CN101080393A - New (5's)-2',4'-dicyclopentyl -3'-9(s)-fluoro(4-(trifluoromethyl) phenyl)methyl)-5',8' -dihydro-6'h-spiro(cyclopropane-1,7'-quinoline)- 5'-ol is a cholesterol ester transfer protein inhibitor useful f - Google Patents
New (5's)-2',4'-dicyclopentyl -3'-9(s)-fluoro(4-(trifluoromethyl) phenyl)methyl)-5',8' -dihydro-6'h-spiro(cyclopropane-1,7'-quinoline)- 5'-ol is a cholesterol ester transfer protein inhibitor useful f Download PDFInfo
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- 0 *=C(CC1(CC1)C1)CC1=O Chemical compound *=C(CC1(CC1)C1)CC1=O 0.000 description 4
- LNXIAMWHYDACNV-UKTHLTGXSA-N N/C(/C1CCCC1)=C/C(c1ccc(C(F)(F)F)cc1)=O Chemical compound N/C(/C1CCCC1)=C/C(c1ccc(C(F)(F)F)cc1)=O LNXIAMWHYDACNV-UKTHLTGXSA-N 0.000 description 1
- DMHJVPIJLQFOMT-UHFFFAOYSA-N O=C(C(C1C2CCCCC2)=C(C2CCCC2)NC(CC2(CC2)C2)=C1C2=O)c1ccc(C(F)(F)F)cc1 Chemical compound O=C(C(C1C2CCCCC2)=C(C2CCCC2)NC(CC2(CC2)C2)=C1C2=O)c1ccc(C(F)(F)F)cc1 DMHJVPIJLQFOMT-UHFFFAOYSA-N 0.000 description 1
- KVFDZFBHBWTVID-UHFFFAOYSA-N O=CC1CCCCC1 Chemical compound O=CC1CCCCC1 KVFDZFBHBWTVID-UHFFFAOYSA-N 0.000 description 1
- WDRGEJKXDHSNOW-QHCPKHFHSA-N O[C@@H]1c2c(C3CCCCC3)c(C(c3ccc(C(F)(F)F)cc3)=O)c(C3CCCC3)nc2CC2(CC2)C1 Chemical compound O[C@@H]1c2c(C3CCCCC3)c(C(c3ccc(C(F)(F)F)cc3)=O)c(C3CCCC3)nc2CC2(CC2)C1 WDRGEJKXDHSNOW-QHCPKHFHSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/06—Ring systems of three rings
- C07D221/16—Ring systems of three rings containing carbocyclic rings other than six-membered
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Abstract
The invention relates to novel derivatives of tetrahydroquinoline with a general formula (I); wherein, R <1> represents cyclohexyl or cyclopentyl, and R <2> and R <3> represent methyl or the R <2> and R <3> form cyclobutane together, and R <4> represents cyclopentyl or isopropyl, salt, solvate and the salt solvate. The invention also discloses a production method for the derivatives, the application of the derivative or the combination for curing or preventing diseases and the produced medicine, in particular to an inhibitor as cholesteryl ester transfer protein (CETP) which cures/prevents cardiovascular diseases, in particular hypolipoproteinemia, abnormal fat blood sickness, hypertriglyceridemia,hyperlipidemia hypercholesterolemia and arteriosclerosis. .
Description
The present invention relates to new tetrahydroquinoline derivative, its preparation method, it is for treatment and/or self or the applied in any combination that ward off disease, and it is for the application of preparation medicine, particularly as treating and/or prevent cardiovascular disorder, particularly hypolipoproteinemia, dyslipidemia, hypertriglyceridemia, hyperlipidaemia, the inhibitor of hypercholesterolemia and arteriosclerotic cholesteryl ester transfer protein (CETP).
