aqueous solution cessation reaction is with DCM (3 * 200 milliliters) extraction.Concentrate organic facies, grind with ether then.Obtain impure slightly white solid 5-acetyl-amino-3-(2,3, the 5-three-O-acetyl group-β-D-nuclear benzofuran sugar yl) thiazole of 12.5 grams (103%) also-246.7-248.1 ℃ of [4,5-d] pyrimidine-2,7 (6H)-diketone 74:mp; R
f=0.20 (SiO
2, 50%EtOAc-CHCl
3);
1H NMR (400MHz, d
6-DMSO) 12.23 (s, 1H), 11.85 (s, 1H), 5.97 (m, 2H), 5.48 (t, J=6,1H), and 4.35-4.40 (m, 1H), 4.25-4.31 (m, 1H), 4.08-4.18 (m, 1H), 2.49 (s, 3H), 2.07 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H).
Embodiment 29:5-amino-3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl) thiazole is [4,5-d] pyrimidines-2,7 (6H)-diketone (75) also
Step 1: preparation 5-amino-3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl) thiazole is [4,5-d] pyrimidines-2,7 (6H)-diketone (75) also
Under 0 ℃, in acetonitrile (160 milliliters) suspension of 21 (5.00 grams, 15.8 mMs), add Et in succession
3N (11.0 milliliters, 79.0 mMs), DMAP (195 milligrams, 1.59 mMs) and Ac
2O (4.47 milliliters, 47.4 mMs).Under the room temperature this reaction mixture stirring after 2 hours, is concentrated and obtains brown slurry.Residue flash column chromatography purification (silicon dioxide, MeOH/CHCl
3=1-10%), obtain the triacetate of 6.22 gram (89%) white solids 75: mp 198-199 ℃;
1H (400MHz, d
6-DMSO) .11.34 (s, 1H), 7.02 (br s, 2H), 5.90 (m, 2H), 5.51 (t, J=6.0Hz, 1H), 4.36 (dd, J=12.4,3.2Hz, 1H), 4.21 (m, 1H), 4.08 (q, J=6.0Hz, 1H), 2.06 (s, 3H), 2.06 (s, 3H), 2.00 (s, 3H); MS (+)-ES[M+H]
+M/z 443.3.
Embodiment 30:5-amino-7-ethyoxyl-3-β-D-nuclear benzofuran sugar yl-thiazole is [4,5-d] pyrimid-2-ones (77) also
Step 1: preparation 5-acetyl-amino-7-ethyoxyl-3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-thiazole is [4,5-d] pyrimid-2-ones (76) also
Being similar to embodiment 2, the mode of step 1, from 74 and ethanol preparation white foam 76, productive rate 72%:MS (+)-ES[M+H]
+M/z513.R
f=0.45 (75% ethyl acetate-CHCl
3).
Step 2: preparation 5-amino-7-ethyoxyl-3-β-D-nuclear benzofuran sugar yl-thiazole is [4,5-d] pyrimid-2-ones (77) also
Being similar to embodiment 2, the mode of step 2, from the title compound of 76 preparation white solids, productive rate 65%:
1H NMR (400MHz, d
6-DMSO) 6.87 (s, 2H), 5.85 (d, J=4.8Hz, 1H), 5.27 (d, J=5.6Hz, 1H), 4.96 (d, J=5.2Hz, 1H), 4.78 (m, 1H), 4.66 (m, 1H), 4.36 (m, 2H), 4.09 (m, 1H), 3.74 (m, 1H), 3.58 (m, 1H), 3.40 (m, 1H), 1.29 (m, 3H); MS (+)-ES[M+H]
+M/z445, [2M+H]
+M/z689.R
f=0.2 (50%THF-CHCl
3). elementary analysis C
12H
16N
4O
6S0.25H
2O: value of calculation: C, 41.31; H, 4.77; N, 16.06; S, 9.19. measured value: C, 41.24; H, 4.71; N, 15.89; S, 9.06.
Embodiment 31:5-amino-7-methoxyl group-3-β-D-nuclear benzofuran sugar yl-thiazole is [4,5-d] pyrimid-2-ones (79) also
Step 1: preparation 5-acetyl-amino-7-methoxyl group-3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-thiazole is [4,5-d] pyrimid-2-ones (78) also
Being similar to embodiment 2, the mode of step 1, from 74 and methanol prepare 77 of white foam, productive rate 65%:MS (+)-ES [M+H]
+499.R
f=0.5 (75% ethyl acetate-CHCl
3).
Step 2: preparation 5-amino-7-methoxyl group-3-β-D-nuclear benzofuran sugar yl-thiazole is [4,5-d] pyrimid-2-ones (79) also
Being similar to embodiment 2, the mode of step 2, from the title compound of 78 preparation white solids, productive rate 78%:
1H NMR (400MHz, d
6-DMSO) 6.91 (s, 2H), 5.86 (d, J=5.2Hz, 1H), 5.28 (d, J=5.2Hz, 1H), 4.96 (d, J=5.2Hz, 1H), 4.77 (m, 1H), 4.66 (m, 1H), 4.09 (m, 1H), 3.90 (s, 3H), 3.75 (m, 1H), 3.56 (m, 1H), 3.43 (m, 1H); MS (+)-ES[M+H]
+331.R
f=0.2 (50%THF-CHCl
3). elementary analysis C
11H
14N
4O
6S0.25H
2O: value of calculation: C, 39.46; H, 4.37; N, 16.73; S, 9.58. measured value: C, 39.59; H, 4.17; N, 16.55; S, 9.52.
Embodiment 32:(5-amino-2-oxygen-3-β-D-nuclear benzofuran sugar yl-2,3-dihydro-thiazole is [4,5-d] pyrimidin-7-yl oxygen methyl also)-urethanes (82)
Step 1: preparation 5-amino-3-(2 ', 3 ', 5 '-three-O-triethylsilyl-β-D-nuclear benzofuran sugar yl)-thiazole is [4,5-d] pyrimidine-2 also, 7-diketone (80)
Under the room temperature, in DMF (20 milliliters) suspension of 21 (1.00 grams, 3.16 mMs), add imidazoles (753 milligrams, 11.06 mMs), DMAP (39 milligrams, 0.32 mM) and chloro triethyl-silicane (1.64 milliliters, 9.80 mMs) in succession.Under the room temperature reaction mixture stirring after 2 hours, is used saturated NaHCO
3Aqueous solution (20 milliliters) cessation reaction.Mixed liquor CHCl
3(3 * 20 milliliters) extraction, MgSO
4Drying concentrates.Residue flash column chromatography (silicon dioxide, MeOH/CHCl
3=1-5%) purification obtains the chemical compound 80 of 1.91 gram white solids (92%):
1H (400MHz, d
6-DMSO) .5.99 (s, 1H), 5.62 (br s, 2H), 5.19 (dd, J=4.4,6.0Hz, 1H), 4.35 (dd, J=2.8,4.4Hz, 1H), 3.99 (m, 1H), 3.77 (dd, J=7.6,10.8Hz, 1H), 3.68 (dd, J=4.8,10.4Hz, 1H), 1.10 (t, J=7.1Hz, 3H), 0.96 (t, J=7.1Hz, 3H), 0.89 (t, J=7.1Hz, 3H), 0.68 (q, J=7.1Hz, 2H), 0.61 (q, J=7.1Hz, 2H), 0.54 (m, 2H); MS (+)-ES[M+H]
+M/z 660.0.
