CN1753666A - Halogenated triptolide derivatives as immunomodulators and anticancer agents - Google Patents

Halogenated triptolide derivatives as immunomodulators and anticancer agents Download PDF

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CN1753666A
CN1753666A CNA200480005153XA CN200480005153A CN1753666A CN 1753666 A CN1753666 A CN 1753666A CN A200480005153X A CNA200480005153X A CN A200480005153XA CN 200480005153 A CN200480005153 A CN 200480005153A CN 1753666 A CN1753666 A CN 1753666A
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choh
radix tripterygii
tripterygii wilfordii
fluorine
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CN100408583C (en
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D·戴
J·H·马瑟
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Pharmagenesis Inc
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Abstract

Compounds having the structure (I): are useful for inducing cell death (apoptosis) and in immunosuppression. In structure I, CR<1>R<2> is selected from CHOH, C=O, CHF, CF2 and C(CF3)OH; CR<6> and CR<13> are selected from CH, COH and CF; CR<7>R<8>, CR<9>R<10> and CR<11>R<12> are selected from CH2, CHOH, C=O, CHF and CF2; and CR<3>R<4>R<5> is selected from CH3, CH2OH, C=O, COOH, CH2F, CHF2 and CF3; such that: at least one of R<1>-R<13> comprises fluorine; no more than two of CR<3>R<4>R<5>, CR<6>, CR<7>R<8>, CR<9>R<10>, CR<11>R<12>, and CR<13> comprises fluorine or oxygen; and, when CR<1>R<2> is CHOH, CR<3>R<4>R<5> is not CH2F.

Description

Halogenated triptolide derivatives as immunomodulator and anticarcinogen
Invention field
The present invention relates to chemical compound as immunosuppressant, antiinflammatory and anticarcinogen.
List of references
Gleichmann，E.et?al.，Immunol?Taday?5：324(1984).
He，Q.et?al.，BeijingDaXueXueBao35：252-5(Jun?2003).
Jung，M.J.et?al.，US?5,972,998(1999).
Jung，M.J.et?al.，US?6,004,999(1999).
Korngold，R.and?Sprent，J.，J.Exp.Med.148：1687(1978).
Krishna，G.et?al.，Am.J.of?Pathology?158(3)：997-1004(Mar2001).
Kupchan，S.M.et?al.，J.Am.Chem.Soc.94：7194(1972).
Kupcha?n，S.M.et?al.，U.S.Patent?No.4,005,108(1977).
Lipsky?et?al.，U.S.Patent?No.5,294,443(1994).
Ma?et?al.，J.Chin.Pharm.Sci.1：12(1992).
Murase，N.et?al.，Transplantation?55：701(1993).
Ono?and?Lindsey，J.Thor.Cardiovasc.Surg.57(2)：225-29(1969).
Panchagnula，R.and?Thomas，N.S.Intl?J?of?Pharmaceutics?201(2)：131-150(2000).
Pu，L.et?al.，zhongguo?YaoliXuebao?11：76(1990).
Qi，Y.et?al.，U.S.Patent?5,663,335(1997).
Qi，Y.et?al.，U.S.Patent?5,962,516(1999).
Qian，S.et?al.，U.S.Patent?No.US?5,430,054(1995).
Wang，J.and?Morris，R.E.，Transplantation?Proc.23：699(1991).
Wang，X.et?al.，PCT?Pubn.No.WO?2002/17931(2002).
Wiedmann，T.et?al.，U.S.Patent?No.5,843,452(1998).
Zhou，Y.X.et?al.，Ai?Zheng?21(10)：1108-8(Oct?2002).
Background of invention
Immunosuppressant is widely used for treating autoimmune disease and is used for the treatment of or prevents transplant rejection, comprises treatment graft versus host disease (GVHD).Common immunosuppressant comprises azathioprine, corticosteroid, cyclophosphamide, methotrexate, Ismipur, vincristine and Ciclosporin A.Generally speaking, it is in full force and effect not having a kind of in these medicines, and great majority are subjected to the restriction of serious toxicity.For example, widely used Ciclosporin A has tangible toxicity to kidney.In addition, effectively treating required dosage may make the patient that the sensitivity that various opportunistic invaders infect is increased.
Identified from Chinese medicinal plant Radix Tripterygii Wilfordii (Tripterygium wilfordii) (TW) deutero-chemical compound lot have immunosuppressive activity, for example, can be used for treating autoimmune disease and be used for the treatment of or prevent transplant rejection, comprise treatment graft versus host disease (GVHD).Report that also pure and mild some derivant of Triptolide and prodrug thereof demonstrate active anticancer.For example referring to Kupchan et al., 1972,1977, and total United States Patent (USP) 6,620,843 (in JIUYUE, 2003), they are incorporated herein by reference.
Generally speaking, find that the biological activity of triptolide alcohol derivative itself is less than natural Radix Tripterygii Wilfordii lactone alcohol.Yet with respect to natural Radix Tripterygii Wilfordii lactone alcohol, these chemical compounds are often showing well aspect pharmacokinetics or bio distribution, and this is because they cause in the difference aspect lipid or the water solublity and/or as the activity of prodrug.For example referring to, Jung etal., United States Patent (USP) 5,972,998 and 6,004,999, Kupchan et al., United States Patent (USP) 4,005,108, Lipsky et al., United States Patent (USP) 5,294,443 and Qian et al., United States Patent (USP) 5,430,054, and total United States Patent (USP) 6,150,539 (the Radix Tripterygii Wilfordii lactone alcohol prodrug has very high water solublity), 5,962,516 (immunosuppressive compounds and methods), 5,843,452 (immunotherapy compositions and methods), 5,759,550 (being used to suppress the method that xenograft repels), 5,663,335 (immunosuppressive compounds and methods) and 5,648,376 (immunosuppressant diterpene compounds), and the list of references that wherein is cited.
Summary of the invention
On the one hand, the invention provides the chemical compound that can be used for immunosuppressant, antiinflammatory and anticancer therapy.These chemical compounds are triptolide alcohol derivatives of representing with structural formula I:
Figure A2004800051530000081
Wherein:
CR 1R 2Be selected from CHOH, C=O, CHF, CF 2Or C (CF 3) OH;
CR 6And CR 13Be selected from CH, COH or CF;
CR 7R 8, CR 9R 10And CR 11R 12Be selected from CH 2, CHOH, C=O, CHF or CF 2
CR 3R 4R 5Be selected from CH 3, CH 2OH, C=O, COOH, CH 2F, CHF 2Or CF 3
Wherein: at R 1-R 13In have at least one to comprise fluorine; At CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10, CR 11R 12And CR 13In have two and preferably have one at the most and comprise fluorine or oxygen at the most; In addition, work as CR 1R 2When being CHOH, CR 3R 4R 5Not CH 2F.
Preferably, about CR 7R 8Spatial chemistry be: work as CR 7R 8When being CHOH, have the beta-hydroxy configuration, work as CR 7R 8When being CHF, has α-fluorine configuration.Similarly, about CR 9R 10Spatial chemistry be preferably: work as CR 9R 10When being CHOH, have the beta-hydroxy configuration, and work as CR 9R 10When being CHF, has α-fluorine configuration.
In the preferred embodiment of structural formula I, CR 1R 2Be CHF, have α-fluorine configuration.
Preferred embodiment also comprises following those chemical compounds, wherein is selected from CR 1R 2, CR 3R 4R 5, CR 6, CR 7R 8, CRR 9R 10Or CR 11R 12Carbon center in just have one to comprise fluorine.More preferably, at CR 1R 2, CR 6, CR 7R 8, CR 9R 10And CR 11R 12In just have one to comprise fluorine.
In optional embodiment, has only CR 1R 2Comprise fluorine.Correspondingly, CR in these embodiments 1R 2Be selected from CF 2, CHF or C (CF 3) OH.About CR 1R 2Spatial chemistry preferably: work as CR 1R 2Be C (CF 3) during OH, have the beta-hydroxy configuration, work as CR 1R 2When being CHF, has α-fluorine configuration.