The coronary heart disease that is caused by arteriosclerosis is one of dead main cause of modern society.In big quantity research, the low plasma concentration that shows the HLD cholesterol is important risk factor [Barter and Rye, the Atherosclerosis that arteriosclerosis forms
121, 1-12 (1996)].Except LDL (low-density lipoprotein) and VLDL (very low-density lipoprotein), (high-density lipoprotein (HDL)) is a class lipoprotein, and its most important function is the transportation of fat in blood, for example cholesterol, cholesteryl ester, triglyceride level, lipid acid or phosphatide.High LDL cholesterol concentration (＞160mg/dl) and the low HDL cholesterol concentration (＜40mg/dl) facilitated arteriosclerotic formation [ATP III Guidelines, Report of the NCEP ExpertPanel] basically.Except coronary heart disease, disadvantageous HLD/LDL ratio has also promoted the formation of peripheral vascular disease and apoplexy.Therefore, the novel method that improves HDL cholesterol in the blood plasma be prevent and treat arteriosclerosis and with the treatment of its diseases associated on useful progress.
The cholesteryl ester in cholesteryl ester transfer protein (CETP) the adjusting blood between different lipoprotein and exchange [Tall, the J.Lipid Res. of triglyceride level
34, 1255-74 (1993)].Here particularly importantly cholesteryl ester is by the transfer of HDL to LDL, and it has caused the minimizing of blood plasma HDL cholesterol concentration.Gu this, the inhibition of CETP should cause the minimizing of the rising of plasma concentration of HDL cholesterol and LDL cholesterol blood plasma concentration and therefore cause in the blood plasma influence [McCarthy, Medicinal Res.Rev. useful in the treatment of lipid in nature
13, 139-59 (1993); Sitori, Pharmac.Ther.
67, 443-47 (1995); Swenson, J.Biol.Chem.
264, 14318 (1989)].
Tetrahydroquinoline with pharmacological activity is by EP-A-818 448, and WO 99/14215, and WO99/15504 and WO 03/028727 are known.Substituted-tetrahydro quinoline with pharmacological activity is known by WO 99/14174.
The purpose of this invention is to provide control disease, the new material of cardiovascular disorder particularly, this material has improved curative properties.
The invention provides the have structural formula compound of (I)
R wherein
1Represent cyclohexyl or cyclopentyl, R
2And R
3Represent methylidene or form tetramethylene, R together separately
4Represent cyclopentyl or sec.-propyl,
With their salt, the solvate of solvate and salt.
The present invention provide especially and had systematic naming method (5 ' S)-4 '-cyclohexyl-2 '-cyclopentyl-3 '-{ (S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-5 '-compound of pure and mild structural formula (Ia)
With its salt, the solvate of solvate and salt.
The present invention also provide especially and had systematic naming method (5 ' S)-2 ', 4 '-two cyclopentyl-3 '-{ (S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-5 '-compound of pure and mild structural formula (Ib)
With its salt, the solvate of solvate and salt.
The present invention also provide especially and had systematic naming method (5 ' S)-4 '-cyclopentyl-3 '-{ (S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-2 '-sec.-propyl-5 ', 8 '-dihydro-6 ' H-spiral shell [tetramethylene-1,7 '-quinoline]-5 '-compound of pure and mild structural formula (Ic)
With its salt, the solvate of solvate and salt.
The present invention also provides especially has systematic naming method (5S)-2,4-two cyclopentyl-3-{ (S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-7,7-dimethyl-5,6,7, the compound of the pure and mild structural formula of 8-tetrahydroquinoline-5-(Id)
With its salt, the solvate of solvate and salt.
The present invention also provides especially has systematic naming method (5S)-4-cyclopentyl-3-{ (S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-2-sec.-propyl-7,7-dimethyl-5,6,7, the compound of the pure and mild structural formula of 8-tetrahydroquinoline-5-(Ie)
With its salt, the solvate of solvate and salt.
Hereinafter, the compound that has according to formula of the present invention (Ia)-(Ie) is called the compound that has according to formula of the present invention (I) with odd number.
Also can exist according to compound of the present invention with stereomeric form (enantiomer, diastereomer).The present invention includes all enantiomers, diastereomer and their mixtures separately.From the mixture of this enantiomer and/or diastereomer, can separate stereomeric homogeneous component in known manner.Show in the preferred formula (I) at C-5 ' with at the S-of C-3 ' a configuration.