Step 2: preparation 5-amino-3-(2 ', 3 ', 5 '-three-O-triethylsilyl-β-D-nuclear benzofuran sugar yl)-2,3-dihydro-thiazole is [4,5-d] pyrimidin-7-yl oxygen methyl also)-urethanes (81)
Being similar to the mode of step 1 among the embodiment 2, from 80 and N-ethyl carbamic acid ethyl ester prepare the chemical compound 81 of white solid, productive rate 31%:[M+H]
+760.5;
1H NMR (400MHz, CDCl
3) δ 6.43 (br s, 2H), 6.09 (t, J=7.6Hz, 1H), 5.94 (d, J=6.0Hz, 1H), 5.31 (d, J=4.8Hz, 2H), 5.19 (dd, J=6.0,4.8Hz, 1H), 4.35 (dd, J=4.8,2.8Hz, 1H), 4.19 (q, J=6.4Hz, 2H), 3.98 (m, 1H), 3.76 (dd, J=10.8,7.6Hz, 1H), 3.68 (dd, J=10.4,4.8Hz, 1H), 1.29 (t, J=6.8Hz, 3H), 1.02 (t, J=8.0Hz, 3H), 0.96 (t, J=7.6Hz, 3H), 0.90 (t, J=8.0Hz, 3H), 0.69 (q, J=8.0Hz, 2H), 0.61 (q, J=8.0Hz, 2H), 0.55 (m, 2H); [M+H]
+760.5.
Step 3: preparation (5-amino-2-oxygen-3-β-D-nuclear benzofuran sugar yl-2,3-dihydro-thiazole is [4,5-d] pyrimidin-7-yl oxygen methyl also)-urethanes (82)
Under the room temperature, with 81 (244 milligrams, 321 μ mol), the pyridine of 5M HF (321 microlitres, 1.60 mMs) and THF (3.20 milliliters) solution stirring 5 hours.Solvent removed in vacuo is with gained residue purified by flash chromatography (SiO
2, 10%MeOH-CHCl
3), obtain white solid 82 (119 milligrams, 90%):
1H NMR (400MHz, d
6-DMSO) δ 8.43 (br s, 1H), 7.76 (br s, 2H), 5.82 (d, J=5.2Hz, 1H), 5.78 (s, 2H), 5.32 (d, J=5.6Hz, 1H), 5.24 (dd, J=6.0,4.8Hz, 1H), 5.00 (d, J=5.6Hz, 1H), 4.82 (q, J=5.6Hz, 1H), 4.68 (t, J=6.0,1H), 4.11 (q, J=5.2Hz, 1H), 4.09 (q, J=7.2Hz, 2H), 3.78 (q, J=5.6Hz, 1H), 3.60 (m, 1H), 3.46 (m, 1H), 1.21 (t, J=7.2Hz, 3H); [M+H]
+418.2.
Embodiment 33:(5-amino-2-oxygen-3-β-D-nuclear benzofuran sugar yl-2,3-dihydro-thiazole is [4,5-d] pyrimidin-7-yl oxygen methyl also)-methyl-urethanes (84)
Step 1: preparation (5-amino-2-oxygen-3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-2,3-dihydro-thiazole is [4,5-d] pyrimidin-7-yl oxygen methyl also)-methyl-urethanes (83)
Being similar to embodiment 2, the mode of step 1, from 75 and N-methyl-N-(methylol) urethanes prepare the chemical compound 83 of white solid, productive rate 24%:R
f=0.4 (33%EtOAc-CHCl
3);
1H NMR (400MHz, CDCl
3) δ 11.49 (br s, 1H), 6.08 (d, J=4.0Hz, 1H), 5.75 (t, J=6.0Hz, 1H), 5.53 (s, 2H), 4.49 (dd, J=13.5,8.4Hz, 1H), 4.30 (m, 5H), 3.62 (q, J=7.2Hz, 2H), 2.30 (s, 3H), 2.12 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 1.36 (t, J=6.8Hz, 3H), 1.20 (t, J=6.8Hz, 3H); [M+H]
+614.2.
Step 2: preparation (5-amino-2-oxygen-3-β-D-nuclear benzofuran sugar yl-2,3-dihydro-thiazole is [4,5-d] pyrimidin-7-yl oxygen methyl also)-methyl-urethanes (84)
Being similar to embodiment 1, the mode of step 4, from the title compound of 83 preparation white solids, productive rate 20%:
1H NMR (400MHz, d
6-DMSO) δ 7.86 (br s, 2H), 5.82 (d, J=4.8Hz, 1H), 5.47 (s, 2H), 5.31 (d, J=5.2Hz, 1H), 5.00 (d, J=5.6Hz, 1H), 4.82 (q, J=5.2Hz, 1H), 4.67 (q, J=5.6Hz, 1H), 4.18 (q, J=6.4Hz, 2H), 4.12 (m, 1H), 3.78 (q, J=6.0Hz, 1H), 3.60 (m, 1H), 3.47 (m, 1H), 3.30 (s, 3H), 1.27 (t, J=6.8Hz, 3H); [M+H]
+432.3.
Embodiment 34:5-amino-7-(5-methyl-2-oxygen-[1,3] Dloxole-4-ylmethoxy)-3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-thiazole is [4,5-d] pyrimid-2-ones (85) also
Step 1: preparation 5-amino-7-(5-methyl-2-oxygen-[1,3] Dloxole-4-ylmethoxy)-3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-thiazole is [4,5-d] pyrimid-2-ones (85) also
Under 0 ℃, in THF (50 milliliters) solution of the triacetate 75 (1.55 grams, 3.50 mMs), adding polymer support-triphenyl phasphine (4.95 grams, 10.50 mMs, Argonaut).In this mixed liquor, add 4-methylol-5-methyl-[1,3] Dloxole-2-ketone (0.91 gram, 7.00 mMs) (according to Alepegiani, Syn.Comm., 22 (9), the described method preparation of 1277-82 (1992)).Dropwise add diethylazodicarboxylate's (0.73 milliliter, 4.60 mMs) then.Under the room temperature gained mixed liquor was stirred 48 hours, filter, with MeOH and CHCl
3Washing.Concentrated filtrate, flash column chromatography purification (silicon dioxide, acetone/CHCl
3=10-20%), obtain the Dloxole ketone derivatives 85 (1.38 grams, 71%) of white solid:
1H (400MHz, d
6-DMSO); 7.06 (s, 2H), 6.00 (d, J=4.0Hz, 1H), 5.92 (dd, J=6.6,4.4Hz, 1H), 5.56 (t, J=6.4Hz, 1H), 5.30 (s, 2H), 4.38 (dd, J=11.6,3.6Hz, 1H), 4.25 (t, J=3.6Hz, 1H), 4.10 (q, J=6.0Hz, 1H), 2.23 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.00 (s, 3H); MS (+)-ES[M+H]
+M/z555.3.C
21H
22N
4O
12SMe
2The elementary analysis of CO, value of calculation: C, 47.06; H, 4.61; N, 9.15; S, 5.23. measured value: C, 47.25; H, 4.37; N, 9.53; S, 5.52.
