CN103755767A - CDDO ethyl ester polymorphic substance and application thereof - Google Patents
CDDO ethyl ester polymorphic substance and application thereof Download PDFInfo
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Abstract
The invention relates to a polymorphism shape of a compound disclosed as Formula I (ethyl 2-cyano-3,12-dioxooleanane-1,9(11)diolefin-28-ester (CDDO ethyl ester)) and a method thereof for treating various diseases, such as cancers and inflammation accompanied by the cancers.
Description
The related application of cross reference
The application requires the right of priority of the PCT international application that the international application no of submission on September 28th, 2012 is PCT/CN2012/082278, and the full content of this earlier application is incorporated to herein by reference at this.
Technical field
The application relates to triterpenoid, ethyl 2-cyano group-3,12-dioxo volatile oil-1, the polymorphs form of 9 (11) dienes-28-ester (CDDO ethyl ester), and utilize their at least one polymorphs form treatment various disease states, be generally the method with the morbid state of inflammation-related.
Background technology
Triterpenoid is to carry out biosynthesizing by the cyclization of squalene in plant.Although triterpenoid is candidate's medical compounds, the molecule of these natural generations shows relatively weak biological activity.Therefore, chemists have attempted the similar compound that synthetic effect improves people such as (, 1997 and 1998) Honda.
It is reported, again the forming of the derivable nitricoxide synthase of cytokine (iNOS) and cyclooxygenase-2 (COX-2) in the scavenger cell that some synthetic similar compounds have suppressed to be stimulated by IFN-γ or LPS (people such as Suh, 1998; The people such as Honda, 2002).Wherein, 2-cyano group-3,12-dioxo volatile oil-1,9(11) diene-28-ester (CDDO) demonstrates anti-inflammatory and antiproliferative activity (people such as Honda, 1998 and 2000).As described in, triterpenoid is as the inhibition of iNOS activity, the particularly inhibiting research of the generation to NO has shown the efficient (IC of CDDO and CDDO methyl ester
50<lnM level).Referring to the people such as Honda (2000).In any case, believe that their treatment potential is not also utilized completely, CDDO ethyl ester still less up to now.
In order to be familiar with the treatment potential suc as formula the CDDO ethyl ester shown in I, the inventor has studied the polymorphs form of this compound.Therefore, contriver has disclosed three kinds of crystal formation forms of CDDO ethyl ester, and these three kinds of crystal formation forms have some favourable character (for example, pharmacokinetic performance and higher system expose preferably), and this makes them become desirable candidate's new drug.
Summary of the invention
On the one hand, the invention provides the polymorphic form of the compound shown in formula I, and/or its hydrate or solvate.
Wherein, these polymorphic forms comprise at least two kinds of compound shown in formula I pure crystal habit and a kind of pure glassy solids forms substantially substantially.For convenient, these two kinds of crystal habits are in this called after form I and form II.
In some embodiments, polymorphic form is the glassy solids form of compound shown in formula I, and its feature is that Tg is 52 ℃ ± 10 ℃.
In other embodiments, polymorphic form is the glassy solids form of compound shown in formula I, and its feature is that its X-ray powder diffraction pattern is about 14.3 ° at diffraction angle 2 θ and locates to have characteristic peak.
In other embodiments, the feature of the glassy solids form of compound shown in formula I is, its purity >=85%(: be at least 85%).In other embodiment, its purity is at least 95% or 99%.
In other embodiments, this polymorphic form is the crystal habit I of compound shown in formula I, and its feature is that its X-ray powder diffraction pattern is about 10.3 ° at diffraction angle 2 θ, locates to have characteristic peak for 14.1 ° and 14.6 °.
In other embodiments, this polymorphic form is the crystal habit I of compound shown in formula I, and its feature is, its X-ray powder diffraction pattern is about 10.3 ° at diffraction angle 2 θ, 14.1 °, and 14.6 °, 15.8 °, locate to there is characteristic peak for 16.6 ° and 19.6 °.
In other embodiments, this polymorphic form is the crystal habit I of compound shown in formula I, and its feature is that its X-ray powder diffraction pattern has characteristic peak, represents that spacing is
with
In other embodiments, this polymorphic form is the crystal habit I of compound shown in formula I, and its feature is that its X-ray powder diffraction pattern has characteristic peak, represents that spacing is
with
Another part of the embodiment of polymorphic form of the present invention is crystal habit I.
In some such embodiments, the feature of the crystal habit I of compound shown in formula I is, its X-ray powder diffraction pattern is about 10.3 ° at diffraction angle 2 θ, 14.1 °, and 14.6 °, 15.7 °, and 16.6 ° located to have characteristic peak.
In other embodiments, the crystal habit I of compound shown in formula I, in its X-ray powder diffraction pattern, is about 9.3 ° and 19.6 ° at diffraction angle 2 θ and locates to have extra characteristic peak.