In other optional embodiment of structural formula I, CR 9R 10Or CR 3R 4R 5Comprise fluorine, and CR 1R 2Comprise oxygen; Preferred CR 1R 2Be C=O, perhaps be more preferably CHOH (beta-hydroxy).In these embodiments, for example, CR 9R 10Be selected from CF 2Or CHF (preferred α-fluorine), perhaps CR 3R 4R 5Be selected from CHF 2Or CF 3
In the embodiment that structural formula I further selects, CR 7R 8Or CR 11R 12Comprise fluorine, and CR 1R 2Comprise oxygen; Preferred CR 1R 2Be C=O, perhaps be more preferably CHOH (beta-hydroxy).In these embodiments, for example, CR 7R 8Be selected from CF 2Or CHF (preferred α-fluorine), perhaps CR 11R 12Be selected from CF 2Or CHF.
In others, the invention provides the method that is used to influence immunosuppressant method and inducing cell inner cell apoptosis, thereby can be used for resisting rheumatoid treatment, particularly anticancer therapy effectively.Described method comprise with effective dose, as herein described have compound in structural formula I be administered into respectively this treatment of needs the patient or with described cells contacting.In addition, but the present invention includes and have compound in structural formula I and can influence the purposes of immunosuppressant or inducing cell inner cell apoptosis medicine in preparation.Typically, this chemical compound provides pharmaceutical acceptable carrier.The particular of described method and purposes can adopt any particular about structural formula I described herein.
When reading in conjunction with the accompanying drawings following detailed description of the present invention, above and other objects of the present invention and feature will become more apparent.
Description of drawings
Fig. 1 demonstrates with Radix Tripterygii Wilfordii lactone alcohol (being appointed as PG490) and compares, and The compounds of this invention 14-deoxidation-1-α-fluoro-Radix Tripterygii Wilfordii lactone alcohol (being appointed as PG763) is in the intracellular cytotoxin influence of Jurkat (embodiment 2);
Fig. 2 demonstrates with Radix Tripterygii Wilfordii lactone alcohol and compares, The compounds of this invention (PG763) in the Jurkat cell to the inhibition (embodiment 4) of IL-2 product;
Fig. 3 A-B demonstrates with Radix Tripterygii Wilfordii lactone alcohol and compares, and (is epitriptolide by control compounds 14-Alpha-hydroxy Radix Tripterygii Wilfordii lactone alcohol; Be appointed as PG524) and 14-β-(methyl mercapto) methyl Radix Tripterygii Wilfordii lactone alcohol (being appointed as PG691) inductive apoptosis of dose dependent (embodiment 3) in the Jurkat cell; And
Fig. 4 A-B demonstrates with Radix Tripterygii Wilfordii lactone alcohol and compares, by the inhibition (embodiment 4) to the IL-2 product in the Jurkat cell of control compounds 14-Alpha-hydroxy Radix Tripterygii Wilfordii lactone alcohol (being epitriptolide) and 14-β-(methyl mercapto) methyl Radix Tripterygii Wilfordii lactone alcohol.
Detailed Description Of The Invention
I. triptolide alcohol derivative
In order to realize purpose disclosed by the invention, use following numbering plan that the pure and mild triptolide alcohol derivative of Triptolide is numbered:
Figure A2004800051530000101
As further described herein, The compounds of this invention is for passing through Radix Tripterygii Wilfordii lactone alcohol C1, C2, C5, C14, C15, C16 and/or C19 position, the one or more hydroxyls or the hydrogen atom halogen atom of preferred C2, C14 or C16 position, preferred chlorine or fluorine, and fluorine most preferably replaces and the derivant that obtains.More specifically, The compounds of this invention is represented with following structural formula I:
Figure A2004800051530000102
Wherein:
CR 1R 2Be selected from CHOH, C=O, CHF, CF 2Or C (CF 3) OH;
CR 6And CR 13Be selected from CH, COH or CF;
CR 7R 8, CR 9R 10And CR 11R 12Be selected from CH 2, CHOH, C=O, CHF or CF 2
CR 3R 4R 5Be selected from CH 3, CH 2OH, C=O, COOH, CH 2F, CHF 2Or CF 3
Wherein, at R 1-R 13In have at least one to comprise fluorine; At CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10, CR 11R 12And CR 13In have at the most two and preferably have at the most one comprise fluorine or oxygen and; Work as CR 1R 2When being CHOH, CR 3R 4R 5Not CH 2F.
Preferably, about CR 7R 8And CR 9R 10Spatial chemistry be: CHOH has the beta-hydroxy configuration, and CHF has α-fluorine configuration.
In the preferred embodiment of structural formula I, CR 1R 2Be CHF (α).(as used herein, symbol " CHX (α) " or " CHX (β) " represent that respectively non-hydrogen substituent X has α or beta comfiguration).More preferably, in these embodiments, CR only 1R 2Comprise fluorine.Still more preferably, in these embodiments, CR only 9R 10And/or CR 3R 4R 5Comprise oxygen; For example, CR 9R 10And/or CR 3R 4R 5In one or two, preferred one is that CHOH is (for CR 9R 10, preferred CHOH (β)).Alternatively, in these embodiments, CR 3R 4R 5, CR 6, R 7R 8, CR 9R 10, CR 11R 12And CR 13Do not comprise oxygen.
The preferred embodiment of structural formula I also comprises these chemical compounds, they be selected from CR 1R 2, CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10Or CR 11R 12In carbon center just comprise a fluorine.More preferably, CR 1R 2, CR 7R 8, CR 9R 10And CR 11R 12In just have one to comprise fluorine.Preferably, in these embodiments, CR only 9R 10And/or CR 3R 4R 5Comprise oxygen; CR for example 9R 10And/or CR 3R 4R 5In one or two, preferred one is that CHOH is (for CR 9R 10, preferred CHOH (β)).Alternatively, in these embodiments, CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10, CR 11R 12And CR 13Do not comprise oxygen.
In the select embodiment of structural formula I, CR only 1R 2Comprise fluorine.Correspondingly, CR 1R 2Be selected from CF 2, CHF or C (CF 3) OH.Preferably, CR 1R 2Spatial chemistry be: C (CF 3) OH is C (α-CF 3) β-OH, perhaps CHF is CH (α-F).
In other optional embodiment of structural formula I, CR 9R 10Or CR 3R 4R 5Comprise fluorine and CR 1R 2Comprise oxygen; Preferred CR 1R 2Be C=O, perhaps CHOH (beta-hydroxy) more preferably.In these embodiments, CR 9R 10Be CF 2Or CHF (preferred α-fluorine), CR 3R 4R 5Be preferably CHF 2Or CF 3
In the further selectable embodiment of structural formula I, CR 7R 8Or CR 11R 12Comprise fluorine and CR 1R 2Comprise oxygen; Preferred CR 1R 2Be C=O, perhaps CHOH (beta-hydroxy) more preferably.In these embodiments, CR 7R 8Be CF 2Or CHF (preferred α-fluorine), CR 11R 12Be CF 2Or CHF.
The present invention also comprises the chemical compound similar to compound in structural formula I, wherein, fluorine with different halogen atoms such as chlorine, bromine or iodine particularly chlorine replace; For example, 14-deoxidation-14 α-chlorine Radix Tripterygii Wilfordii lactone alcohol.Other selectable and embodiment preferred of this chemical compound is corresponding to the fluoric compound with structural formula I described above.
A. Preparation
The compounds of this invention can be from Radix Tripterygii Wilfordii lactone alcohol or the preparation of its hydroxylation derivative.The latter comprises Tripdiolide (2-hydroxyl Radix Tripterygii Wilfordii lactone alcohol) or 16-hydroxytriptolide, and they and Radix Tripterygii Wilfordii lactone alcohol can obtain from the root xylem of Chinese medicinal plant Radix Tripterygii Wilfordii (TW) or obtain from other known source together.Found the TW plant in Fujian China province and other province, south China; The TW plant material can obtain in China usually, or buys through commercial channel from the U.S..Be used to obtain that the method for Radix Tripterygii Wilfordii lactone alcohol, Tripdiolide and 16-hydroxytriptolide is known in the art, and at for example Kupchan et al. (1972,1977); Lipsky et al. (1994); Pu et al. (1990); Be described among the Ma et al. (1992).