Embodiment 35:5-amino-7-(5-methyl-2-oxygen-[1,3] Dloxole-4-ylmethoxy)-3-β-D-nuclear benzofuran sugar yl-thiazole is [4,5-d] pyrimid-2-ones (87) also
Step 1: preparation 5-amino-7-(5-methyl-2-oxygen [1,3] Dloxole-4-ylmethoxy)-3-(2 ', 3 ', 5 '-three-O-triethylsilyl-β-D-nuclear benzofuran sugar yl)-thiazole is [4,5-d] pyrimid-2-ones (86) also
Being similar to the mode of embodiment 34, from 80 and 4-methylol-5-methyl-[1,3] Dloxole-2-ketone prepare the chemical compound 86 of white solid, productive rate 45%:
1H NMR (400MHz, CDCl
3) δ 6.06 (d, J=6.0Hz, 1H), 5.21 (dd, J=6.0,4.8Hz, 1H), 5.18 (d, J=3.2Hz, 2H), 4.94 (br s, 2H), 4.38 (dd, J=4.8,2.8Hz, 1H), 4.00 (m, 1H), 3.79 (dd, J=11.2,8.0Hz, 1H), 3.69 (dd, J=10.8,5.2Hz, 1H), 2.23 (s, 3H), 1.02 (t, J=8.0Hz, 3H), 0.96 (t, J=7.6Hz, 3H), 0.89 (t, J=8.4Hz, 3H), 0.70 (q, J=7.6Hz, 2H), 0.61 (q, J=8.0Hz, 2H), 0.53 (m, 2H); [M+H]
+771.5.
Step 2: preparation 5-amino-7-(5-methyl-2-oxygen-[1,3] Dloxole-4-ylmethoxy)-3-β-D-nuclear benzofuran sugar yl-thiazole is [4,5-d] pyrimid-2-ones (87) also
To be similar to the mode of step 3 among the embodiment 32, from the title compound of 86 preparation white solids, productive rate 89%:
1H NMR (400MHz, d
6-DMSO) δ 7.03 (br s, 2H), 5.90 (d, J=5.2Hz, 1H), 5.33 (s, 2H), 5.02 (d, J=4.8Hz, 1H), 4.83 (q, J=5.6Hz, 1H), 4.71 (t, J=6.0Hz, 1H), 4.14 (q, J=5.2Hz, 1H), 3.80 (q, J=4.8Hz, 1H), 3.62 (m, 1H), 3.47 (m, 1H), 2.27 (s, 3H); [M+H]
+429.2.
Embodiment 36:5-amino-3-β-D-nuclear benzofuran sugar yl-3H-thiazole also-[4,5-d] pyrimid-2-ones (90)
Step 1: preparation 5-amino-7-sulfo--3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-thiazole is [4,5-d] pyrimid-2-ones (88) also
Under the room temperature, in pyridine (50 milliliters) solution of 75 (1 gram, 2.26 mMs), add P
2S
5(2.13 grams, 4.79 mMs).This solution of light and slow backflow (bath temperature 130-140 ℃) 29 hours.Be evaporated to this reaction mixture dried in the vacuum.60 ℃ down by adding H
2O (40 milliliters) makes too much P
2S
5Decompose.Under 60 ℃ this mixed liquor stirring after 1 hour, is cooled to room temperature.Use CHCl
3(3 * 40 milliliters) extract this mixed liquor.(the MgSO of evaporation drying
4) organic layer, obtain slurry, flash column chromatography purification (silicon dioxide, acetone/CHCl
3=15%), obtain (90%) 88 of 0.93 gram yellow solid:
1H (400MHz, d
6-DMSO) .12.50 (s, 1H), 7.35 (br s, 2H), 5.89 (m, 2H), 5.51 (t, J=6.4Hz, 1H), 4.36 (dd, J=12.0,4.0Hz, 1H), 4.24 (m, 1H), 4.10 (q, J=6.0Hz, 1H), 2.07 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H); MS (+)-ES[M+H]
+M/z 459.3.
Step 2: preparation 5-amino-3-(2 ', 3 ', 5 '-three-O-acetyl group-β-D-nuclear benzofuran sugar yl)-3H-thiazole is [4,5-d] pyrimid-2-ones (89) also
With Raney
_(3 big scrapers are used H to 2800 nickel in advance
2O, MeOH and washing with acetone) acetone (50 milliliters) suspension returning stirred 1 hour.Under refluxing, 88 triacetate (0.93 gram, 2.03 mMs) is joined in the above-mentioned suspension then.This mixed liquor was stirred 5 minutes, and postcooling was to room temperature in 30 minutes.With H
2S (gram) feeds 2h in the mixed liquor, cessation reaction.With gained mixed liquor filtration passing through Celite
_Short filler also washs with EtOH.Concentrated filtrate, flash column chromatography purification (silicon dioxide, MeOH/CHCl
3=1-2%), obtain 121-123 ℃ of (60%) 89:mp of 0.52 gram white solid;
1H (400MHz, d
6-DMSO) 8.38 (s, 1H), 6.93 (s, 2H), 6.03 (d, J=3.6Hz, 1H), 5.93 (dd, J=6.4,3.6Hz, 1H), 5.58 (t, J=6.0Hz, 1H), 4.38 (dd, J=11.6,3.6Hz, 1H), 4.26 (m, 1H), 4.11 (q, J=6.0Hz, 1H), 2.08 (s, 3H), 2.07 (s, 3H), 2.00 (s, 3H); MS (+)-ES[M+H]
+M/z 427.2.C
16H
18N
4O
8The elementary analysis value of calculation of S: 0.5CH
3OH0.25H
2O:C, 44.34; H, 4.62; N, 12.54; S 7.17. measured value: C, 44.54; H, 4.88; N, 12.16; S, 7.17.
Step 3: preparation 5-amino-3-β-D-nuclear benzofuran sugar yl-3H-thiazole is [4,5-d] pyrimid-2-ones (90) also
In MeOH (20 milliliters) solution of 89 (0.52 gram, 1.22 mMs), add K
2CO
3(25 milligrams, 0.18 mM).Under the room temperature reactant liquor stirring is spent the night, use AcOH (21 microlitres, 0.36 mM) neutralization then.Under the room temperature gained mixed liquor is continued to stir 30 minutes, concentrate, use H
2O (2 milliliters) grinds, and obtains the chemical compound 90 (89%) of 0.33 gram white solid: 220 ℃ of mp (Dec);
1H (400MHz, d
6-DMSO) .8.34 (s, 1H), 6.85 (s, 2H), 5.90 (d, J=4.8Hz, 1H), 5.31 (d, J=5.6Hz, 1H), 4.98 (d, J=5.6Hz, 1H), 4.81 (q, J=5.2Hz, 1H), 4.67 (t, J=6.0Hz, 1H), 4.11 (q, J=5.2Hz, 1H), 3.77 (dd, J=10.8,4.8Hz, 1H), 3.58 (m, 1H), 3.44 (m, 1H); MS (+)-ES[M+H]
+M/z301.1.C
10H
12N
4O
5The elementary analysis value of calculation of S: 0.3H
2O:C, 39.29; H, 4.15; N, 18.33; S 10.49. measured value: C, 39.51; H, 4.18; N, 17.95; S, 10.27.
Embodiment 37:5-amino-3-(2 ', 3 '-two-O-acetyl group-β-D-nuclear benzofuran sugar yl)-3H-thiazole is [4,5-d] pyrimid-2-ones (93) also
Step 1: preparation 5-amino-3-(the 5 '-O tert-butyl group-dimetylsilyl-β-D-nuclear benzofuran sugar yl)-3H-thiazole is [4,5-d] pyrimid-2-ones (91) also
In DMF (10 milliliters) solution of 90 (0.68 gram, 2.28 mMs), add imidazoles (0.54 gram, 7.93 mMs) and tert-butyldimethylsilyl chloride (0.68 gram, 4.56 mMs) in succession.Under the room temperature this reaction mixture stirring after 2 hours, is concentrated flash column chromatography purification (silicon dioxide, MeOH/CHCl
3Gradient=5-20%), obtain (52%) 91 of 0.49 gram white solid:
1H (400MHz, d
6-DMSO) .8.33 (s, 1H), 6.87 (s, 2H), 5.90 (d, J=4.0Hz, 1H), 5.33 (d, J=5.6Hz, 1H), 5.00 (d, J=5.2Hz, 1H), 4.79 (q, J=5.2Hz, 1H), 4.16 (q, J=5.2Hz, 1H), 3.77 (m, 2H), 3.64 (dd, J=12.0,7.2Hz, 1H), 0.84 (s, 9H), 0.00 (s, 6H); MS (+)-ES[M+H]
+M/z415.4.