In other embodiment, the feature of the crystal habit I of compound shown in formula I is that fusing point is 174-177 ℃.
In other embodiments, the feature of the crystal habit I of compound shown in formula I is, its purity >=85%(: be at least 85%).In other embodiment, its purity is at least 95% or 99%.
Another part of the embodiment of polymorphic form of the present invention is crystal habit II.
In some such embodiments, the feature of the crystal habit II of compound shown in formula I is that its X-ray powder diffraction pattern is about 10.4 ° at diffraction angle 2 θ, locates to have characteristic peak for 12.1 ° and 13.4 °.
In other embodiments, the crystal habit II of compound shown in formula I, in its X-ray powder diffraction pattern, is about 15.4 ° at diffraction angle 2 θ, locates to have extra characteristic peak for 17.8 ° and 18.8 °.
In other embodiments, the feature of the crystal habit II of compound shown in formula I is that its X-ray powder diffraction pattern has characteristic peak, represents that spacing is
with
In other embodiments, the crystal habit II of compound shown in formula I has extra characteristic peak, represents that spacing is
with
In other embodiment, the feature of the crystal habit II of compound shown in formula I is that fusing point is 209-212 ℃.
In other embodiments, the purity of the crystal habit II of compound shown in formula I is for example not less than 85%(, and its purity is at least 95% or is at least 99%).
On the other hand, the invention still further relates to the preparation method of the crystal polymorphic form of compound shown in formula I.
An embodiment of described method comprises the steps: excessive compound to be added to pulp in solvent, and described solvent is CH
2cl
2, ethyl acetate, acetonitrile, ethanol, methyl alcohol, heptane or its mixture, at least through 24 hours, reclaim the crystal polymorphic form or the glassy solids form that generate.
In some embodiments, the crystal habit of generation can be form I or form II.
In other embodiments, compound shown in formula I under the condition of room temperature or 50 ℃, pulp in mixed solvent.
In other embodiments, the pulp at least 48 hours in solvent of compound shown in formula I.
Described solvent can be the mixture of ethyl acetate/heptane, the mixture of ethanol/heptane.For example, in the mixture of described ethyl acetate/heptane, ethyl acetate and the weight ratio of heptane or the ratio of volume ratio are 1:10.
The invention provides a kind of polymorphic form, compound shown in crystallization formula I in suitable solvent system, described solvent system comprises at least one solvent, polymorphic form can obtain by following method: natural sedimentation (evaporation), cooling, or add anti-solvent (solubleness of compound of the present invention in anti-solvent is relatively low), to obtain the supersaturation of solvent system.Crystallization can also by with or with crystal seed, do not realize, described crystal seed is suitable for crystallization compound of the present invention.
In other embodiments, shown in formula I of the present invention, compound is dissolved in solvent heptane at ambient temperature, obtains desired crystal habit I afterwards by natural sedimentation, and the fusing point of this crystal habit I is 174-177 ℃.
In other embodiments, under room temperature or 50 ℃ of conditions, add mixed solvent to stir compound shown in excessive formula I of the present invention, described mixed solvent is ethyl acetate/heptane (weight ratio or volume ratio are 1:10), at least 48 hours, obtain crystal habit II, the fusing point of this crystal habit II is 209-212 ℃.
In other embodiments, compound shown in excessive formula I of the present invention is dissolved in to dichloromethane solvent under room temperature or 50 ℃ of conditions, by evaporating solvent, obtain afterwards the glassy solids form of compound shown in formula I, the Tg scope of this glassy solids form is 52 ℃ ± 10 ℃.
The present invention further provides the purposes of the polymorphic form (comprising crystal kenel I and II and glassy solids form) of compound shown in formula I, be used for the treatment of the uncomfortable or state of an illness, especially with those state of an illness of inflammation, or for the preparation of the medicament that is used for treating uncomfortable or the state of an illness, for example, these states relate to serious or chronic oxidative stress and inflammation, and especially those Partial Feature are that crossing of derivable nitricoxide synthase (iNOS) or derivable epoxidase (COX-2) expressed.
Therefore, the invention still further relates to pharmaceutical composition or medicament, described pharmaceutical composition or medicament comprise crystalline form I of the present invention or Form II or the glassy solids form of at least treating significant quantity, and medicine acceptable vehicle, auxiliary material or carrier.Selectable, described composition or medicament may further include at least one additional active ingredient.
Pharmaceutical composition of the present invention or medicament can be to be suitable for needing the state of an illness for the treatment of or formulation or the form (for example, tablet or capsule) of disease, and with mode administration well known in the prior art, for example, oral.
Pharmaceutical composition of the present invention or medicament can comprise the polymorphic form (for example, crystal habit I, crystal habit II, or glassy solids form) of for example, shown in the formula I of 1-99 % by weight (, 1-70 % by weight, 10-30 % by weight) compound.