The 5-hydroxy derivatives of Radix Tripterygii Wilfordii lactone alcohol can make by selenium dioxide oxidation Radix Tripterygii Wilfordii lactone alcohol, as being described in 60/532,702 in total U.S. Provisional Application series number.In brief, in typical preparation method, Radix Tripterygii Wilfordii lactone alcohol and about 2.2 normal selenium dioxide are dissolved in the dioxane, this solution under about 90 ℃, N 2The middle stirring 72 hours.
(Tetrahedron 59 (23): 4209-4213 as L.Ning et al., 2003) described, cultivate Radix Tripterygii Wilfordii lactone alcohol with cunninghamella blakesleana bacterium (Cunninghamella blakesleana) and obtain above-mentioned hydroxy derivatives and 1 beta-hydroxy Radix Tripterygii Wilfordii lactone alcohol, triptolidenol (15-hydroxyl Radix Tripterygii Wilfordii lactone alcohol), the pure and mild 19 beta-hydroxy Radix Tripterygii Wilfordii lactone alcohols of 19 Alpha-hydroxy Triptolides.Adopt standard method to separate the said goods, promptly use the filtering culture fluid of ethyl acetate extraction, concentrate and residue is carried out silica gel chromatography.
Having compound in structural formula I can prepare by Radix Tripterygii Wilfordii lactone alcohol, its hydroxy derivatives or its oxidized derivatives, by with the hydroxyl on C1, C2, C5, C14, C15, C16 and/or the C19 position or contain the oxygen base and fluorization agent reaction, on described carbon, form CHF or CF respectively 2Group.
List of references as Chemistry of Organic Fluorine CompoundsII(editorsM.Hudlicky and A.E.Pavlath; ACS Monograph 187,1995) and Organofluorine Chemistry:Principles and Commercial Applications(editorsR.E.Banks, B.E.Smart and J.C.Tatlow; Plenum Press, 1994) in these fluorization agents are described.For example, the reagent that is suitable for hydroxyl (C-OH) is converted into C-F comprises the HF-amine complex, as HF-pyridine (Olah ' s reagent) or HF-2,4,6-trimethylpyridine, sulfur tetrafluoride (SF 4) and various SF 4Derivant comprises that (diethylamino) sulfur trifluoride (DAST), (dimethylamino) sulfur trifluoride (methyl D AST), morpholine quinoline are for sulfur trifluoride (morph-DAST) and [two (2-methoxy ethyl) amino] sulfur trifluoride (Deoxo-Fluor TM).Hydroxyl can at first be converted into leaving group such as trifluoromethane sulfonic acid root, its subsequently by the nucleophilic fluoride reagents as three (dimethylamino) sulfur (three silicyls) difluoride (TAS-F), 4-normal-butyl ammonium difluoro triphenyl silicate or inorganic salt such as KF, CsF or Bu 4NF replaces.Spatial chemistry can take place and transform in these reactions usually, as described below.
For example, as described in Example 1, have the natural Radix Tripterygii Wilfordii lactone alcohol of beta-hydroxy in the C14 position, can be by being converted into 14-deoxidation-1-α-fluorine Radix Tripterygii Wilfordii lactone alcohol (this paper is appointed as PG763) with the DAST reaction.Alternatively, epitriptolide (14-α-Radix Tripterygii Wilfordii lactone alcohol, this paper are appointed as control compounds PG524) can be used for preparing 14-deoxidation-14-β-fluorine Radix Tripterygii Wilfordii lactone alcohol.
Similarly, Tripdiolide (having beta-hydroxy at C14 and C2 position) can be converted into 14-deoxidation-2 α, 14 α-difluoro Radix Tripterygii Wilfordii lactone alcohol.If use limited amount fluorization agent, this chemical compound can be used as and obtains (vide infra) with the mixture of-α-fluorine Radix Tripterygii Wilfordii lactone alcohol.
Figure A2004800051530000131
16-hydroxytriptolide and stoichiometry or excessive fluorization agent reaction generates 14-deoxidation-14 α, 16-difluoro Radix Tripterygii Wilfordii lactone alcohol, as shown below.
Figure A2004800051530000132
As above cited, sulfur tetrafluoride and derivant thereof also are the most frequently used fluorization agents, are used for oxo group (for example aldehydes or ketones) is converted into gem difluorinated thing.The same reagent that is fit to is 2,2-two fluoro-1,3-methylimidazole quinoline (DFI).
Can be by being that ketone or aldehyde radical form with one or more hydroxyl oxidizes at the oxo group of Radix Tripterygii Wilfordii lactone alcohol C2, C14 and/or C16 position.At list of references such as M.Hudlicky, Oxidations in Organic Chemistry(ACS Monograph Series 186,1990), R.C.Larock, Comprehensive Organic Transformations(the 2nd edition, Wiley, 1999), or J.March, Advanced Organic ChemistryThe method of having described oxidant and being applicable to selective oxidation alcohol in (the 4th edition, Wiley, 1992).Should avoid highly acid or strong alkaline condition.If desired, can adopt, for example HPLC separates required product with any side-product.
For example, the secondary alcohol of Radix Tripterygii Wilfordii lactone alcohol C14 position can adopt for example chromic acid-pyridine complex, CrO 2Cl 2/ aluminium oxide or corresponding oxidant, thus be oxidized to ketone (generating the compound known triptonide).For example, generate 14-deoxidation-14 with the DAST reaction, 14-difluoro Radix Tripterygii Wilfordii lactone alcohol, as follows.
Figure A2004800051530000141
Two kinds of secondary alcohol of Tripdiolide (2-hydroxyl Radix Tripterygii Wilfordii lactone alcohol) also can all adopt described oxidizer oxygen to turn to ketone.Generate tetrafluoro compound as follows with DAST or another kind of suitable reagent reacting.When used amount of reagent has in limited time, also can obtain 2, the 2-difluoro compound, as follows.
Figure A2004800051530000142
Alternatively, under the condition of gentleness, adopt the oxidant as noted above of stoichiometry, can optionally be oxidized to ketone by the alcohol that the steric hindrance of Tripdiolide C2 position is less.For the secondary alcohol and the primary alconol of oxidation 16-hydroxytriptolide, preferred employing can be not further with the reagent of product formoxy-ization, for example DMSO, pyridinium chlorochromate (Corey ' reagent) or cerium ammonium nitrate are to generate ketone-aldehyde intermediate.Keto-aldehyde and DAST or other suitable reagent reacting generate tetrafluoro compound shown below.Equally,, also can obtain 14-oxygen-16 when the consumption of fluorization agent has in limited time, the 16-difluoro compound, as follows.
Adopt reagent such as RuCl 2(PPh 3) 3, (Me 3SiO) 2/ catalytic RuCl 2(PPh 3) 3, DMSO/ClCOCOCl/Et 3N or DMSO/ pyridine .SO 3/ i-Pr 2NEt (referring to above-cited Larock), the also primary alconol of oxidation 16-hydroxytriptolide optionally.This aldehyde can be converted into 16 subsequently, and 16-difluoro Radix Tripterygii Wilfordii lactone alcohol is as follows.