Step 2: preparation 5-amino-3-(2 ', 3 '-two-O-acetyl group, the 5 '-O tert-butyl group-dimetylsilyl-β-D-nuclear benzofuran sugar yl)-3H-thiazole is [4,5-d] pyrimid-2-ones (92) also
Under 0 ℃, in acetonitrile (5 milliliters) solution of 91 (0.20 gram, 0.48 mMs), add Et in succession
3N (0.26 milliliter, 1.86 mMs) and Ac
2O (91 microlitres, 0.96 mM).Under the room temperature this reaction mixture stirring after 24 hours, is concentrated flash column chromatography purification (silicon dioxide, acetone/CHCl
3: gradient=5-10%), obtain (92%) 92 of 0.22 gram white solid:
1H (400MHz, d
6-DMSO) .8.36 (s, 1H), 6.90 (s, 2H), 6.00 (m, 2H), 5.57 (t, J=6.0Hz, 1H), 4.07 (q, J=5.2Hz, 1H), 3.77 (m, 2H), 2.07 (s, 3H), 2.06 (s, 3H), 0.83 (s, 9H), 0.00 (d, J=2.4Hz, 6H); MS (+)-ES[M+H]
+M/z 499.5.
Step 3: preparation 5-amino-3-(2 ', 3 '-two-O-acetyl group-β-D-nuclear benzofuran sugar yl)-3H-thiazole is [4,5-d] pyrimid-2-ones (93) also
In plastic test tube, in THF (5 milliliters) solution of 92 (0.22 gram, 0.44 mMs), add HF/ pyridine (0.70 milliliter).This reactant liquor was stirred 2h hour, concentrate flash column chromatography purification (silicon dioxide, MeOH/CHCl
3: gradient=5-10%), obtain (100%) title compound of 0.17 gram white solid: mp 109-111 ℃;
1H (400MHz, d
6-DMSO) .8.37 (s, 1H), 6.91 (s, 2H), 6.00 (m, 2H), 5.48 (t, J=6.0Hz, 1H), 4.91 (t, J=6.0Hz, 1H), 4.04 (dd, J=10.4,6.0Hz, 1H), 3.64 (m, 1H), 3.52 (m, 1H), 2.08 (s, 3H), 2.05 (s, 3H); MS (+)-ES[M+H]
+M/z385.3.C
14H
16N
4O
7S0.5CH
3The elementary analysis of OH0.2, value of calculation: C, 41.61; H, 4.32; N, 13.21; S 7.56: measured value: C, 41.73; H, 4.29; N, 12.86; S, 7.33.
Embodiment 38:[2-ethoxyl methyl-1-(2-hydroxy-2-methyl-propyl group)-1H-imidazo [4,5-c] quinolyl-4]-urethanes (39)
Step 1: preparation [2-ethoxyl methyl-1-(2-hydroxy-2-methyl-propyl group)-1H-imidazo [4,5-
Except that using MeOH as the solvent, to be similar to the mode of step 1 among the embodiment 8, from 1-(4-amino-2-ethoxyl methyl-imidazo [4,5-c] quinoline-1-yl)-2-methyl-propan-2-ol (38) (according to the described method preparation of international publication number WO 94/17043) and pyrocarbonic acid diethyl ester prepare the oily title compound, productive rate 39%:
1H NMR (400MHz, CDCl
3) δ 8.36 (d, J=8.0Hz, 1H), 8.05 (d, J=8.0Hz, 1H), 7.70 (t, J=7.2Hz, 1H), 7.61 (t, J=8.0Hz, 1H), 4.96 (br s, 2H), 4.80 (s, 2H), 4.39 (q, J=7.2Hz, 2H), 3.62 (q, J=7.2Hz, 2H), 1.40 (t, J=7.2Hz, 3H), 1.36 (br s, 6H), 1.24 (t, J=6.8Hz, 3H); MS (+)-ES[M+H]
+387.4m/z.
6.4TLR7 the masking action of part prodrug
Typical experiment will be used the human peripheral blood mononuclear cell (PBMC) who separates and place the multiple hole of cell culture from healthy donors; Generally, inoculate 2 * 10 in every hole
6To 5 * 10
6Cell.37 ℃, contain 5%CO
2Wet environment in, PBMC was cultivated 24 hours under the condition that does not contain test compound, to stablize condition of culture, then with 100 micromole's isatoribines, the TLR7 part joins respectively in each hole of containing from identical donor PBMC with corresponding TLR7 part prodrug; Comprise untreated in the same old way.The concentration that can change TLR7 part and TLR7 part prodrug to be to be fit to particular experiment, then with the PBMC culture 37 ℃, contain 5%CO
2Wet environment in cultivated 2-48 hour.Cell culture medium supernatant sampling between culture period.ELISA. the generation of the analysis of cells factor.The TLR7 part residual when finishing and the amount of TLR7 part prodrug are hatched in the analysis of LC-MS method.To the generation in the same old way, calculate production of cytokines with respect to isatoribine, deduct production of cytokines in the untreated control sample then.The result of thinner intracellular cytokine is to determine the active degree of TLR7 part greater than corresponding TLR7 part prodrug.
Therefore, if after similar time of contact and concentration, the TLR7 part produces the more interferon-ALPHA of multiple dose (cytokine of measuring easily) than corresponding TLR7 part prodrug, thinks that then TLR7 part prodrug is the TLR7 part prodrug of " sheltering ".The cytokine that constitutes " sheltering " produces the amplitude that reduces can be little of reducing by 25% than parent TLR, therefore, can correspondingly increase dosage for given tolerance level.
The data show that table 9-14 provides can be sheltered the TLR7 part of number of chemical type.Illustrated embodiment shows to have suitable masking action with respect to parent TLR7 part.Shown in chemical substituent group be exemplary, never be limitation of the present invention, so other chemical substituent group also can show masking action and is included in the scope of the present invention.Substituent group realization masking action is introduced in a plurality of positions that can be provided at the TLR7 part, and it is as shown in the table can comprise many chemical bonds.Should be understood that preferred substituted can be different with chemical bond for different parent TLR7 parts.
Table 9: the sheltering of isatoribine prodrug
| Parent molecule and prodrug thereof | Compound number | With respect to the amount of the inductive INFa of 100 μ M isatoribines, % |
| Parent molecule: isatoribine prodrug: amino-acid ester prodrug: deoxidation prodrug: 6-ethyoxyl prodrug: 6-methoxyl group prodrug: aminal prodrug: aminal prodrug: Dloxole ketone | 21 24 93 77 79 84 82 85 | 100 1 0 0 0 0 0 0 |
Available PBMC experimental study isatoribine prodrug shelter character.Result's (table 9) of PBMC test has shown the amount of parent compound and the INFa that prodrug discharged after (valine-isatoribine, 24) or 24 hours (other prodrug) in 8 hours thereof of contact initial concentration 100 μ M.Under the 100 μ M, in identical blood sample, under identical time of contact, the amount of the INFa that discharges is calibrated to the inductive amount of 100 μ M isatoribines.