All polymorphic forms of the present invention (comprising crystal habit or glassy solids form) substantially or approximate be pure.As used herein, term " being pure substantially " or " approximate is pure " (for example refer at least 85 % by weight, at least 95 % by weight, or at least 99 % by weight) formula I shown in compound be present in polymorphic form of the present invention, especially in crystal habit I or crystal habit II.
The main peak of above-mentioned crystal and glassy solids form polymorphic form is reproducible, and in limit of error (prescribed value ± 0.2).
In the present invention, " the X-ray powder diffraction pattern shown in Fig. 1 " refers to as shown in Figure 1, main peak shown in X-ray powder diffraction pattern, wherein, main peak refers to that in Fig. 1, those are using climax as reference (relative intensity at climax is appointed as 100%), and relative intensity is greater than 10%, is preferably greater than 30% peak.Same, in the present invention, X-ray powder diffraction pattern shown in Fig. 2 refers to as shown in Figure 2, main peak shown in X-ray powder diffraction pattern, wherein, main peak refers to that in Fig. 2, those are using climax as reference (relative intensity at climax is appointed as 100%), and relative intensity is greater than 10%, is preferably greater than 30% peak.
The present invention also comprises the method for compound shown in preparation formula I, and described method comprises step as shown below:
The present invention also provides compound shown in formula I, or its polymorphic form or be selected from crystal habit I, crystal habit II, the purposes for the preparation of medicine of the form of glassy solids form, described medicine is for suppressing the generation of the NO being induced by IFN-γ in scavenger cell, or be used for the treatment of cancer, or be used for the treatment of the disease with inflammation, uncomfortable, or the state of an illness, for example lupus, or rheumatic arthritis, Crohn disease or ulcerative colitis, cardiovascular disorder, diabetes, one or more complication of diabetes, wherein, described complication is selected from following symptom: obesity, hypertension, atherosclerosis, coronary heart disease, apoplexy, peripheral vascular disease, ephrosis, neuropathy, myonecrosis, retinopathy, metabolic syndrome, X chromosome fragility syndromes, or the combination of above-mentioned two or more symptoms.
At this term used, " treatment significant quantity " refers to the amount of compound, when being a kind of disease for the treatment of, or a kind of disease or at least one uncomfortable clinical symptom and during to body administration one by one, be enough used for affecting the result for the treatment of of these diseases, discomfort or symptom.Described " treatment significant quantity " can be according to compound, disease, discomfort and/or disease or uncomfortable symptom, the seriousness of disease, discomfort and/or disease or uncomfortable symptom, the individual age for the treatment of, and/or treat individual weight and change.Suitable amount in any specific example is obvious to those skilled in the art, maybe can determine by normal experiment.In the situation of composite treatment, (that is, except polymorphic form of the present invention, have at least another kind of active ingredient), term " treatment significant quantity " refers to effectively treat disease, the total amount of the composition of discomfort or situation.
The pharmaceutical composition that comprises compound of the present invention can, by oral, suck, rectum, and injection or topical are to the individual administration of needs treatment.For Oral administration, described pharmaceutical composition can be conventional solid dosage, for example tablet, powder, small-particle, capsule etc., for example water of liquid dosage form or oil suspension or such as syrup of other liquid dosage form, solution, suspension etc.; For drug administration by injection mode, described pharmaceutical composition can be solution, the aqueous solution, oil-based suspension concentrates, lyophilized powder etc.Preferably, the formulation of described pharmaceutical composition is selected from tablet, coated tablet, capsule, suppository, nasal spray or injection, more preferably tablet or capsule.Described pharmaceutical composition can be the single cell administration with exact dosage desired.In addition, described pharmaceutical composition may further include additional active ingredient.
Whole formulations of pharmaceutical composition of the present invention can prepare by the ordinary method of pharmaceutical field.For example, described active ingredient can with one or more mixed with excipients, then make need formulation.Described " medicine acceptable carrier " refers to the conventional pharmaceutical carrier of the pharmaceutical dosage form that is suitable for requirement, for example: thinner, communication media is water such as, various organic solvents, etc.; Filler is starch such as, sucrose, etc.; Tamanori is derivatived cellulose such as, alginate, gelatin and polyvinylpyrrolidone (PVP); Wetting agent is glycerine such as; Disintegrating agent is agar such as, calcium carbonate, sodium bicarbonate; Absorption enhancer, as quarternary ammonium salt compound; Tensio-active agent is hexadecanol such as; For example kaolin of sorbent material and bentonite; Lubricant is mica such as, calcium stearate, and Magnesium Stearate, polyoxyethylene glycol, etc.In addition, described pharmaceutical composition further comprises such as dispersion agent of the acceptable vehicle of other medicines, stablizer, thickening material, complexing agent, buffer reagent, penetration enhancer, polymer, perfume compound, sweeting agent, and staining agent.Preferably, described vehicle is suitable for formulation and the administering mode of requirement.