Figure A2004800051530000152
Uncle's methylol (CH of C16 position 2OH) can be carboxylic acid also, then with SF by initial oxidation 4Thereby reaction is converted into trifluoromethyl, shown in the following reaction mechanism mechanism of reaction:
Figure A2004800051530000161
Can be in the C14 position or contain the center of the second month in a season or primary hydroxyl in addition, for example Cl, C2, C16 or C19 position are ketone by initial oxidation, then and the reaction of trifluoromethyl trimethyl silane, thereby introduce trifluoromethyl group.Shown in the following reaction mechanism mechanism of reaction:
Similar compounds can adopt the similar approach preparation usually, and wherein, fluorine replaces with different halogen atoms.C-OH is converted into the method for C-X is known in the art, wherein X is Cl, Br or I.Be used to prepare muriatic reagent and comprise for example SOCl 2Or PCl 5, POCl 3Deng.Equally, react with halide reagent subsequently for hydroxyl is converted into leaving group such as toluene fulfonate or trifluoromethane sulfonic acid root in the path of recommendation.For example referring to, Larock, Comprehensive Organic Transformations(VCH, New York, 1989), p.360.Also can use and SF 4Similarly mode is used PCl 5, so that form together with dichloride from aldehydes or ketones.
B. biological activity
As mentioned above, the multiple derivant of Radix Tripterygii Wilfordii lactone alcohol and analog are known in the art, and many be pharmaceutically useful.With respect to natural Radix Tripterygii Wilfordii lactone alcohol, these chemical compounds are because deliquescent variation and/or as the activity of aspects such as prodrug in lipid or water often can show the advantage of aspects such as pharmacokinetics or bio distribution.Yet generally speaking, the biological activity of these derivants and analog itself is less than natural Radix Tripterygii Wilfordii lactone alcohol.
Particularly, this area it has been generally acknowledged that at 14, preferred 14 β positions, and the existence of hydrogen donor group is important for the biological activity of keeping triptolide alcohol derivative.For example referring to the comparing data among this paper Fig. 3 A-B and the 4A-B.Shown in Fig. 3 A and 4A, 14-α-Radix Tripterygii Wilfordii lactone alcohol has the Alpha-hydroxy spatial chemistry in the C14 position, has immunosuppressant and apoptosis activity simultaneously, and its activity obviously is weaker than Radix Tripterygii Wilfordii lactone alcohol, and (in immunosuppressant was analyzed, effect was approximately little 30 times; In apoptosis was analyzed, effect was little 25 times).Have 14 β spatial chemistry in the C14 position and have the negative electricity substituent group but be not in the chemical compound of hydrogen donor group, i.e. 14 β-(methyl mercapto) methyl Radix Tripterygii Wilfordii lactone alcohol, can see with respect to Radix Tripterygii Wilfordii lactone alcohol, biological activity decline big (Fig. 3 B and 4B) is (in immunosuppressant is analyzed, approximately little 60 times; In apoptosis is analyzed, little 40 times).
Therefore, in view of the present situation of this area, the very high biological activity that 14-deoxidation of the present invention-14 α-fluorine compounds (PG763) are had is surprising, because it lacks the hydrogen donor group at 14.Shown in Fig. 1-2, in the analysis of assessment cytotoxicity and IL-2 inhibition, the approximate Radix Tripterygii Wilfordii lactone alcohol that is equal to of the activity of PG763.
Therefore, 14-deoxidation-14 α-fluorine compounds are structural formula I particularly preferred embodiment disclosed herein.In specific embodiment, chemical compound is PG763.
II. therapeutic combination
The preparation that contains triptolide alcohol derivative of the present invention can adopt solid, semisolid, lyophilized powder form, liquid dosage form, as tablet, capsule, powder, sustained release formulation, solution, suspension, emulsion, ointment, washing liquid or aerosol, preferably be applicable to the unit dosage forms of accurate dosage, simple administration.Typically, said composition comprises conventional pharmaceutical carriers or excipient, and can comprise other medicament, carrier or adjuvant.
Preferably, said composition contains one or more The compounds of this invention of the 0.5%-75 weight % that has an appointment, and all the other are made up of suitable pharmaceutically acceptable excipient.For oral administration, excipient comprises mannitol, lactose, starch, magnesium stearate, saccharin sodium, Talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate and other analog of pharmaceutical grade.If desired, said composition can also contain a spot of nontoxic auxiliary material, for example wetting agent, emulsifying agent or buffer agent.
Said composition can oral administration, transdermal or through parenterai administration to the patient, for example, by intravenous, subcutaneous, intraperitoneal or intramuscular injection.Use with oral liquid, said composition can be made solution, suspension, emulsion or syrup, with liquid form or be applicable to the dried forms administration of hydration in water or normal saline.For parenterai administration, injectable parenterai administration compositions typically contains and is dissolved in suitable parenteral solutions, for example the triptolide alcohol derivative in the physiological saline solution solution.
By with triptolide alcohol derivative (about 0.5%-about 20%) and the dissolving of optional pharmaceutically acceptable adjuvant or be dispersed in pharmaceutically acceptable carrier, for example in saline solution, D/W, glycerol or the ethanol, form solution or suspension and prepare fluid composition.
This chemical compound also can pass through inhalation, adopts the form of solid or liquid aersol particle, preferably has the granularity that can suck.This granule little to once suck can through port and larynx enter bronchus and alveolar.Usually, the granularity that can suck is about 1-10 micron, preferably less than about 5 microns.The fluid composition of inhalation contains and is dispersed in aqueous carrier, for example the active substance in the water of the saline solution of aseptic no pyrogen or aseptic no pyrogen.If desired, said composition can be mixed spraying and the formation aerosol to help said composition with propeller.
The method for preparing this dosage form is known, perhaps it will be apparent to those skilled in the art that; For example referring to Remington ' s Pharmaceutical Sciences(the 19th edition, Williams ﹠amp; Wilkins, 1995).This administration composition contains the selected compounds of certain effective dose, is used for influencing patient's immunosuppressant or the apoptosis of target cell.
Described as Panchagnula et al. (2000), the partition coefficient of pharmaceutical agents or logP can influence its suitability for various route of administration, comprise oral administration biaavailability.Chemical compound described herein is by having replaced one or more hydroxyls with fluorine, expection has the logP value of calculation higher than parent compound Radix Tripterygii Wilfordii lactone alcohol, thereby makes them become the better drug candidate of oral availability.
III. immunomodulating and anti-inflammatory treatment
As shown in Figure 2, has compound in structural formula I, 14-deoxidation-1-α-fluorine Radix Tripterygii Wilfordii lactone alcohol (being appointed as PG763) suppresses IL-2 product (referring to embodiment 4) in the dose dependent mode in the Jurkat cell, the concentration of its use is equivalent to the similar analysis that adopts Radix Tripterygii Wilfordii lactone alcohol to carry out.Therefore, the present invention comprises the purposes of The compounds of this invention as immunosuppressant, for example auxiliary treatment that is used for graft procedure or is used for autoimmune disease.
Show, immunoregulatory abnormality is present in various autoimmune and the chronic inflammatory disease, comprises whole body lupus erythematosus, chronic rheumatic arthritis, I and type ii diabetes, enteritis, biliary cirrhosis, uveitis, multiple sclerosis and other disorder as crohn, ulcerative colitis, pemphigoid, sarcoidosis, psoriasis, ichthyosis, graves' ophthalmopathy and asthma.Although the potential pathogeny of these symptoms can be obviously different, they all have identical multiple autoantibody and the performance of autoreaction lymphocyte.This autoreaction may partly be that normal immune system is moved under this balance owing to lost homoiostasis control.
Similarly, after bone marrow transplantation or other hematopoietic stem cell transplantation are carried out in the donor tissue source of containing mature lymphocyte, the lymphocyte of transfer is external with the host tissue antigen recognition.These cells become activated and the host is initiated may be fatal attack (graft versus host reaction).In addition, after the organ transplantation, antibody-mediated immunoreation (host is to graft reaction) in the concurrent kinetocyte of external organization's antigen of host's lymphocyte identification organ graft causes the impaired and repulsion of graft.
A kind of result of autoimmune or rejection is exactly by the caused tissue damaged of the amboceptor of inflammatory cells and release thereof.Antiinflammatory such as NSAID mainly work by influence or the secretion of blocking these amboceptors, but do not change the amynologic basis of disease.On the other hand, cytotoxic agent works in the non-specific mode normal and autoimmune response of blocking-up simultaneously as cyclophosphamide.In fact, adopt the patient of this nonspecific immunosuppressive agent treatment, as they can die from autoimmune disease, might die from infection.