In the context of the present invention, preferred salt is the physiological acceptable salt according to compound of the present invention.The present invention also comprises and itself is not suitable for medicinal application, but can for example be used for the salt of isolated or purified according to compound of the present invention.
Physiological acceptable salt according to compound of the present invention comprises mineral acid, the acid salt of carboxylic acid and sulfonic acid, spirit of salt for example, Hydrogen bromide, sulfuric acid, phosphoric acid, methanesulfonic, ethane sulfonic acid, toluenesulphonic acids, Phenylsulfonic acid, naphthalene disulfonic acid, acetate, trifluoroacetic acid, propionic acid, lactic acid, tartrate, oxysuccinic acid, citric acid, fumaric acid, toxilic acid and benzoic salt.
The salt that also comprises conventional base according to the physiological acceptable salt of compound of the present invention, for example with preferred as alkali salt (for example sodium salt and sylvite), alkaline earth salt (for example calcium salt and magnesium salts) and derived from ammonia or have the organic amine of 1-16 carbon atom, for example with preferred ethamine, diethylamine, triethylamine, ethyl diisopropylamine, Monoethanolamine MEA BASF, diethanolamine, trolamine, dicyclohexylamine, dimethylaminoethanol, PROCAINE HCL, PHARMA GRADE, dibenzyl amine, N-methylmorpholine, arginine, Methionin, the ammonium salt of quadrol and N-methyl piperidine.
In the context of the present invention, solvate refers to those forms of compound of the present invention, and its state with solid or liquid forms title complex by the coordination with solvent molecule.Hydrate is the particular form of solvate, and wherein coordination is to carry out with water.In the context of the present invention, preferred solvate is a hydrate.
And the present invention also comprises the prodrug (Prodrug) according to compound of the present invention.Term " prodrug " comprise itself can be biological activity or nonactive but its only in health the retention period transformed the compound that (for example metabolism or hydrolysis) becomes compound of the present invention.
In the context of the present invention, (C
1-C
4)-alkyl represent has the straight chain or the branched alkyl groups of 1-4 carbon atom.For example and preferably can mention following groups: methyl, ethyl, just-and propyl group, sec.-propyl, just-and butyl, different-butyl, the second month in a season-butyl and tert-butyl.
The present invention also provides and has prepared the compound method that has according to formula of the present invention (I), wherein R
1, R
2, R
3And R
4Show as defined above and for its mode of the compound with formula (Ia) separately, be characterised in that the have formula compound of (II) with embodiment
After it
[A] changes into the have formula compound of (IV) by introducing hydroxy-protective group
Wherein
PG representation hydroxy blocking group preferably has formula-SiR
1R
2R
3Group, wherein
R
1, R
2And R
3Be identical or different and representative (C
1-C
4)-alkyl,
Change into compound by the cis-selectivity reduction afterwards with formula V
Wherein
PG as defined above,
Or the opposite order of reaction sequence
[B] at first produces the compound with formula (IV) by the cis-selectivity reduction
Introducing hydroxy-protective group PG by regioselectivity after it changes into the compound with formula V,
Use the fluorizating agent reaction to produce compound after having the formula V compound with formula (VII)
Wherein PG as defined above,
Remove hydroxy-protective group PG again by ordinary method afterwards and produce compound with formula (I)
The compound that randomly has a formula (I) is with suitable (i) solvent and/or (ii) alkali or acid change into its solvate, the solvate of salt and/or salt.
Compound with formula (II) can be by having formula (VIII) with the 3-component reaction under the condition that exists at protonic acid or Lewis acid, (IX) and compound (X)
Reaction produces the compound with formula (XI) each other
This compound of oxidation afterwards becomes to have the compound of formula (II).
It is commercially available to have formula (VIII) and compound (X), known or be similar to the known method of document preparation (referring to WO 99/14215 and WO 03/028727) by document.
Compound with formula (IX) can obtain by the witig reaction of acid catalyzed cyclopropanone acetal and 1-(the triphenyl phosphorane is pitched base) acetone (1-(triphenylphosphoranylidene) acetone) to produce 1-ring propylidene benzylacetone and to react (square case 1 with malonic ester subsequently; Referring to WO 03/028727 and I.Kortmann, B.Westermann, Synthesis1995,931-933).