Used at this, term " solvate " refers to the chemical substance being formed by solvent and Compound Phase mutual effect.Suitable solvate is the acceptable solvate of medicine, and for example hydrate, comprises monohydrate and semihydrate.
Used at this, term " active ingredient " is used to refer to has bioactive chemical substance.In some embodiments, " promoting agent " is the compound with medicinal effectiveness.For example, promoting agent can be anticancer therapeutic agent.
Term " disease " or " discomfort " or " state of an illness " refer to any disease, discomfort, disease, symptom or sign.
Accompanying drawing explanation
Fig. 1 is the X-ray powder diffraction pattern of the crystal habit I of compound shown in formula I;
Fig. 2 is the X-ray powder diffraction pattern of the crystal habit II of compound shown in formula I;
Fig. 3 is the X-ray powder diffraction pattern of the glassy solids form of compound shown in formula I;
Fig. 4 is heat flux (w/g) figure of glassy solids form;
Fig. 5 is that the crystal habit II of compound shown in formula I (CDDO ethyl ester) suppresses the effect that stimulates nitrogen protoxide to produce by IFN-g in mouse macrophage 17.
Embodiment
X-ray powder diffraction pattern (XRPD) shown in Fig. 1, Fig. 2 and Fig. 3 is generated by the PANalytical X-ray diffraction system with sharp shadow supervisory control desk.Diffraction peak position is the silicon single crystal calibration of 28.443 degree by 2 θ values.Use the k-α radiation of sharp shadow Cu LEF X-ray tube as the light source of X ray.
The present invention further illustrates by the following examples, but is not to limit.In an embodiment of the present invention, described technology or method, unless stated otherwise, be technology or the method for routine well known in the prior art.
Compound shown in embodiment 1, synthesis type I
Step 1 is synthesized APSN13B-1
| Title | M.W. | Amount | mol | Equivalent |
| Oleanolic Acid | 456 | 1000g | 2.2mol | 1 |
| EtI | 156 | 376g | 2.4mol | 1.1 |
| K 2 CO 3 | 138 | 604g | 4.4mol | 2 |
| DMF | / | 12L | / | / |
To Oleanolic Acid (1000g, 2.2mol) and salt of wormwood (604g, 4.4mol), be dissolved in DMF(12L) solution in add iodoethane (376g, 2.4mol).Gained mixture stirs and spends the night under the condition of 45 ℃.After can't detect Oleanolic Acid with HPLC, described mixture is cooled to room temperature and pours in water (120L).The suspension agitation obtaining 30 minutes.By whizzer, collect solid, water (1L) washs and 50 ℃ of vacuum-dryings, obtains 976g APSN13B-1 for subsequent step.Productive rate is 92%.
Step 2, synthetic APSN13B-2
| Title | M.W. | Amount | mol | Equivalent |
| APSN13B-1 | 484 | 975g | 2mol | 1 |
| Ac 2 O | 102 | 612g | 6mol | 3 |
| Pyridine | 79 | 474g | 6mol | 3 |
| THF | / | 6L | / | / |
| DMAP | 122 | 24.4g | 0.2mol | 0.1 |
At 45 ℃ to APSN13B-1(975g, 2mol), pyridine (474g), DMAP(24.4g, 0.2mol) be dissolved in THF(6L) mixture in add diacetyl oxide (612g, 6mol).Described solution stirring is spent the night.After reaction finishes, described solution is poured in water (60L).Solid is collected by whizzer and 45 ℃ of vacuum-dryings, is obtained 847g APSN13B-2 for subsequent step.Productive rate is 80%.
Step 3, synthetic APSN13B-3
| Title | M.W. | Amount | mol | Equivalent |
| APSN13B-2 | 526 | 846g | 1.6mol | 1 |
| H 2 O 2 | 34 | 136g | 4mol | 2.5 |
| HCOOH | 46 | 1L | / | / |
| DCM | / | 5L | / | / |
At ambient temperature, to APSN13B-2(846g, 1.6mol), formic acid (1L) is dissolved in DCM(5L) in solution in slowly to add mass concentration be 30% aqueous hydrogen peroxide solution (the 453g aqueous solution, 4mol hydrogen peroxide) and stir and spend the night.To adding water (2L) in gained solution and extracting with DCM.The full NaHCO closing for organic phase
3solution washing, is neutral to water, then, washes and use anhydrous Na SO with strong brine
4dry.Filter organic phase, filtrate is evaporated under vacuum condition, obtains 871gAPSN13B-3, productive rate 100%.This material, for next step reaction, does not carry out further purifying.