Table 10: the sheltering of loxoribine prodrug
| Parent molecule and prodrug thereof | Compound number | With respect to the amount of the inductive INFa of 100 μ M isatoribines, % |
| Parent molecule: loxoribine prodrug: 6-ethyoxyl prodrug: deoxidation prodrug: L-valine ester | 17 45 43 68 | 50 0 0 0 |
Available PBMC experimental study loxoribine prodrug shelter character.Result's (table 10) of PBMC test has shown the amount of parent compound and the INFa that prodrug discharged after 24 hours thereof of contact initial concentration 100 μ M.Under 100 μ M, in the identical blood sample, under identical time of contact, the amount of the INFa that discharges is calibrated to the inductive amount of 100 μ M isatoribines.
Table 11: the sheltering of imiquimod prodrug
| Parent molecule and prodrug thereof | Compound number | With respect to the amount of the inductive INFa of 100 μ M isatoribines, % |
| Parent molecule: imiquimod prodrug: amyl carbamate prodrug: urethanes | 31 34 50 | 60-76 * 0 0 |
*Three different donors are carried out result of experiment twice
Available PBMC experimental study imiquimod prodrug shelter character.Result's (table 11) of PBMC test has shown the amount of parent compound and the INFa that prodrug discharged after 24 hours thereof of contact initial concentration 100 μ M.Under 100 μ M, in the identical blood sample, under identical time of contact, the amount of the INFa that discharges is calibrated to the inductive amount of 100 μ M isatoribines.
Table 12: the sheltering of resiquimod prodrug
Available PBMC experimental study resiquimod prodrug shelter character.Result's (table 12) of PBMC test has shown contact initial concentration 1 or the parent compound of 100 μ M and the amount of the INFa that prodrug discharged after 24 hours thereof.Under 100 μ M, in the identical blood sample, under identical time of contact, the amount of the INFa that discharges is calibrated to the inductive amount of 100 μ M isatoribines.
Table 13: the sheltering of bropirimine prodrug
| Parent molecule and prodrug thereof | Compound number | With respect to the amount of the inductive INFa of 100 μ M isatoribines, % |
| Parent molecule: bropirimine prodrug: deoxidation prodrug: ethyoxyl prodrug: urethane ester prodrugs: amyl carbamate | 35 48 37 36 49 | 22 0 0 0 0 |
Available PBMC experimental study bropirimine prodrug shelter character.Result's (table 13) of PBMC test has shown the amount of parent compound and the INFa that prodrug discharged after 24 hours thereof of contact initial concentration 100 μ M.Under 100 μ M, in the identical blood sample, under identical time of contact, the amount of the INFa that discharges is calibrated to the inductive amount of 100 μ M isatoribines.
Table 14: the sheltering of adenine prodrug
| Parent molecule and prodrug thereof | Compound number | With respect to the amount of the inductive INFa of 100 μ M isatoribines, % |
| Parent molecule prodrug: methoxyl group prodrug: ethyoxyl prodrug: deoxidation prodrug: ethyl carbonate prodrug: amyl carbonate | 29 65 64 62 51 54 | 1280.1μM 0100μM 010μM 00.1μM 1832μM 1510μM |
Available PBMC experimental study adenine prodrug shelter character.Result's (table 14) of PBMC test has shown the amount of the INFa that the parent compound that contacts different initial concentrations and prodrug thereof discharged after 24 hours.Under 100 μ M, in the identical blood sample, under identical time of contact, the amount of the INFa that discharges is calibrated to the inductive amount of 100 μ M isatoribines.
Also can be converted into reactive precursor TLR7 part and carry out in-vitro evaluation TLR7 part prodrug.This can measure by prodrug is hatched in blood, blood plasma or hepatocellular cell culture.At selected time sampling, with the residual quantity of mensuration prodrug and the generation of TLR7 part.By using analytical tool known in the art such as LC-MS easily to measure.The TLR7 part prodrug that mensuration is sheltered is converted into the degree of parent TLR7 part, is used for decryption, and wherein in described PBMC test, incubation time is short more then to be sheltered obviously, and incubation time length is then sheltered disappearance.Measure the conversion rate of prodrug to the TLR7 part, significantly increase to guarantee the cytokine result, this is owing to the contact prodrug, rather than because contact prodrug under experimental condition transforms the TLR7 part of generation fast.
6.5TLR7 the biological test of part prodrug
Oral availability increases and side effect reduces
Oral availability
Estimate the bioavailability that TLR7 part prodrug improves by in vivo test.In this experiment, with candidate's prodrug oral administration mice, rat, monkey and/or Canis familiaris L., at interval blood sampling seclected time.Prodrug in the analysis blood sample and required TLR7 part.Other blood or liver sample can be used for analyzing the interferon of TLR7 approach function activation in the expression body and the existence of other cytokine.Measure based on mole, required candidate compound will show blood contact prodrug and show that the blood contact amount of required TLR7 part is the 10%-99% of institute's application dose.
Representative example is the result who obtains with TLR7 part prodrug valine-isatoribine (24), the parent TLR7 part isatoribine (21) that produces significant quantity in the blood of mice and Canis familiaris L. as described below.Referring to, Application No. 10/305,061 (including this paper in as a reference).
The concentration of interferon-ALPHA in the mice (Mu-IFN-α)
Normal mouse provides useful system, is used to estimate the improvement degree of material of the present invention to 21 (isatoribine) oral delivery.Not only can measure the plasma concentration of isatoribine behind the oral described prodrug, and the research of carrying out in the mice of immunology widely provides and has been applicable to the reagent of measuring interferon-ALPHA (a kind of reflect a kind of cells of interest factor in the required biologic activity of isatoribine) level.
Use the Mus system in a series of experiments, show 24, promptly 21 (valine-isatoribines), 5 '-L-valine ester can be induced and be substantially exceeded the interferon response that gives isatoribine itself.
Table 15 has write down orally give concentration 50 mg/kg, is mixed with the isatoribine twice of bicarbonate form, the result of the test of Mus interferon-ALPHA in mice plasma.The result shows detection less than interferon, even after 4 hours repeat administration also detect less than.
Table 15: the isatoribine of interval twice oral 50 mg/kg dosage after 4 hours, the plasma concentration (pg/ml) of interferon-ALPHA in the mice (Mu-IFN-α)
BQL
n-be lower than the metering upper limit＜n pg/mL
Table 16 has write down and has at first given bicarbonate, and after 4 hours orally give concentration 50 mg/kg, be mixed with the isatoribine of bicarbonate form, the result of the test of Mus interferon-ALPHA in mice plasma.There is interferon in the blood plasma of four mices, comprises two mices that give the sodium bicarbonate carrier.The all values of record is all lower in this experiment, and all three mices of the interferon level of record and each time point record are inconsistent, and pointing out these information is the errors that cause owing near the analysis lower limit.
Table 16: the carrier dosage and the isatoribine of one time 50 mg/kg dosage after 4 hours, the plasma concentration (pg/ml) of interferon-ALPHA in the mice (Mu-IFN-α)
BQL
n-be lower than the metering upper limit＜n pg/mL.
NR-detect less than
Table 17 has write down orally give valine-isatoribine, is dissolved in the bicarbonate, is equivalent to the isatoribine of 50 mg/kg, the result of the test of Mus interferon-ALPHA in mice plasma based on the dosage of mole.Clearly, can easily detect interferon in 1.0 hours, 1.5 hours and 2.0 hours after the administration.Put all mices and all detect interferon preset time, and prompting gives the reliability that acts on behind valine-isatoribine.Therefore, single gives valine-isatoribine and is better than single dose or multiple dose isatoribine.