The present composition can be used for proving that Radix Tripterygii Wilfordii lactone alcohol and prodrug and other derivant thereof are during effectively those are used, and for example is used for immunosuppressant therapy as treatment autoimmune disease, prevention transplant rejection, treatment or prevention graft versus host disease (GVHD).For example referring to total United States Patent (USP) 6,150,539, it is incorporated herein by reference.The derivant of Radix Tripterygii Wilfordii lactone alcohol and this paper also is used for the treatment of other inflammatory diseases for example wound inflammation and male fertility decline effectively.
Said composition can be used for suppressing organa parenchymatosum's graft, tissue grafts or from the repulsion of the cellular transplant of people's donor of mutual repulsion, thus the life span and the function that have prolonged graft, and the life span of receptor.This purposes will comprise, but be not limited to organa parenchymatosum's graft (as heart, kidney and liver), tissue grafts (as skin, intestinal, pancreas, gonad, bone and cartilage) and cellular transplant (as cell) from pancreas, brain and nervous tissue, muscle, skin, bone, cartilage and liver.
Said composition also is used to suppress xenograft (between kind) effectively and repels, promptly prevent repulsion from non-human animal's organa parenchymatosum's graft, tissue grafts or cellular transplant, though its be on constituting or on the biological engineering (heredity is controllable) can the expressing human gene, RNA, protein, peptide or other non-born heterologous molecule, or born gene, RNA, protein, the peptide that can not express animal on the biological engineering lack and express or the molecule of other normal expression.The present invention also comprises the purposes that compositions as mentioned above is used to prolong the life span of this organa parenchymatosum's graft, tissue grafts or the cellular transplant that come from the non-human animal.
The present invention also comprises the treatment autoimmune disease or has the method for the disease of autoimmune performance, as Addison disease, autoimmune hemolytic anemia, autoimmune thyroid, crohn, diabetes (I type), Graves disease, Ji-Ba syndrome, whole body lupus erythematosus (SLE), lupus nephritis, multiple sclerosis, myasthenia gravis, psoriasis, primary biliary cirrhosis, rheumatic arthritis and uveitis, asthma, arteriosclerosis, thyroiditis, allergic encephalomyelitis, glomerulonephritis and various anaphylaxis.
Other purposes can comprise treatment and prevention inflammation and high outgrowth dermatosis and the immune-mediated disease of cutaneous manifestations, as psoriasis, hereditary irritated dermatitis, pemphigus, rubella, skin eosinophilia, acne and bareheaded speckle; Various oculopathy, as conjunctivitis, uveitis, keratitis, and sarcoidosis; Mucus and vascular inflammatory, the vessel lesion that causes as gastric ulcer, by ischemic diseases and thrombosis, ischemic enteropathy, inflammatory enteropathy and downright bad enterocolitis; Enteritis/Sensitive disease is as coeliac disease and ulcerative colitis; Nephropathy is as a matter nephritis, Goodpasture, hemolytic uremic syndrome and diabetic nephropathy; Hematopoietic disease is as sudden thrombocytopenia purpura and autoimmune hemolytic anemia; Dermatosis is as dermatomyositis and cutaneous T cell lymphoma; The blood circulation disease is as arteriosclerosis and arteriosclerosis; Nephropathy, the insufficient and chronic renal insufficiency as the ischemic acute kidney; And behcet disease.
The compositions and methods of the invention also are used for the treatment of inflammation effectively, as has the asthma of endogenous and exogenous performance, for example, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, particularly chronic or habitual asthma (for example, old age asthma and air flue high response).Described compositions and method also can be used for the treatment of other inflammatory disease, comprise inflammation in traumatic inflammation, the Lyme arthritis disease, chronic bronchitis (chronic infection pneumonopathy), sepsis and pulmonary's sarcoidosis that chronic sinusitis is relevant with adult respiratory distress syndrome.For treatment respiratory symptom such as asthma, described compositions is preferably by inhalation, but the route of administration of any routine also is available.
When treatment autoimmune symptom, to the regular administration of patient, for example take said composition 1-2 time weekly with the dosage that is enough to mitigation symptoms and improves patient's comfort level.Particularly for treatment rheumatic arthritis, said composition can or be injected directly in the joint of infection by intravenous injection.Can be in several weeks after the disease of patient symptom takes place, with at least 24 hours intervals to patient's repetitive therapy.The dosage scope is preferably 1-25mg/kg weight in patients/sky, and lower dosage is preferred for parenterai administration, the preferred oral administration administration of higher dosage.According to the known method of this area, can determine optimal dose by routine test.
For the transplant rejection treatment, this method especially comprises the repulsion of treatment heart, kidney, liver, cell and bone marrow graft, and can be used for treating GVHD.For acute transplant rejection, treatment normally transplant operation not long ago or after the operation soon, in peri-operation period (perioperatively) beginning, and implement dosage regimen every day, continue to carry out the treatment in several at least weeks.During treating, can be by for example, comprise the lymphocytic mixed lymphocyte reaction of homotransplantation or by transplanted tissue is carried out tissue biopsy, regularly the immunosuppressant level to the patient detects.
In addition, said composition can also be prevented transplant rejection or treatment transplant rejection acute attack in late period by long term administration.As mentioned above, dosage is preferably 1-25mg/kg weight in patients/sky, and lower dosage is preferably through parenterai administration, the preferred oral administration administration of higher dosage.Resist the ability of infection according to patient during reaction and the treatment, this dosage can suitably increase or reduce.
Come from when stem cell receptor coupling or unmatched bone marrow, splenocyte, tire tissue, Cord blood or fixed or other collection is transplanted the graft versus host disease that causes in treatment or prevention, the preferred 0.25-2mg/kg body weight/day of this disease dosage, and preferred 0.5-1mg/kg/ days, oral administration or through parenterai administration.
Scope of the present invention also comprises the combined treatment of the immunosuppressant with compound in structural formula I and one or more routines.These immunosuppressant that are in the scope of the invention comprise, but be not limited to, IMUREK (Imurek (Burroughs Wellcome) .), brequinar sodium, SPANIDIN (the gusperimus trihydrochloride also claims the deoxidation spermatin), mizoribine (also claiming bredinin), cell CEPT (mycophenolate mofetil), NEORAL (Ciclosporin A; The different preparations that on the market with the trade mark are SANDIMMUNE are sold), PROGRAF (blood flow spectrum, also claim FK-506), RAPIMMUNE (sirolimus, also claim rapamycin), leflunomide (also claiming HWA-486), ZENAPAX, adrenocortical hormone, as andrographolide and derivant thereof; Antibody such as quadrature clone (OKT3) and antithymyocyte globulin such as thymus globulin.When being used for the immunosuppressive drug administration simultaneously of immunosuppressant therapy as mentioned above with another kind, this chemical compound also can be used as reinforcing agent effectively.Therefore, amount that can be with well below individually dosed this chemical compound the time is carried out administration (for example 20%-50% of standard dose) to the immunosuppressive drug of above-mentioned routine.Alternative dosage is to make The compounds of this invention and immunosuppressive drug better than use this medicine or The compounds of this invention gained immunosuppressive effect separately with same dose with the resulting immunosuppressive effect of this amount administration.Typically, immunosuppressive drug and synergist are with at least 2 weeks of interval administration of routine.
Compositions of the present invention also can be with one or more anti-inflammatory agent administrations of routine, and wherein, the dosage of this medicine or medicine itself is invalid for inducing suitable inhibition or suppressing inflammation.