Step 4, synthetic APSN13B-4
| Title | M.W. | Amount | Mol | Equivalent |
| APSN13B-3 | 542 | 871g | 1.6mol | 1 |
| HBr | 81 | 30ml | / | / |
| Br 2 | 160 | 768g | 4.8mol | 3 |
| HAc | / | 7L | / | / |
At 45 ℃, to APSN13B-3((871g, 1.6mol) be dissolved in the solution of acetic acid (5L) and add Hydrogen bromide (30ml) to be dissolved in the solution of acetic acid (40%), then slowly add bromine simple substance (256g) to be dissolved in the solution of acetic acid (700ml).Gained mixture stirs 30 minutes at 45 ℃.At ambient temperature, slowly add another part of bromine simple substance (512g) to be dissolved in the solution of acetic acid (1.3L), and continue to stir and spend the night.When reaction finishes, gained mixture is poured in cold water (35L).Collect gained solid, with full, close sodium sulfite solution washing and be dried in a vacuum, obtain yellow solid 841g APSN13B-4, productive rate is 97%.
MS-ESI(m/z):541[M+H]
+
| Title | M.W. | Amount | mol | Equivalent |
| APSN13B-4 | 540 | 840g | 1.56mol | 1 |
| KOH | 56 | 263g | 4.7mol | 3 |
| EtOH | / | 4.5L | / | / |
By APSN13B-4(840g, 1.56mol) and KOH(263g, 4.7mol) be dissolved in EtOH(4.5L) vlil 30 minutes.Remove in a vacuum after EtOH the HCl acidified aqueous solution that the mixture obtaining is 6N by concentration.Ethyl acetate for solution layer (3L) extraction.The full NaHCO closing for the organic phase obtaining
3solution washing, strong brine washing, and use anhydrous Na SO
4dry.Filter organic phase, filtrate is evaporated under vacuum condition, obtains 928g APSN13B-5, productive rate 100%.
MS-ESI(m/z):499[M+H]
+.
Step 6, synthetic APSN13B-6
| Title | M.W. | Amount | mol | Equivalent |
| APSN13B-5 | 498 | 927g | 1.8mol | 1 |
|
|
100 | 400ml | 1.8mol | 1 |
| Acetone | / | 5L | / | / |
To APSN13B-5((927g, 1.8mol in ice bath) be dissolved in the solution of acetone (5L) and drip Jones reagent (400ml).Gained mixture at room temperature stirs, until can't detect APSN13B-5 with TLC.Remove after acetone, in gained mixture, add water.Gained water mixture extracts with DCM.The full NaHCO that closes for the organic phase obtaining
3solution washing, strong brine washing, uses anhydrous Na SO
4dry, and filter.Filtrate is evaporated under vacuum condition, obtains thick APSN13B-6.By recrystallization in oil and ethyl acetate, obtain the pure APSN13B-6 of 498g, productive rate 54%.
MS-ESI(m/z):497[M+H]
+
Step 7, synthetic APSN13B-7
| Title | M.W. | Amount | mol | Equivalent |
| APSN13B-6 | 496 | 496g | 1mol | 1 |
| CH 3 ONa | 54 | 216g | 4mol | 4 |
| HCOOEt | 74 | 185g | 2.5mol | 2.5 |
| Dry toluene | / | 2L | / | / |
To APSN13B-6(496g, 1mol) be dissolved in the solution of dry toluene (2L) and add ethyl formate (185g, 2.5mol) and CH
3oNa(216g, 4mol).Gained mixture at room temperature stirs 2 hours.Then, gained ethyl acetate (1L) dilution for mixture, and with 5% HCl solution washing (3 times).Water extracts by ethyl acetate again, and merge organic phase with strong brine, wash, use anhydrous Na SO
4dry, and filter.Filtrate is evaporated under vacuum condition, obtains 497gAPSN13B-7, productive rate 95%.
MS-ESI(m/z):525[M+H]
+
| Title | M.W. | Amount | mol | Equivalent |
| APSN13B-7 | 524 | 496g | 0.94mol | 1 |
| NH 2 OH?HCl | 69 | 98g | 1.4mol | 1.5 |
| EtOH | / | 2.5L | / | / |
| Water | / | 200ml | / | / |
To APSN13B-7(496g, 0.94mol) be dissolved in EtOH(2.5L) and the solution of water (200ml) in add oxammonium hydrochloride (98g, 1.4mol).Gained mixture is heated and reflux 2 hours.Gained mixture is concentrated under vacuum condition, and adds water (2L).EA extraction (3 times) for gained mixture.The organic phase water that gained merges and strong brine washing, use anhydrous Na SO
4dry, and filter.Filtrate is evaporated under vacuum condition, obtains 453g APSN13B-8, productive rate 92%.