Table 17: after single gives the valine-isatoribine of 73.0 mg/kg dosage, the plasma concentration (pg/ml) of interferon-ALPHA in the mice (Mu-IFN-α)
BQL-is lower than metering limit＜12.5pg/mL
BQL
n-be lower than the metering upper limit＜n pg/mL
NR-detect less than
Also can be from the angle consideration table 15,16 and 17 of the incidence rate of the interferon level that can measure institute's column data.In the test of isatoribine, have only in 114 mices in 4 the blood plasma and can measure interferon, and give valine-isatoribine, have in 30 mices in 10 the blood plasma and can measure interferon.Therefore, prodrug can increase to 30% from 4% with the mice ratio that shows the interferon response, and average and peak response value all increase twice (100%).
In other embodiments, intravenous route gives isatoribine, measure the blood plasma level of isatoribine and interferon-ALPHA in the mice, the level of isatoribine and interferon-ALPHA behind these blood plasma levels and the orally give valine-isatoribine is compared.These data are summarised among Fig. 1.As shown in the figure, oral valine-isatoribine (" val-isator ") (50 mg/kg isatoribine molar equivalent) the level of inductive interferon-ALPHA be similar to the level that vein gives 25 mg/kg isatoribines (" isator ").Therefore, the isatoribine that provides of oral valine-isatoribine and interferon level approximately are that vein gives isatoribine originally viewed after one's death 50%.
The Beagle Canis familiaris L.
After having studied orally give beagle Canis familiaris L., the effect of prodrug (valine-isatoribine, 24) isatoribines (21) whole body level.In sodium bicarbonate solution, prepare isatoribine.Valine-isatoribine and isatoribine are prepared into following preparation, select these preparations to guarantee dissolubility:
Preparation 1: the sodium bicarbonate solution of isatoribine, 1 and 4 mg/ml.
Preparation 2: the phosphate buffered saline (PBS) of valine-isatoribine, 1.62 and 6.48 mg/ml in mole, are equivalent to the isatoribine of 1 and 4 mg/ml.
When beginning research use heavy 15-27kg, about 1-2 the year big four male and four female adult beagle Canis familiaris L.s.Animal is divided into 2 groups, and every group 2 male 2 female.Raised by force in the 1st day and the 8th day and give substances, have 7 days removing phase between giving for twice.After each administration, the blood sample (2 milliliters) of predose, 15 minutes, 30 minutes, 1,2,3,4,6,8 and 10 hour following every animal is collected in the lithium calparine pipe.With plasma freezing at-70 ℃ up to analysis.Isatoribine in the HPLC-MS/MS method analysed for plasma.
Pharmacokinetic parameters from the isatoribine of isatoribine or valine-isatoribine in every Canis familiaris L. is summarised in table 18 and 19.The ratio of the crucial pharmacokinetic parameters of the total amount that area (AUC) under the Cmax (Cmax) of the bicarbonate solution of prodrug and 50 mg/kg dosage and the time-concentration curve is calculated is summarised in the table 20.For prodrug 24, the Cmax ratio be 2.98 ± 0.695 and the AUC ratio be 2.38 ± 0.485.Results suggest is under 50 mg/kg dosage, and prodrug valine-isatoribine has significantly higher Cmax and bigger bioavailability than the bicarbonate solution of isatoribine.
The Cmax of the bicarbonate solution of prodrug and 10 mg/kg dosage and AUC ratio are summarised in the table 21.For prodrug, the Cmax ratio is 2.24 ± 0.249, and the AUC ratio is 1.82 ± 0.529.These results suggest, under 10 mg/kg dosage, prodrug valine-isatoribine has higher Cmax and bigger bioavailability than the bicarbonate solution of isatoribine.
Therefore, under 10 and 50 mg/kg dosage, compare with isatoribine itself, the Cmax of isatoribine doubles at least behind orally give prodrug valine-isatoribine, and the whole body level of isatoribine improves about 2 times.
The pharmacokinetic parameters of isatoribine in the Canis familiaris L. under the table 18:50 mg/kg dosage
The pharmacokinetic parameters of isatoribine in the Canis familiaris L. under the table 19:10 mg/kg dosage
The ratio of the pharmacokinetic parameters of isatoribine in the Canis familiaris L. under the table 20:50 mg/kg dosage
The ratio of isatoribine pharmacokinetic parameters in the Canis familiaris L. under the table 21:10 mg/kg dosage
Preferred prodrug valine-isatoribine is based on following reason.The first, prepare this prodrug easily so that the activating agent of height ratio to be provided.This feasible Caplet that can prepare given dose, favourable for oral drugs.The second, under test dose, the isatoribine plasma concentration that valine-isatoribine provided behind the oral administration drops in the scope of required biological effect well, and is not like this for isatoribine itself.
Stump-tailed macaque
Use the only male or female stump-tailed macaque of 2-4 in this animal experiment.Test compound is formulated in is suitable in animal is oral or vein gives the vehicle.The vehicle of using is aqueous buffer solution or the solution that contains polyoxyethylene castor oil.Every animal is raised by force or the intravenous injection administration by oral.Collect blood sample (about 0.5 milliliter) at preset time point (usually, after predose, the administration 15 minutes, 30 minutes, 45 minutes, 1,1.5,2,2.5,3,4,8 and 24 hour), blood sample is placed the test tube that contains the EDTA disodium.Place sample on the ice bath after the sampling and separated plasma as quickly as possible.Plasma sample is dispensed in the single developmental tube, and-20 ℃ of freezing preservations are up to be transported to the promoter on dry ice approximately.After the administration about 4 hours, administration animal food and water.
Adopt known LCMS/MS quantitative technique, by triple quadrupole device such as Sciex API3000, the sampling of analysed for plasma sample and parent compound.Use orally give parent compound itself or its prodrug of orally give to send the quantitative result of parent compound, calculate from the area under curve (AUC) of time zero to 24 hour and be worth (PO AUC0-24h).The comparison parent compound is sent into the AUC value of the parent compound of systemic circulation and the AUC value of prodrug, calculates the oral relatively availability of prodrug.The results are shown in Table 22-26.If vein gives parent compound originally its AUC data (AUCIV) of sending parent compound are known after one's death,, calculate absolute oral administration biaavailability by with the PO AUC (0-24h) of prodrug IV AUC (0-24h) divided by parent molecule.
Table 22: the oral administration biaavailability of isatoribine and prodrug thereof in the monkey
| Tuo Libin prodrug: amino-acid ester prodrug: deoxidation prodrug: 6-ethyoxyl prodrug: 6-methoxyl group prodrug: aminal prodrug: aminal prodrug: Dloxole ketone | 24 93 77 79 84 82 85 | 7-9 * 80 28 21 14 4 17 |
*The meansigma methods of repeatedly testing under the various dose
Table 23: the oral administration biaavailability of loxoribine and prodrug thereof in the monkey
| Loxoribine prodrug: 6-ethyoxyl prodrug: deoxidation | 45 43 | 9 13 |
Table 24: the oral administration biaavailability of imiquimod and prodrug thereof in the monkey
| Parent molecule and prodrug thereof | Compound number | Oral administration biaavailability in the stump-tailed macaque, % |
| Parent molecule: imiquimod prodrug: amyl carbamate prodrug: urethanes | 31 34 50 | 100 AUC(0-24h)＝9.0 555 AUC(0-24h)＝50 234 AUC(0-24h)＝21.1 |
Table 25: the oral administration biaavailability of bropirimine and prodrug thereof in the monkey
*Oral administration biaavailability surpasses 100% may be relevant with sex difference, because parent compound is research and prodrug is to study in female monkey in male monkey.