For the suitable inert solvent of each method steps is ether for example, ether for example, Di Iso Propyl Ether, two alkane, tetrahydrofuran (THF), ethylene glycol dimethyl ether or diethylene glycol dimethyl ether, hydrocarbon, for example, benzene for example, toluene, dimethylbenzene, hexane, hexanaphthene or mineral oil component, or halohydrocarbon, for example methylene dichloride, trichloromethane, tetracol phenixin, 1, the 2-ethylene dichloride, trieline, or phenalgin.Also may use the mixture of the solvent of mentioning.
At method steps (II) → (III), (IV) → (V) and (III) → (V) reduction in uses the reductive agent that is suitable for ketone is reduced into oxy-compound to carry out usually.These are particularly including complexing aluminum hydride or hydroborate, lithium hydride for example, sodium hydride, potassium hydride KH, zinc borohydride, lithium aluminum hydride, diisobutyl aluminium hydride (DIBAH), dihydro two (2-methoxy ethoxy) aluminium sodium, trialkylboron lithium hydride or hydrogenation tri-alkoxy aluminium lithium, or borane complexes, for example borine tetrahydrofuran (THF), borine dimethyl thioether or borine N, N-diethylbenzene amine complex.
Asymmetric reduction in method steps (II) → (III) the corresponding isomer of catalytic amount (0.01-0.3 molar equivalent) pure (1R, 2S)-carry out under the condition that 1-aminoidan-2-alcohol exists as the chiral induction body.The preferred for this purpose reductive agent that uses is a borine, N, N-diethylbenzene amine complex.Usually in a kind of ether listed above or in toluene, preferably in carrying out tetrahydrofuran (THF), at-80 ℃-+50 ℃, preferred 0 ℃-+30 ℃ temperature range is carried out in reaction.
For reduction (IV) → (V) and the preferred diisobutyl aluminium hydride of reductive agent (DIBAH) that (III) → (V) uses.Usually in a kind of ether listed above or in toluene, preferably in carrying out tetrahydrofuran (THF) or toluene, at-80 ℃-+50 ℃, preferred-60 ℃-+30 ℃ temperature range is carried out in reaction.
For method steps (III) → (IV) or (VI) → (V) preferred hydroxy-protective group is silyl-group, for example trimethyl silyl, triethylsilyl, triisopropyl silyl or tert-butyl dimetylsilyl.Preferred especially tert-butyl dimetylsilyl.Silyl-group is usually at alkali, triethylamine for example, N, N-diisopropyl ethyl amine, pyridine, 2,6-lutidine or 4-N, under the condition that N-dimethyl aminopyridine (DMAP) exists at listed above a kind of hydrocarbon as solvent, halohydrocarbon, ether or in dimethyl formamide, add.
In method steps (III) → (IV), the silylating agent of use preferably with as 2 of alkali, 6-lutidine bonded trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester.Preferably in methylene dichloride or toluene, at-40 ℃-+40 ℃, preferred-20 ℃-+30 ℃ temperature range is carried out in reaction.
In method steps (VI) → (V), the silylating agent of use preferably with triethylamine and DMAP bonded tert-butyl dimetylsilyl muriate as alkali.Reaction is preferably in dimethyl formamide, and at 0 ℃-+100 ℃, preferred+temperature range of 20 ℃-+80 ℃ is carried out.
Fluoridizing usually at a kind of hydrocarbon listed above or halohydrocarbon or acetonitrile in method steps (VI) → (VII) preferably carries out in toluene or methylene dichloride, uses diethylamino sulfur trifluoride (DAST) or morpholine sulfur trifluoride as fluorizating agent.
The removing usually in acid of silyl blocking group in method steps (VII) → (I) for example under the help of spirit of salt or trifluoroacetic acid, or at fluorochemical, for example carried out under the help of hydrogen fluoride or tetrabutyl ammonium fluoride (TBAF).Suitable inert solvent is an ether listed above, alcohol, for example methyl alcohol or ethanol, or the mixture of solvent listed above.Removing common use TBAF carries out in as the tetrahydrofuran (THF) of solvent.Reaction is usually at-20 ℃-+60 ℃, and preferred 0 ℃-+30 ℃ temperature range is carried out.