MS-ESI(m/z):522[M+H]
+
Step 9, synthetic APSN13B-9
| Title | M.W. | Amount | mol | Equivalent |
| APSN13B-8 | 521 | 452g | 0.86mol | 1 |
| CH 3 ONa | 54 | 56g | 1.04mol | 1.2 |
| EtOH | / | 1.5L | / | / |
To APSN13B-8(452g, 0.86mol under condition of ice bath) be dissolved in EtOH(1.5L) solution in add CH
3oNa(56g, 1.04mol).Gained mixture at room temperature stirs 1 hour.Then, gained mixture is concentrated under vacuum condition, and adds ethyl acetate (2L).5% HCl solution washing (3 times) for gained mixture.Water extracts with EA again, and merge organic layer with strong brine, wash, use anhydrous Na SO
4dry, and filter.Filtrate is evaporated under vacuum condition, obtains 429g APSN13B-9, productive rate 95%.
MS-ESI(m/z):522[M+H]
+
| Title | M.W. | Amount | mol | Equivalent |
| APSN13B-9 | 521 | 428g | 0.82mol | 1 |
| DDQ | 227 | 205g | 0.90mol | 1.1 |
| Toluene | / | 2.5L | / | / |
Spend the night and heat and reflux APSN13B-9(428g, 0.82mol) and DDQ(205g, 0.90mol) be dissolved in the mixture of toluene.After removing by filter insolubles, filtrate is evaporated and is obtained thick solid under vacuum condition.Gained solid obtains 341g APSN13B-10 by rapid column chromatography, productive rate 80%.Products therefrom is further purified by recrystallization in ethyl acetate and sherwood oil, obtains the product of chemical purity.The maximum contaminant content of finished product is lower than 0.1%.
MS-ESI(m/z):520[M+H]
+
1H-NMR(CDCl
3):δ8.04(1H,S),5.96(1H,S),4.17(2H,t,J=6.9Hz),3.05(1H,d,J=13.5Hz),2.95(1H,d,J=4.5Hz),2.04～0.07(21H,m).
The crystal habit I of compound shown in embodiment 2, preparation formula I
Compound as shown in formula I prepared by method as described in above-described embodiment 1, is dissolved in heptane solvent in room temperature, obtains desired crystal habit I afterwards by natural sedimentation, and its fusing point is 174-177 ℃.
Its X-ray powder diffraction pattern shown in Fig. 1, is summarized in table 1.
table 1
The crystal habit II of compound shown in embodiment 3, preparation formula I
Compound as shown in formula I prepared by method as described in above-described embodiment 1, excessive described compound is added to the mixed solvent of ethyl acetate and heptane, and (ratio of ethyl acetate and heptane is 1:10, weight ratio or volume ratio) in, stirring makes its pulp, under room temperature or 50 ℃ of conditions, stir at least 48 hours, obtain crystal habit II, its fusing point is 209-212 ℃.
Its X-ray powder diffraction pattern shown in Fig. 2, is summarized in table 2.
table 2
The glassy solids form of compound shown in embodiment 4, preparation formula I
Compound as shown in formula I prepared by method as described in above-described embodiment 1, under room temperature or 50 ℃ of conditions, the slurry suspension that contains excessive described compound is dissolved in methylene chloride, obtains afterwards the glassy solids form of compound shown in formula I by evaporating solvent, its Tg is 52 ℃ ± 10 ℃.
Fig. 3 has shown the X-ray powder diffraction pattern of the glassy solids form of compound shown in formula I; Fig. 4 has shown heat flux (w/g) figure of glassy solids form.
The preliminary pharmacokinetics of the polymorphic form of compound shown in embodiment 5, formula I
Female CD-1 mouse, three kinds of polymorphics of compound (CDDO ethyl ester) shown in abdominal injection 10mg/kg formula I and methyl 2-cyano group-3,12-dioxo volatile oil-1,9 (11) dienes-28-ester (CDDO methyl ester) is dissolved in DMSO-polyoxyethylenated castor oil-PBS(1:1:8, weight ratio or volume ratio) solution.After administration, respectively at 0.083,0.25,0.5,1,2,4,8, and 24 hours blood sample collections.Utilize HPLC/MS to detect the level in blood sample, compound is added in blank blood sample as standard.
Pharmacokinetics result is as shown in table 3.The system of three kinds of polymorphic forms of the ethyl ester of Compound C DDO shown in formula I exposes higher than CDDO methyl ester.It is their order below: the crystal habit I>CDDO methyl ester of the crystal habit II>CDDO ethyl ester of the glassy solids form >CDDO ethyl ester of CDDO ethyl ester
table 3
The mensuration of the stability of embodiment 6, crystal habit
The crystal habit II of compound shown in the formula I that as described in Example 3 prepared by method detects respectively under 25 ℃/60% relative humidity and 40 ℃/75% relative humidity condition.The XRD figure of all samples does not significantly change.In this research, similarly use other polymorphic form of the present invention.In this research, compared with other polymorphic form of the present invention, the crystal habit II of compound shown in formula I shows to have optimum thermodynamic stability.