Animal model known in the art by using, as to have set up, immunosuppressive activity in the body that can assessing compound.This analysis can be used for estimating the relative potency of immunosuppressive compounds, and can be used to estimate to carry out the suitable dosage of immunosuppressant therapy.These analyses for example comprise the rat model system of allograft by Ono and the described complete characterization of Lindsey (1969), wherein, the heart of transplanting is related by blood greatly with the abdominal part that alloplast is accepted animal, the survival ability of the ability mensuration heart transplant of beating in receptor by heart.By the xenograft models that Wang (1991) and Murase (1993) describe, wherein receptor is different kind system.The model of assessing anti-GVHD effect comprises the splenocyte to normal Fi injected in mice father and mother; Mice produces the GVHD syndrome, it is characterized by splenomegaly and immunosuppressant (Korngold, 1978; Gleichmann, 1984).Spleen by individuality prepares single-cell suspension liquid, and sets up the degree of micropore (microwell) culture with the response of evaluation mitogenesis in the concanavalin A existence with not.
IV. anticancer therapy
As shown in Figure 1, has compound in structural formula I, 14-deoxidation-1-α-fluorine Radix Tripterygii Wilfordii lactone alcohol (being appointed as PG763) has cytotoxin (referring to embodiment 2) in the dose dependent mode to the Jurkat cell, and concentration is equivalent to the similar analysis that adopts Radix Tripterygii Wilfordii lactone alcohol to carry out.Therefore, the present invention comprises the purposes of The compounds of this invention as cytotoxic agent, in particular for the treatment cancer.Refer to all types of cancers or tumor or the malignant tumor particularly found among the people mammal comprise leukemia, sarcoma, cancer and melanoma as " cancer " used herein.
Term " leukemia " general reference blood forms the cumulative bad malignant disease of organ, generally it is characterized by the interior leukocyte of blood and bone marrow and the undesired hypertrophy and the growth of precursor thereof.Term " sarcoma " refers generally to the tumor that is formed by the material as embryo's conjunctive tissue and is made of the tight tytosis that is embedded in fiber or the isotropic body under normal conditions.Term " melanoma " refers to come from the tumor of the melanocyte system of skin and other organ.Term " cancer " refers to epithelial pernicious new growth, is tending towards permeating surrounding tissue and also shifts.
For example, the cancer that comprises relates to the cell that comes from germinal tissue (as sexual cell and other cell of Sai Ertuolishi (Sertoli) cell, sexual cell, developmental or more sophisticated spermatogonium, spermatid or spermatocyte and trophocyte, ovary), lymph or immune system (as Hodgkin and non-Hodgkin lymphoma), hemopoietic system and epithelial cell (as skin, comprise malignant melanoma and gastrointestinal tract), organa parenchymatosum, nervous system such as glioma (referring to Y.X.Zhou et al., 2002) and flesh skeletal tissue.This chemical compound can be used for treating various cancerous cell types, comprise but be not limited to, brain comprises medulloblastoma, head and neck, breast, colon, minicell lung, maxicell lung, thyroid, testis, bladder, prostate, liver, kidney, pancreas, esophagus, stomach, ovary, cervix uteri or lymphoma.Especially wish to treat breast, colon, lung and prostate tumor.
Can said composition be administered into the patient who tormented by cancer and/or leukemia by aforesaid conventional route of administration.This method can be slowed down tumor growth, prophylaxis of tumours growth, the local decline of induced tumor effectively and be caused that tumor fails fully until complete obiteration.This method also is used to prevent to come from the transforming growth of solid tumour effectively.
The present composition can be used as independent curative and comes administration, or carries out with other complementary or therapeutic treatment of no anticancer effect in the patient.This method also comprises the present composition with one or more conventional cancer therapy drugs or bioprotein agent, to have the effective dose administration of expection anticancer effect in the patient.Wherein, the amount of this medicine or reagent itself suppresses invalid to inducing suitable growth of cancers.These cancer therapy drugs comprise radiating streptozotocin D, camptothecine, carboplatin, cisplatin, cyclophosphamide, arabinose cytidine, daunomycin, amycin, etoposide, fludarabine, 5-fluorouracil, hydroxycarbamide, gemcitabine, Mexician scammony, methotrexate, ametycin, rice drags anthraquinone, handkerchief nit west, taxotere, teniposide, hycamtin, vinblastine, vincristine, desacetyl vinblastine amide and vinorelbine.Anticancer bioprotein agent comprises tumor necrosis factor (TNF), the TNF related apoptosis brings out part (TRAIL), other TNF relevant or the TRAIL associated ligands and the factor, interferon, IL-2, other interleukin, other cytokine, the chemistry element alive and the factor, the antibody of tumor correlation molecule or receptor (as anti--BER2 antibody) and with the reagent of these reagent reactings or bonding (TNF as receptor goes up the member of family, other receptor, receptor antagonist and the antibody that is exclusively used in these reagent).
By the animal model of adopt setting up, at United States Patent (USP) 6,620, described in 843, can estimate the anti-tumor in vivo activity of particular composition as Fidler et al.According to the known method of clinician, determine clinical dosage and treatment plan according to severity of disease and patient's factors such as all symptoms.
V. other explanation
The compounds of this invention also can be used for the treatment of some CNS disease.Glutamate, Glu can be realized many physiologic functions, and the bag expansion is playing an important role aspect various neurological and the psychotic pathophysiology.The exitotoxicity and the neurotoxicity of glutamate, Glu in histanoxia, ischemia and damage and chronic neural degeneration or nerve metabolism disease, Alzheimer, Heng Yandun disease and Parkinson disease, have been hinted.Consider that the Radix Tripterygii Wilfordii lactone alcohol of having reported has neuroprotective, particularly can prevent by the inductive cell death of glutamate, Glu (Q.He et al., 2003; X.Wang et al., 2003), so The compounds of this invention is expected to resist the neurotoxicity of glutamate, Glu and thereby may be a kind of new treatment means that is used for this disease.
Up-to-date evidence from rehabilitation period patient MS hints out that change has taken place in the dynamic equilibrium of glutamate, Glu in the brain.The neurotoxic outbreak that occurs in patient MS may be the reason that causes oligodendrocyte and neuronal cell death.In patient MS, the exitotoxicity of the application of the invention compounds for treating antiglutamic acid salt receptor-mediation may have the therapeutics implication.Other CNS disease such as Ji-Ba syndrome, Meniere, polyneuritis, polyneuritis, mononeuritis and radiculopathy also can be treated with The compounds of this invention.
The compounds of this invention also can be used for the treatment of some pneumonopathy.Sudden pulmonary fibrosis (PF) produces fissured pneumonopathy for a kind of accumulation, its cause of disease the unknown.PF is characterized as substrate and collagen excessively dislocation in interstitial tissue of lung in the cell, and replaces bubble, consequently inflammation and cystic fibrosis by scar tissue gradually.Along with advancing of disease, the increase of scar tissue has disturbed oxygen to transfer to the ability of blood flow from lung.Reported the inductive PF of 14-succinimide ester bleomycin capable of blocking (G Krishna et al., 2001) of Radix Tripterygii Wilfordii lactone alcohol.Therefore, The compounds of this invention can be used for the treatment of PF effectively.Think that also this chemical compound can treat other respiratory disorder, as sarcoidosis, fibroid lung and sudden interstitial pneumonia.
Other relates to lung and expects that the disease for the treatment of with The compounds of this invention comprises serious acute respiration syndrome (SARS) and adult respiratory distress syndrome (ARDS).Particularly about SARS, as following pointed, reduce viral level (SARS-CoV) and adopt the effectiveness of corticosteroid treatment to hint out that the life-threatening development that has a strong impact on most of SARS may come from the exaggeration response (immune hyperkinesia) of health to infecting before the peak at disease progression, rather than the influence of virus itself is (referring to common unsettled and total U.S. Provisional Application 60/483,335, it is incorporated herein by reference).Corticosteroid treatment has been used for patient SARS so that suppress a large amount of releases of cytokine, and it has showed the feature in immune superactivity stage, wishes to stop in next stage the progress of pneumonopathy.Corticosteroid treatment has produced good clinical effectiveness, has reduced some main SARS symptoms.Yet, have several and the relevant side effect of treatment, and be starved of and have more optionally immunosuppressant and/or antiinflammatory.
Embodiment
Following embodiment is used to explain the present invention, and is not to be limitation of the present invention.