Condensation reaction (VIII)+(IX)+(X) → (XI) usually in a kind of ether listed above, at alcohol, methyl alcohol for example, ethanol, just-propyl alcohol or Virahol in, carry out at the mixing species of acetonitrile or the solvent mentioned.The preferred Di Iso Propyl Ether that uses.
The protonic acid that is suitable for this method steps is organic acid normally, acetate for example, trifluoroacetic acid, oxalic acid or right-toluenesulphonic acids, or mineral acid, spirit of salt for example, sulfuric acid, or phosphoric acid.Suitable still Lewis acid, for example aluminum chloride or zinc chloride.Preferred trifluoroacetic acid.
Usually, be reflected at 0 ℃-+120 ℃, preferred+temperature range of 20 ℃-+80 ℃ is carried out.
Oxidation (dehydrogenation) is usually in a kind of halohydrocarbon listed above in method steps (XI) → (II), or chooses wantonly at alcohol, for example in methyl alcohol or the ethanol, carries out in acetonitrile or in water.Suitable oxygenant is, nitric acid for example, ammonium nitrate caesium (IV), 2,3-two chloro-5,6-dicyano-1,4-benzoquinones (DDQ), pyridinium chlorochromate (PCC), perosmic anhydride, Manganse Dioxide or the catalytic dehydrogenation of using platinum dioxide or charcoal to carry platinum.Preferably in as the methylene dichloride of solvent, use the oxidation of DDQ.Oxidation is usually at-50 ℃-+100 ℃, and preferred 0 ℃-+40 ℃ temperature range is carried out.
Each method steps carries out under boost or reduce pressure (for example 0.5-5bar) at normal atmosphere.Usually, method steps under atmospheric pressure carries out.
Following synthetic schemes explanation is carried out and is passed through in the preparation that has the compound of formula (Ib) → (Ie) according to the present invention similarly:
Scheme a1
Scheme a2
Scheme b 1
Scheme b 2
Scheme c
Scheme d
Scheme e
[abbreviation: tBu=tert-butyl; DAST-dimethylamino sulfur trifluoride; DDQ:2,3-two chloro-5,6-dicyano-1,4-benzoquinones; The DIBAH=diisobutyl aluminium hydride; The Et=ethyl; The Me=methyl; The Ph=phenyl; P-TsOH=is right-toluenesulphonic acids; The TBAF=tetrabutyl ammonium fluoride; TBDMSOTf=trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester; TFA=trifluoroacetic acid].
Compound according to the present invention has unpredictalbe useful spectrum of pharmacological activity.Therefore, it is suitable for as the pharmaceutical active compounds that treats and/or prevents the humans and animals disease.
Compound according to the present invention has been opened further treatment and has been selected and represented pharmacological progress.Compare with known and previously used preparation, according to compound exhibits of the present invention improved action spectrum.
It is preferably with huge specificity, and good tolerance and less side effect and the toxicity that reduces and give prominence to are particularly at cardiovascular field with in the liver field.
Advantage according to compound of the present invention is its high reactivity in human plasma.According to the further advantage of compound of the present invention be reduce and metabolic enzyme, particularly cytochrome P 450 enzymes and more especially with the potentiality of P450 3A4 enzyme interacting.In addition, compound according to the present invention has the sedimentary tendency in fatty tissue of minimizing.
The compound that has formula (I) according to the present invention has useful pharmacology performance and can be used to prevent and treat disease.According to compound of the present invention particularly cholesteryl ester transfer protein (CETP) highly efficient depressor and stimulate contrary cholesterol transport.Its HDL cholesterol concentration in the blood that raise.Compound according to the present invention is particularly suitable for treatment and elementary or intermediate coronary heart disease disease, for example myocardial infarction of preventing.In addition, can be used for the treatment of and prevent arteriosclerosis according to compound of the present invention, restenosis, apoplexy and alzheimer's disease.And, also can be used for the treatment of and prevent hypolipoproteinemia, dyslipidemia according to compound of the present invention, hypertriglyceridemia, hyperlipidaemia, hypercholesterolemia, fat (Adipositas), obesity (Obesitas), pancreatitis, insulin-dependent and non insulin dependent diabetes, the diabetes sequela, retinopathy for example, ephrosis and neuropathy, plyability hyperlipidaemia and metabolism syndrome.