The mensuration of the stability of embodiment 7, glassy solids form
Shown in the formula I that as described in Example 4 prepared by method, the glassy solids form of compound is placed one week respectively under the condition of 25 ℃/60% relative humidity and 40 ℃/75% relative humidity, detects afterwards.The XRD figure of all samples does not significantly change.
Testing process general introduction
RAW264.7 cell cultures
1, RAW264.7 cell is cultivated in the DMEM that contains 10%FBS, and condition is 37 ℃, 5%CO
2environment.
2, use microscopic examination culture, evaluate the degree definite bacterium and the fungal contamination of not existing that merge.
3, remove the substratum of using.
4, use the D-PBS washed cell individual layer of preheating.
5, with suction pipe, pipette 3ml Regular Insulin/EDTA and be added on washed cell monolayer, rotary flask, makes Regular Insulin cover individual layer.
6, with microscopy cell and confirm that whole cells are all from wall adrift.
7, with a small amount of substratum containing fresh serum, cell is suspended again so that Regular Insulin inactivation.
8, use centrifugal method collecting cell, centrifugal 5 minutes of 1000rpm at ambient temperature.
9, in 37 ℃ at 5%CO
2environment incubated cell.
In RAW264.7 cell, produce nitrogen protoxide
1, seed cells on 96 porocyte culture plates, number of cells is 1 × 10
5/ hole.
2, at 37 ℃, 5%CO
2under environment after incubated cell 2 hours, absorb not attached cell and add the perfect medium that contains 10ng/ml IFN-γ of firm preparation and the compound of dilution.
3, in 37 ℃ at 5%CO
2incubated cell 48 hours again under environment.
Measure the intracellular nitrogen protoxide of RAW
1, with whizzer with the rotating speed of 1000rpm centrifugal 5 minutes, collect supernatant liquor substratum.
2, by the 0.1M nitrite standardized solution providing according to 1:1, matrix or damping fluid dilution for 000 ratio, preparation 1ml concentration is the nitrite solution of 100 μ M, as laboratory sample.
3, prepare nitrite standardized solution, concentration is respectively 100,50,25,12.5,6.25,3.13,1.56 and 0 μ M, 50 μ l/ holes
4, make sulfanilamide (SN) solution and NED solution equilibria to room temperature (15-30 minute).
5, Xiang Kongzhong adds 50 μ l laboratory samples, duplicate.
In the hole of the nitrite standardized solution that 6, with multichannel pipettor, 50 μ l sulfanilamide (SN) solution is assigned to all laboratory samples and contains a series of dilutions.
7, lucifuge, at room temperature hatches 5-10 minute.
8, with multichannel pipettor by 50 μ lNED solution be assigned to institute porose in.
9, lucifuge, at room temperature hatches 5-10 minute.Formed immediately purple/carmetta.
10,, in 30 minutes, use with absorbing wavelength and measure absorbancy at the plate reader of the optical filtering between 520nm to 550nm.
Fig. 5 has shown that the crystal habit II of compound shown in formula I (CDDO ethyl ester) (passes through IC to the nitric oxide production inhibition being produced by IFN-g induction in mouse macrophage 17 is active
50(nM) value metering).The IC of the crystal habit II of compound shown in formula I (CDDO ethyl ester)
5034nM.The IC of the glassy solids form of compound shown in formula I (CDDO ethyl ester)
5032nM.
Claims (29)
1. the polymorphic form of the compound shown in a formula I
It is characterized in that, described polymorphic form is crystal habit I, and the X-ray powder diffraction pattern of crystal habit I is 10.3 ° at diffraction angle 2 θ, locates to have characteristic peak for 14.1 ° and 14.6 °.
2. polymorphic form according to claim 1, is characterized in that, described polymorphic form is crystal habit I, and its X-ray powder diffraction pattern has characteristic peak, represents that spacing is
with
3. polymorphic form according to claim 1, is characterized in that, described polymorphic form is crystal habit I, and its X-ray powder diffraction pattern is about 10.3 ° at diffraction angle 2 θ, 14.1 °, 14.6 °, 15.8 °, locates to have characteristic peak for 16.6 ° and 19.6 °.
4. polymorphic form according to claim 3, is characterized in that, described polymorphic form is crystal habit I, and its X-ray powder diffraction pattern has characteristic peak, represents that spacing is
with
5. polymorphic form according to claim 1, is characterized in that, described polymorphic form is crystal habit I, and its X-ray powder diffraction pattern is about 10.3 ° at diffraction angle 2 θ, 14.1 °, and 14.6 °, 15.7 °, and 16.6 ° located to have characteristic peak.
6. polymorphic form according to claim 5, is characterized in that, in the X-ray powder diffraction pattern of described polymorphic form, is about 9.3 ° and 19.6 ° locates to have extra characteristic peak at diffraction angle 2 θ.