Embodiment 1. preparation 14-deoxidation-14 α-fluorine Radix Tripterygii Wilfordii lactone alcohols
Figure A2004800051530000251
At N 2Down, in the time of 0 ℃, to PG490 (Radix Tripterygii Wilfordii lactone alcohol, add among the 17.3mg, dichloromethane solution 0.048mmol) (1.0ml) (diethylamino) sulfur trifluoride (DAST, 100 μ l, 0.763mmol).This reactant mixture adds saturated NaHCO in 0 ℃ of stirring after 2 hours 3Solution (0.8ml).With this reactant mixture of 3 * 2ml dichloromethane extraction.The organic layer anhydrous Na SO that merges 4Drying is also carried out vacuum concentration.Quantitatively obtain required product (PG763).
Analyze TLC:Rf=0.78 (ethyl acetate/hexane/methanol 1: 1: 0.1).IR(KBr)：3031.0，2961.2，2942.4，2873.8，1764.6，1680.9，1449.3，1438.3，1172.2，1098.1，1074.5，1057.0，1047.1，1034.2，1018.2，1005.6，987.3，972.3，923.9，909.0，743.6，586.0，566.0，539.8，527.6cm -1。 IHNMR (300MHz, CDCl 3): δ=5.16 (d, 1H, 14-CH), 4.70 (q, 2H, 19-CH 2), 3.80 (d, 1H, 11-CH), 3.73 (d, 1H, 7-CH), 3.50 (t, 1H, 12-CH), 2.70 (m, 1H, 5-CH), 2.34 (d, 1H, 2-CHb), 2.27-2.02 (m, 3H, 6-CHb, 2-CHa and 15-CH), 1.95 (m, 1H, 6-CHa), 1.56 (dd, 1H, 1-CHb), 1.24 (m, 1H, 1-CHa), 1.11 (s, 3H, 20-CH 3), 1.10 (d, 3H, 17-CH 3), 0.91 (d, 3H, 16-CH 3) ppm.
Embodiment 2: cytotoxicity (MTT) is analyzed
Test compounds is dissolved among the DMSO, and concentration is 20mM.(MD) middle (HyClone Laboratories, Logan UT) further dilute with 10% hyclone for GIBCO, Rockville at the RPMI1640 medium.
In the MTT of standard analyzed, (#1 465 007, RocheDianostics, Mannheim, Germany) cytotoxicity of mensuration chemical compound to adopt cell amplification test kit I.In brief, in each dilution point, a series of 3 times of diluents that have test compounds or contain with specimen under the situation of medium of same concentrations DMSO, with HTL (Jurkat) cell (4 * 10 5/ hole) in the tissue culturing plate in 96 holes, cultivated 24 hours.Then, culture also used the lytic agent in 0.1ml/ hole to replenish in additional 4 hours other 16 hours with the MTT reagent in 10 μ l/ holes subsequently.(divide subset, Menlo Park CA) goes up the optical density (OD that measures the 570nm place in the ThermoScan microplate reader 570).Data are expressed as OD with respect to compound concentrations 570Value.Fig. 1 has provided the result of PG763 (14-deoxidation-1-α-fluorine Radix Tripterygii Wilfordii lactone alcohol) with respect to PG490 (Radix Tripterygii Wilfordii lactone alcohol) and medium control group.
Embodiment 3: the annexin V apoptosis is analyzed
Specimen is diluted to 1mM in complete tissue culture medium's (RPMI 1640 media add hyclone, 1%HEPES, 1%pen/strep, 1% glutamate, Glu of 5% thermal denaturation).Thereby being placed on solution on little culture plate and preparing a series of diluents makes ultimate density comprise the scope of the 2-6000nM that increases with semilog.Jurkat people T lymphoma cell line (#TIB-152 from the index increase, available from American Type Culture Collection, Manassas, VA) collecting cell in the culture, eccentric cleaning once, be suspended in once more completely in the tissue culture medium (TCM), and further to be diluted to concentration be 1 * 10 6Cell/ml.With 100 μ (1 * 10 5) the l cell joins in the hole of containing 100 μ l diluted compounds, and with plate at 5%CO 2Cultivate down in 37 ℃ in the couveuse.
After 24 hours, plate is by centrifugal so that cell spheroidization, and the hyclone with 2% thermal denaturation in PBS cleans cell 2 times.(Mountain View CA) adds 500ul binding buffer liquid to each hole for Bio Vision, Inc. according to the annexin V analytical method.Then, the fluorescein isothiocyanate (FITC) that adds 5 μ l annexin Vs to each hole is gripped thing altogether (BioVision Inc.), was cultivated 5 minutes then in the dark.In some were analyzed, (BioVision was Inc.) to check downright bad cell to add the propidium iodide in this stage.Transfer to the material in the hole in the test tube separately and adopt FACSCalibur flow cytometer (BD immunity hemocytometer number system, San Jose, CA) analysis of cells apoptosis.Annexin V is regarded as apoptosis in conjunction with the cell that is positive, and with apoptotic cell percentage calculating data recently.
Data are mapped with respect to apoptotic cell percentage ratio with compound concentration.Fig. 3 A-B has provided two kinds of 14-replacement Radix Tripterygii Wilfordii lactone alcohols, 14-Alpha-hydroxy Radix Tripterygii Wilfordii lactone alcohols and (has been also referred to as epitriptolide; Be appointed as PG524) and 14-β-(methyl mercapto) methyl Radix Tripterygii Wilfordii lactone alcohol (specify PG691) with respect to the comparing data of PG490 (Radix Tripterygii Wilfordii lactone alcohol).
Embodiment 4:IL-2 product analysis
Specimen is being diluted to 1mM in the tissue culture medium (TCM) completely.This solution is placed on (with Jurkat cytositimulation IL-2 product) on the miniature culture plate of anti-cd 3 antibodies coating thus and prepare a series of diluents and make ultimate density can comprise 0.001-10 with the logarithm increase, the scope of 000nM.(#TIB-152 is available from AmericanType Culture Collection from Jurkat human T-cell line that index increases, Manassas, VA) collecting cell in the culture, eccentric cleaning once, being suspended in completely in the tissue culture medium (TCM) once more and being diluted to concentration is 2 * 10 6Cell/ml.With 50 μ l Jurkat cells (1 * 10 5Cell) join in the hole of containing 100 μ l diluted compounds, to each hole add 50 μ l PMA (10ng/ml) and with plate at 5%CO 2In the couveuse in 37 ℃ of cultivations.After 24 hours, plate is taken out 150 μ l supernatant and sample is stored in-20 ℃ by centrifugal so that cell spheroidization from each hole.Adopt Luminex 100 (Luminex Corporation, Austin, TX), the bonded Luminex microsphere of anti-IL-2 capture antibody and the bonded anti-IL-2 of fluorescent dye detect the IL-2 concentration of the supernatant that antibody analysis stores.Data are represented with the pg/ml of IL-2.
Data are mapped with respect to IL-2 concentration with compound concentration.Fig. 2 has provided the result of PG763 (14-deoxidation-1-α-fluorine Radix Tripterygii Wilfordii lactone alcohol) with respect to PG490 (Radix Tripterygii Wilfordii lactone alcohol) and solvent control group.The 14-that Fig. 4 A-B has provided other replaces Radix Tripterygii Wilfordii lactone alcohol, 1-Alpha-hydroxy Radix Tripterygii Wilfordii lactone alcohol (is also referred to as epitriptolide; Be appointed as PG524) and 14-β-(methyl mercapto) methyl Radix Tripterygii Wilfordii lactone alcohol (being appointed as PG691) with respect to the comparing data of PG490 (Radix Tripterygii Wilfordii lactone alcohol).