Table 26: the oral administration biaavailability of adenine prodrug in the monkey
The reduction of GI irritation
TLR7 part prodrug of the present invention shows that also unexpected toxic action reduces greatly, and especially the GI zest reduces.
Be arranged with many immuning tissues (for example, Peyer ' s speckle etc.) on the gastrointestinal tract (" GI ").Along with reagent by the lymphoid tissue in the gastrointestinal tract, TLR7 part prodrug can be sheltered active structure, thereby makes the minimum reduction of adenoid activations GI zest.
Explanations such as Robins, then active disappearance of 5 '-hydroxyl of removing the isatoribine nucleoside.Referring to Robins etc., Adv.Enzyme Regul., 29,97-121 (1989).Be not bound by any particular theory, suppose with the ester substituent group and seal this hydroxyl group sites elimination activity but allow transhipment in the systemic circulation similarly, will remove L-valine ester in the systemic circulation and expose isatoribine.
Find that above-mentioned supposition is proved.In the beagle Canis familiaris L., carry out the toxicity research that vein gives isatoribine and orally give isatoribine and valine-isatoribine.The research that the toxicity result of orally give isatoribine carries out from ICN/Nucleic Acid Research Institute.
The oral toxicity of comparison 21 and 24 and 21 venous toxicity in Canis familiaris L..Observe, compared with oral 21, oral 24 toxicity is more near vein 21.Specifically, oral 3 dose-limiting toxicity is similar to vein 21 in nature, gives 21 backs and takes place during observed level when blood concentration is similar to vein.On the contrary, oral 21 have different restricted toxicity (injury of gastrointestinal tract), and this toxicity can be observed under the toxicity dose that is lower than vein 21 or oral 24.And, under being lower than oral 24 the dosage that causes vomitting, can be observed vomiting in oral 21 Canis familiaris L.s of handling.Referring to table 27.Other system of also known evaluation vomiting, for example ferret gives chemical compound with more oral and vein.For example, referring to Strominger N. etc., Brain Res.Bull, 5,445-451 (2001).
Every animal gives the chemical compound of solution form, by raising by force or by venoclysis.Estimate multiple parameter in routine and the toxicity research.In the research that improves higher potential isatoribine level, the LC/MS method is estimated saying of isatoribine and can be read.To observed GI phenomenon scoring, list in the table 27.
Table 27: in toxicity research, give of the influence of isatoribine (21) or valine-isatoribine (24) back, with whole body level (AUC) score of isatoribine to GI toleration in the Canis familiaris L.
For the orally give isatoribine, main discovery is relevant with the GI toleration, as measuring the GI zest.Clinical manifestation shown in the table 27 is vomiting and/or has loose bowels.These clinical manifestations are more common in 10 mg/kg, and hemafecia also appears in next animal of this dosage.The rough histopathology evaluation of gastrointestinal tract finds that by many erythema that are dispersed in, microscopic evaluation finds that cell is congested and hemorrhage, the existence of prompting local inflammation on 4 intestinal mucosa in following 8 Canis familiaris L.s of 10 mg/kg.The influence of GI determines that NOAEL is 5 mg/kg.
Vein gives isatoribine can be caused vomiting common in the Canis familiaris L. and/or have loose bowels; Under high a lot of application dose this effect is appearring just than orally give isatoribine.Injury of gastrointestinal tract is not all found in anatomic tissue or histopathology evaluation.GI toxicity can not respond NOAEL, and according to other discovery, NOAEL has been defined as 12.5 mg/kg.
Orally give valine-isatoribine demonstration is similar to the toxic characteristic that vein gives isatoribine.Under higher application dose, observe the vomiting and have loose bowels.Do not find the GI damage, though this is the focus of this research evaluation.Give isatoribine for vein, find to determine NOAEL according to other.The whole body level of viewed toxicity and isatoribine is that this institute is concerned about; The thresholding of the isatoribine AUC that observes vomiting and have loose bowels is similar to vein and gives isatoribine and orally give valine-isatoribine (table 27).
Notes of Key Data orally give valine-isatoribine in the table 27 provides the toxic characteristic above the improvement of orally give isatoribine, and consistent with following hypothesis: shelter by can realize the active chemistry of isatoribine at 5 ' of nucleoside-position hydroxy chemical replacement ester.As show shown in the 9-14, can use multiple substituent group chemistry to shelter any TLR7 part.Be eliminated after making above-mentioned substituent group enter in the body, provide the whole body level of compound activity of usefulness and the GI toxicity restriction that do not have the gastrointestinal tract anatomical structure to cause.As show shown in the 22-25, can design the chemical substituent group that can be eliminated after giving on the TLR7 part of sheltering.Like this, the part prodrug of sheltering can be used for any TLR7 part.Compare with only giving the chemical compound that parent " do not shelter ", this makes in the giving dosage and can be much higher than acceptable dosage of mole, produces the stronger effectiveness and the side effect of reduction.
6.6 oral group
Table 28 has shown a collection of preparation and the single dosage unit preparation that contains 100 milligrams of valine-isatoribines
Table 28:100 milligram tablet
| Material | Percentage by weight | Amount (mg/ sheet) | Amount (kg/ criticizes) |
| Valine-isatoribine | 40％ | 100.00 | 20.00 |
| Microcrystalline Cellulose, NF | 53.5％ | 133.75 | 26.75 |
| Pluronic F-68 surfactant | 4.0％ | 10.00 | 2.00 |
| Cross-linking sodium carboxymethyl cellulose A type, NF | 2.0％ | 5.00 | 1.00 |
| Magnesium stearate, NF | 0.5％ | 1.25 | 0.25 |
| Total amount | 100.0％ | 250.00mg | 50.00kg |
Make microcrystalline Cellulose, cross-linking sodium carboxymethyl cellulose and valine-isatoribine component by #30 mesh sieve (about 430 μ-655 μ).Make Pluronic F-68_ (JRH Biosciences, Inc., Lenexa, KS manufacturing) surfactant by #20 mesh sieve (about 457 μ-1041 μ).Pluronic F-68_ surfactant and the 0.5kg cross-linking sodium carboxymethyl cellulose 16qt. bivalve tumble mixer (twin shelltumble blender) of packing into was mixed about 5 minutes.Any mixture being transferred in 3 cubic feet the bivalve tumble mixer adds microcrystalline Cellulose and mixed about 5 minutes.Added the Thalidomide remix 25 minutes.By the drum-type press, the exit of drum-type press has hammer mill, and then gets back in the tumble mixer with this premix.Remaining cross-linking sodium carboxymethyl cellulose and magnesium stearate are joined in the tumble mixer stir about 3 minutes.Final mixture is made compacting at rotary tablet machine, every 250 (200,000 every batch).
6.7 mucosa compositions
Isatoribine and the single fluoromethane of 12.6kg trichlorine are mixed the preparation concentrate in having the sealing rustless steel container of high-shear mixer.Mixed about 20 minutes.Then, with a certain amount of propellants in concentrate and the batch product jar, temperature is controlled at 21-27 ℃ in sealed container, and pressure is controlled at 2.8-4.0, prepares a large amount of suspensions.Design has the 17ml aerosol container of metering valve, so that can suck the present composition 100 times.Each container has following composition:
Valine-isatoribine 0.0120g
Isceon 1.6960g
Dichlorodifluoromethane 3.7028g
Dichlorotetra-fluoroethane 1.5766g
Total amount 7.0000g
6.8 vein compositions
With suitable liquid medium, as water for injection (WFI) or 5% glucose solution, rebuild The compounds of this invention, the preparation intravenous formulations.The liquid medium of an amount of The compounds of this invention and proper volume is rebuild, obtained the desired concn of intravenous formulations.The desired concn of intravenous formulations can offer the patient who needs the intravenous pharmacy preparation, preferred mammal, and more preferably the people treats the The compounds of this invention of effective dose, and keeps the treatment preferred levels of The compounds of this invention in the patient.The treatment effective dose will depend on that intravenous formulations sends into patient's the speed and the concentration of intravenous formulations.