Pharmacotoxicological effect according to compound of the present invention can use CETP inhibition test described below to measure.
The present invention also provides compound according to the present invention for treating and/or warding off disease the particularly application of above-mentioned disease.
The present invention also provides compound according to the present invention to be used for the treatment of for preparation and/or has warded off disease, the particularly application of the medicine of above-mentioned disease.
The present invention also provide use significant quantity according to compounds for treating of the present invention and/or ward off disease the method for above-mentioned disease particularly.
The present invention also provides and has comprised the medicine that is used for the treatment of and/or wards off disease according to compound of the present invention and one or more active compounds.The active compound that is suitable for making up is for example with preferred:
Antidiabetic drug,
Material with anti thrombotic action,
Material for lowering blood pressure,
The lipid metabolism modified material,
The anti-inflammatory material,
Stablize the material of the arteriosclerotic plague (Plaque).
The compound that has formula (I) according to the present invention can be preferably and following one or more combinations of substances
At Roten Liste 2002/II, the antidiabetic drug of mentioning in the 12nd chapter,
Agent with anti thrombotic action, for example with preferred anticoagulant or antithrombotics,
Hypotensor, for example with preferred calcium antagonist, Angiotensin AII antagonist, ACE inhibitor, beta-blocker, phosphodiesterase inhibitor, the stimulant of soluble guanylate cyclase, cGMP reinforce and hydragog(ue), and/or
The active substance of modification lipid metabolism is for example with preferred thryoid receptor agonist, cholesterol synthesis inhibitor, HMG-CoA reductase inhibitor for example, squalene synthetic inhibitor, squalene epoxidase inhibitor or squalene oxide cyclase inhibitor, the ACAT inhibitor, the MTP inhibitor, PPAR agonist, fibrate (Bei Te), lipase inhibitor, cholesterol absorption inhibitor, cholic acid reuptake inhibithors, polymerization cholic acid absorption agent and lipoprotein antagonist.
Antidiabetic drug for example be interpreted as and preferred Regular Insulin or insulin derivates and with the orally active compound of hypoglycemia effect.
Here Regular Insulin or insulin derivates comprise animal, Regular Insulin and its mixture of people or biotechnology origin.
Comprise for example with preferred with the orally active compound of hypoglycemia effect, sulfonylurea, biguanides, lattice row naphthalene class (meglitinide) derivative, diazole diketone, the thiazolidine diketone, alpha-glucosidase inhibitors, glucagon antagonist, GLP-1 agonist, insulin sensitizer, the liver enzyme inhibitors that relates to the stimulation of gluconeogenesis and/or glycogenolysis, the conditioning agent of glucose absorption and potassium channel openers, for example, those that in WO 97/26265 and WO99/03861, disclose.
In the preferred embodiment of the invention, the compound with formula (I) combines with Regular Insulin to be taken.
In the preferred embodiment of the invention, compound and sulfonylurea with formula (I) are for example with preferred tolbutamide, Glyburide (glibenclamide), glimepiride (glimepiride), Glipizide (glipizide) or Ge Lieqite (gliclazide) are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the biguanides of formula (I), for example mix and take with preferred N1,N1-Dimethylbiguanide (Metformin).
In the preferred embodiment of the invention, have formula (I) compound dative row naphthaline derivatives, for example and preferred Rui Gelie naphthalene (repaglinide) or Na Gelie naphthalene (nateglinide) in conjunction with taking.
In the preferred embodiment of the invention, have formula (I) compound and PPAR gamma agonist, for example from thiazolidine two ketones, for example and preferred pioglitazone (pioglitazone), or rosiglitazone (rosiglitazone) is in conjunction with taking.