Claims (23)

1. have compound in structural formula I:
Wherein: CR 1R 2Be selected from CHOH, C=O, CHF, CF 2Or C (CF 3) OH;
CR 6And CR 13Be selected from CH, COH or CF;
CR 7R 8, CR 9R 10And CR 11R 12Be selected from CH 2, CHOH, C=O, CHF or CF 2And CR 3R 4R 5Be selected from CH 3, CH 2OH, C=O, COOH, CH 2F, CHF 2Or CF 3
Wherein, at R 1-R 13In have at least one to comprise fluorine;
At CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10, CR 11R 12And CR 13In have two to comprise fluorine or oxygen at the most;
And, work as CR 1R 2When being CHOH, CR 3R 4R 5Not CH 2F.
2. according to the chemical compound of claim 1, wherein, CR 7R 8And CR 9R 10All independently be selected from CH 2, CHOH (β), C=O, CHF (α) or CF 2
3. according to the chemical compound of claim 2, wherein, at CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10, CR 11R 12And CR 13In have one to comprise fluorine or oxygen at the most.
4. according to the chemical compound of claim 3, wherein, at CR 1R 2, CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10And CR 11R 12In just have one to comprise fluorine.
5. according to the chemical compound of claim 4, wherein, at CR 1R 2, CR 6, CR 7R 8, CR 9R 10And CR 11R 12In just have one to comprise fluorine.
6. according to the chemical compound of claim 5, wherein, CR 1R 2Comprise fluorine.
7. according to the chemical compound of claim 6, wherein, CR 1R 2Be CF 2
8. according to the chemical compound of claim 6, wherein, CR 1R 2Be CHF (α).
9. chemical compound according to Claim 8, wherein, R 3-R 13All be hydrogen.
10. influence the immunosuppressant method, comprise with effective dose, be included in the pharmaceutically acceptable carrier, have an experimenter that compound in structural formula I is administered into this processing of needs
Figure A2004800051530000031
Wherein: CR 1R 2Be selected from CHOH, C=O, CHF, CF 2Or C (CF 3) OH;
CR 6And CR 13Be selected from CH, COH or CF;
CR 7R 8, CR 9R 10And CR 11R 12Be selected from CH 2, CHOH, C=O, CHF or CF 2And CR 3R 4R 5Be selected from CH 3, CH 2OH, C=O, COOH, CH 2F, CHF 2Or CF 3
Wherein, at R 1-R 13In have at least one to comprise fluorine;
At CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10, CR 11R 12And CR 13In have two to comprise fluorine or oxygen at the most;
And, work as CR 1R 2When being CHOH, CR 3R 4R 5Not CH 2F.
11. according to the method for claim 10, wherein, CR 7R 8And CR 9R 10All independently be selected from CH 2, CHOH (β), C=O, CHF (α) or CF 2
12. according to the method for claim 10, wherein, at CR 1R 2, CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10And CR 11R 12In just have one to comprise fluorine.
13. according to the method for claim 12, wherein, CR 1R 2Comprise fluorine.
14. according to the method for claim 13, wherein, CR 1R 2Be CF 2
15. according to the method for claim 13, wherein, CR 1R 2Be CHF (α).
16. according to the method for claim 15, wherein, R 3-R 13All be hydrogen.
17. the method for inducing cell inner cell apoptosis comprises described cell is contacted with chemical compound effective dose, that have structural formula 1:
Figure A2004800051530000041
Wherein: CR 1R 2Be selected from CHOH, C=O, CHF, CF 2Or C (CF 3) OH;
CR 6And CR 13Be selected from CH, COH or CF;
CR 7R 8, CR 9R 10And CR 11R 12Be selected from CH 2, CHOH, C=O, CHF or CF 2And CR 3R 4R 5Be selected from CH 3, CH 2OH, C=O, COOH, CH 2F, CHF 2Or CF 3
Wherein, at R 1-R 13In have at least one to comprise fluorine;
At CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10, CR 11R 12And CR 13In have two to comprise fluorine or oxygen at the most;
And, work as CR 1R 2When being CHOH, CR 3R 4R 5Not CH 2F.
18. according to the method for claim 17, wherein, CR 7R 8And CR 9R 10All be selected from CH separately 2, CHOH (β), C=O, CHF (α) or CF 2
19. according to the method for claim 18, wherein, at CR 1R 2, CR 3R 4R 5, CR 6, CR 7R 8, CR 9R 10And CR 11R 12In just have one to comprise fluorine.
20. according to the method for claim 19, wherein, CR 1R 2Comprise fluorine.
21. according to the method for claim 20, wherein, CR 1R 2Be CF 2
22. according to the method for claim 20, wherein, CR 1R 2Be CHF (α).
23. according to the method for claim 22, wherein, R 3-R 13All be hydrogen.
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WO2007112648A1 (en) * 2006-04-04 2007-10-11 Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences The medicinal compositions comprising triptolide derivatives and its dosage form and application
CN102307881A (en) * 2009-02-05 2012-01-04 药物起源公司 Tripolide C-ring derivatives as anticancer agents and immune modulators
CN102443042A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted methoxy-containing phenyl-14 Beta-aminomethyl epitriptolide derivatives, preparation method and use thereof
CN102443039A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted amino phenyl-14 beta-(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443045A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted phenyl-14 beta -(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443040A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted fluorinated phenyl-14 beta-(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443043A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted-14 beta -(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443041A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted methyl phenyl-14 beta -(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443044A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted ester-containing phenyl-14 Beta-aminomethyl epitriptolide derivatives, preparation method and use thereof
CN103204861A (en) * 2012-01-12 2013-07-17 上海汇伦生命科技有限公司 Fluoro-substituted triptolide derivative, its preparation method and application
CN104513290A (en) * 2013-10-08 2015-04-15 中国医学科学院药物研究所 Triptolidenol derivative and application thereof

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WO2007112648A1 (en) * 2006-04-04 2007-10-11 Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences The medicinal compositions comprising triptolide derivatives and its dosage form and application
CN102307881A (en) * 2009-02-05 2012-01-04 药物起源公司 Tripolide C-ring derivatives as anticancer agents and immune modulators
CN102307881B (en) * 2009-02-05 2014-12-24 药物起源公司 Tripolide C-ring derivatives as anticancer agents and immune modulators
CN102443044A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted ester-containing phenyl-14 Beta-aminomethyl epitriptolide derivatives, preparation method and use thereof
CN102443044B (en) * 2010-10-14 2013-11-13 中国科学院上海药物研究所 N-substituted ester-containing phenyl-14 Beta-aminomethyl epitriptolide derivatives, preparation method and use thereof
CN102443040A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted fluorinated phenyl-14 beta-(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443043A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted-14 beta -(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443041A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted methyl phenyl-14 beta -(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443039A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted amino phenyl-14 beta-(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443042A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted methoxy-containing phenyl-14 Beta-aminomethyl epitriptolide derivatives, preparation method and use thereof
CN102443045A (en) * 2010-10-14 2012-05-09 中国科学院上海药物研究所 N-substituted phenyl-14 beta -(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443042B (en) * 2010-10-14 2013-11-13 中国科学院上海药物研究所 N-substituted methoxy-containing phenyl-14 Beta-aminomethyl epitriptolide derivatives, preparation method and use thereof
CN102443039B (en) * 2010-10-14 2013-11-13 中国科学院上海药物研究所 N-substituted amino phenyl-14 beta-(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443043B (en) * 2010-10-14 2013-11-13 中国科学院上海药物研究所 N-substituted-14 beta -(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN102443041B (en) * 2010-10-14 2013-11-13 中国科学院上海药物研究所 N-substituted methyl phenyl-14 beta -(aminomethyl) triptodiolide derivative and preparation method and application thereof
CN103204861A (en) * 2012-01-12 2013-07-17 上海汇伦生命科技有限公司 Fluoro-substituted triptolide derivative, its preparation method and application
CN103204861B (en) * 2012-01-12 2016-07-06 上海汇伦生命科技有限公司 Fluorine replaces triptolide derivatives and its preparation method and application
CN104513290A (en) * 2013-10-08 2015-04-15 中国医学科学院药物研究所 Triptolidenol derivative and application thereof
CN104513290B (en) * 2013-10-08 2019-01-01 中国医学科学院药物研究所 Triptolidenol derivative and its application

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