For example, a bottle is contained compositions (for example, 50mg The compounds of this invention/bottle) rebuild with 5% glucose solution (glucose solution/bottle of 14ml 5%), common property is given birth to 25mL solution.The solution of rebuilding is mixed in the glucose solution of transfusion bag,, obtain being suitable for that venoclysis gives contains 1mg/ml The compounds of this invention solution quantitatively to 50 milliliters.Liquid medium, the preferred concentration of The compounds of this invention is about the 0.001-3 mg/ml in the transfusion bag, preferably is about 0.75-1mg/ml.
Aforementioned content has been set forth relevant and key character of the present invention.Do not deviate from spirit and scope of the invention, can carry out many changes and variation to the present invention, as those skilled in the art understand.Specific embodiments described herein only is exemplary, and the present invention only is subjected to the restriction of appended claims and the desired full scope of equivalents of these claim.
The content of all lists of references that this paper quotes is included into this paper as a reference, and similarly, specific and each publication mentioned independently or the content of patent or patent application are included into this paper as a reference.
Claims (29)
1.TLR7 the purposes of part in the medicine of preparation treatment infection with hepatitis C virus, wherein, described TLR7 part is selected from following:
2. purposes as claimed in claim 1 is characterized in that described medicine is used for the people.
3. purposes as claimed in claim 1, described medicine also comprises pharmaceutically acceptable excipient, carrier or vehicle.
4. purposes as claimed in claim 1, described medicine also comprises other therapeutic agent.
5. purposes as claimed in claim 4 is characterized in that, described other therapeutic agent is an antiviral agent.
6. purposes as claimed in claim 1 is characterized in that, the treatment of described TLR7 part or prevention effective dose are 0.001-100 mg/kg every day.
7. purposes as claimed in claim 1 is characterized in that, the treatment of described TLR7 part or prevention effective dose are 0.01-50 mg/kg every day.
8. purposes as claimed in claim 1 is characterized in that, the treatment of described TLR7 part or prevention effective dose are 0.1-20 mg/kg every day.
9. purposes as claimed in claim 1 is characterized in that parenteral route gives described medicine.
10. purposes as claimed in claim 1 is characterized in that intravenous gives described medicine.
11. purposes as claimed in claim 1 is characterized in that, the described medicine of orally give.
12. purposes as claimed in claim 1 is characterized in that, through mucous membrane gives described medicine.
13. treatment or pharmaceutically the TLR7 part prodrug of sheltering of effective dose or the purposes of its pharmaceutically acceptable salt in the medicine of preparation treatment infection with hepatitis C virus, the described TLR7 part prodrug of sheltering is selected from:
14. purposes as claimed in claim 13 is characterized in that, by effective plasma level concentration in the body of the described medicine acquisition of orally give TLR7 part, its 10%-500% for contacting in effective body that only orally give TLR7 part obtains.
15. purposes as claimed in claim 13 is characterized in that, by effective plasma level concentration in the body of the described medicine acquisition of orally give TLR7 part, its 50%-200% for contacting in effective body that only orally give TLR7 part obtains.
16. purposes as claimed in claim 13 is characterized in that, obtains the treatment effective plasma level concentration of corresponding TLR7 part and does not produce GI irritation by the described medicine of orally give.
17. purposes as claimed in claim 13 is characterized in that, compares with the side effect of only orally give TLR7 part generation, the described medicine of orally give reduces the adverse side effect among the patient.
18. purposes as claimed in claim 13 is characterized in that, compares 50% adverse side effect among the described medicine reduction of the orally give patient with the side effect of only orally give TLR7 part generation.
19. purposes as claimed in claim 18 is characterized in that, described side effect is a GI irritation.
20. purposes as claimed in claim 18 is characterized in that, described stimulation is hemorrhage.
21. purposes as claimed in claim 18 is characterized in that, described stimulation is damage.
22. purposes as claimed in claim 18 is characterized in that, described stimulation is vomiting.
23. purposes as claimed in claim 13 is characterized in that, described medicine is used for the people.
24. purposes as claimed in claim 13, described medicine also comprises pharmaceutically acceptable excipient, carrier and vehicle.
25. purposes as claimed in claim 13, described medicine also comprises other therapeutic agent.
26. purposes as claimed in claim 25 is characterized in that, described other therapeutic agent is an antiviral agent.
27. purposes as claimed in claim 13 is characterized in that, described treatment or prevention effective dose are 0.001-100 mg/kg every day.
28. purposes as claimed in claim 13 is characterized in that, described treatment or prevention effective dose are 0.1-25 mg/kg every day.
29. purposes as claimed in claim 13 is characterized in that, described treatment or prevention effective dose are 0.1-20 mg/kg every day.
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| US50033903P | 2003-09-05 | 2003-09-05 | |
| US60/500,339 | 2003-09-05 | ||
| US51899703P | 2003-11-10 | 2003-11-10 | |
| US51899603P | 2003-11-10 | 2003-11-10 | |
| US60/518,996 | 2003-11-10 | ||
| US60/518,997 | 2003-11-10 | ||
| PCT/US2004/028236 WO2005025583A2 (en) | 2003-09-05 | 2004-09-01 | Tlr7 ligands for the treatment of hepatitis c |
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| KR20120030436A (en) * | 2009-05-21 | 2012-03-28 | 아스트라제네카 아베 | Novel pyrimidine derivatives and their use in the treatment of cancer and further diseases |
| CN103254304A (en) * | 2012-05-30 | 2013-08-21 | 深圳大学 | Immune stimulant couplet, preparation method thereof and antineoplastic application of the immune stimulant couplet |
| WO2019196918A1 (en) * | 2018-04-13 | 2019-10-17 | 罗欣药业(上海)有限公司 | Five-membered heterocyclo-pyrimidine compound, pharmaceutical composition and use thereof |
| WO2020073942A1 (en) * | 2018-10-11 | 2020-04-16 | 江苏恒瑞医药股份有限公司 | Tlr7 agonist prodrug, preparation method therefor and medical use thereof |
| CN111665309A (en) * | 2020-07-21 | 2020-09-15 | 南京品生医疗科技有限公司 | Kit for detecting nucleoside antiviral drugs in serum and application thereof |
| CN111830162A (en) * | 2020-07-21 | 2020-10-27 | 南京品生医学检验实验室有限公司 | Method for detecting concentration of nucleoside antiviral drug in serum |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4880784A (en) * | 1987-12-21 | 1989-11-14 | Brigham Young University | Antiviral methods utilizing ribofuranosylthiazolo[4,5-d]pyrimdine derivatives |
| US5041426A (en) * | 1987-12-21 | 1991-08-20 | Brigham Young University | Immune system enhancing 3-β-d-ribofuranosylthiazolo[4,5-d]pyridimine nucleosides and nucleotides |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4880784A (en) * | 1987-12-21 | 1989-11-14 | Brigham Young University | Antiviral methods utilizing ribofuranosylthiazolo[4,5-d]pyrimdine derivatives |
| US5041426A (en) * | 1987-12-21 | 1991-08-20 | Brigham Young University | Immune system enhancing 3-β-d-ribofuranosylthiazolo[4,5-d]pyridimine nucleosides and nucleotides |
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