7. according to the polymorphic form one of claim 1-6 Suo Shu, it is characterized in that, the fusing point of described polymorphic form is 174-177 ℃.
8. according to the polymorphic form one of claim 1-6 Suo Shu, it is characterized in that purity >=85% of described polymorphic form.
9. polymorphic form according to claim 8, is characterized in that, purity >=95% of described polymorphic form.
10. polymorphic form according to claim 9, is characterized in that, purity >=99% of described polymorphic form.
The method of the polymorphic form of compound shown in 11. 1 kinds of preparation formula I, the method comprises the steps: excessive compound to be added to pulp in solvent, and described solvent is CH
2cl
2, ethyl acetate, acetonitrile, ethanol, methyl alcohol, heptane or its mixture, at least through 24 hours, reclaim the crystal polymorphic generating.
12. methods according to claim 11, is characterized in that, the polymorphic form of compound shown in described formula I is crystal habit I.
13. methods according to claim 11, is characterized in that, compound shown in described formula I under the condition of room temperature or 50 ℃, pulp in mixed solvent.
14. methods according to claim 11, is characterized in that, the pulp at least 48 hours in solvent of compound shown in described formula I.
15. methods according to claim 11, is characterized in that, described solvent is CH
2cl
2, ethyl acetate, acetonitrile, ethanol, methyl alcohol, heptane, ethanol/heptane mixture, ethyl acetate/heptane mixture, or its mixture.
16. methods according to claim 12, is characterized in that, described ethanol/heptane mixture contains ethanol and heptane, and the weight ratio of ethanol and heptane or the ratio of volume ratio are 1:10.
The method of the polymorphic form of compound shown in 17. 1 kinds of preparation formula I, the method comprises the steps: described compound to be dissolved at ambient temperature the mixed solvent of ethanol and heptane, compound natural sedimentation forms crystal polymorphic, reclaims the crystal polymorphic generating.
18. 1 kinds of pharmaceutical compositions, is characterized in that, said composition contains treats the described polymorphic form of one of claim 1-10 of significant quantity, and medicine acceptable vehicle, auxiliary material or carrier.
19. pharmaceutical compositions according to claim 18, is characterized in that, described composition further comprises additional active ingredient.
20. according to the pharmaceutical composition described in claim 18 or 19, it is characterized in that, described composition is by oral way administration.
21. pharmaceutical compositions according to claim 18, is characterized in that, described composition is used with tablet or capsule form.
22. according to the pharmaceutical composition described in claim 18 or 19, it is characterized in that, described composition comprises the polymorphic form of 1-99 % by weight.
23. pharmaceutical compositions according to claim 22, is characterized in that, described composition comprises the polymorphic form of 1-70 % by weight.
24. pharmaceutical compositions according to claim 23, is characterized in that, described composition comprises the polymorphic form of 10-30 % by weight.
The purposes of one of 25. claim 1-10 described polymorphic form, for the preparation of the medicine of the generation that is used for suppressing the NO being induced by IFN-γ in scavenger cell.
The purposes of the described polymorphic form of one of 26. claim 1-10, for the preparation of being used for treating cancer, or with the medicine of the state of an illness of inflammation.
The purposes of 27. polymorphic forms according to claim 26, is characterized in that, the described state of an illness with inflammation is lupus, or rheumatic arthritis, Crohn disease or ulcerative colitis, cardiovascular disorder, diabetes, or the complication of diabetes.
The purposes of 28. polymorphic forms according to claim 27, is characterized in that, the complication of described diabetes is fat, hypertension, atherosclerosis, coronary heart disease, apoplexy, peripheral vascular disease, ephrosis, neuropathy, myonecrosis, retinopathy, metabolic syndrome, X chromosome fragility syndromes, or the combination of above-mentioned symptom.
Prepare the method for the polymorphic form of claim 1 formula shown I compound, comprise step as shown below for 29. 1 kinds:
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Non-Patent Citations (5)
| Title |
|---|
| TADASHI HONDA, ET AL.: "A Novel Dicyanotriterpenoid, 2-Cyano-3,12- dioxooleana-1,9(11)-dien-28-onitrile, Active at Picomolar Concentrations for Inhibition of Nitric Oxide", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| TADASHI HONDA, ET AL.: "A Novel Dicyanotriterpenoid, 2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile, Active at Picomolar Concentrations for Inhibition of Nitric Oxide Production", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| TADASHI HONDA, ET AL.: "Synthetic oleanane and Ursane Triterpenoids with Modified Rings A and C : A Series of Highly Active Inhibitors of Nitric oxide Production in Mouse Macrophages", 《JOURNAL OF MEDICINAL CHEMISTRY》 * |
| TADASHI HONDA，ET AL.: "Extremely potent triterpenoid inducers of the phase 2", 《PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA》 * |
| 苏德森，王思玲 主编: "《物理药剂学》", 30 July 2004, 化学工业出版社 * |
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