In the preferred embodiment of the invention, have formula (I) compound and PPAR α/gamma agonist, for example and preferred GI-262570 (farglitazar), GW 2331, GW409544, and AVE 8042, AVE 8134, and AVE 0847, MK-0767 (KRP-297) or or AZ-242 in conjunction with taking.
Agent with anti thrombotic action preferably is interpreted as the compound that is selected from anticoagulant, for example with preferred acetylsalicylic acid, clopidogrel, ticlopidine or Dipyridamole, or antithrombotics.
In the preferred embodiment of the invention, compound and thrombin inhibitors with formula (I), for example with preferred Xi Meijia group (ximelagatran), Melagatran (melagatran), Bivalirudin (bivalirudin) or gram are filled in (clexane) combination and are taken.
In the preferred embodiment of the invention, have the compound and the GP II b/IIIa antagonist of formula (I), for example and preferably take for Luo Feiban or ReoPro combination.
In the preferred embodiment of the invention, have the compound and the factor Xa inhibitor of formula (I), for example with preferred DX 9065a, DPC 906, and JTV 803 or BAY 59-7939 are in conjunction with taking.
In the preferred embodiment of the invention, the compound with formula (I) combines with heparin or low-molecular-weight (LMW) heparin derivatives to be taken.
In the preferred embodiment of the invention, have the compound and the vitamin K antagonist of formula (I), for example take with preferred tonka bean camphor (coumarine) combination.
Hypotensor for example is interpreted as and preferably from the compound of calcium antagonist, for example with the preferred compound nifedipine, and ammonia oxygen Horizon, nitrendipine, nisoldipine, verapamil or diltiazem grass, Angiotensin A II antagonist, ACE inhibitor, beta-blocker and hydragog(ue).
In the preferred embodiment of the invention, the compound with formula (I) combines with the antagonist of α 1 acceptor to be taken.
In the preferred embodiment of the invention, have the compound and the serpentine of formula (I), minoxidil, diazoxide, two hydralazines, hydralazine and with nitrogen oxide-h substance, for example and preferred pannonit or Sodium Nitroprusside in conjunction with taking.
In the preferred embodiment of the invention, compound and Angiotensin A II antagonist with formula (I), for example with preferred losartan, valsartan, Kan Deshatan, telmisartan, embursatan, irbesartan, Olmesartan, Tasosartan or Saprisartan (saprisartan) are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the ACE inhibitor of formula (I), for example with preferred enalapril, captopril, Ramipril, Yipingshu, fosinopril, quinapril (quinopril), perindopril or Trolapril are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the beta-blocker of formula (I), for example take with preferred Propranololum or atenolol USP 23 combination.
In the preferred embodiment of the invention, have the compound and the hydragog(ue) of formula (I), for example take with preferred furosemide combination.
Lipid metabolism properties-correcting agent is interpreted as, for example with preferably from following compound: thryoid receptor agonist, cholesterol synthesis inhibitor, for example HMG-CoA reductase inhibitor or squalene synthetic inhibitor, the ACAT inhibitor, the MTP inhibitor, PPAR agonist, fibrate (Bei Te), cholesterol absorption inhibitor, the cholic acid reuptake inhibithors, lipase inhibitor, poly-cholic acid absorption agent and lipoprotein antagonist.
In the preferred embodiment of the invention, have the compound and the thryoid receptor agonist of formula (I), for example with preferred D-thyroxine, 3,5,3 '-triiodothyronine (T3), CGS 23425 or axitirome (CGS 26214) are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the squalene synthetic inhibitor of formula (I), for example take with preferred BMS-188494 or TAK-475 combination.
In the preferred embodiment of the invention, have the compound and the ACAT inhibitor of formula (I), for example with preferred avasimibe, eflucimibe or CS-505 are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the cholesterol absorption inhibitor of formula (I), for example with preferred ezetimibe, tiquinamide or pamaquine are in conjunction with taking.
In the preferred embodiment of the invention, have the compound of formula (I) and cholic acid absorption agent inhibitor again, for example and preferred barixibat, AZD 7508, and SC 435, SC 635 S-8921,264W94 or HM 1453 are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the MTP inhibitor of formula (I), for example with preferred implitapide, BMS-201038 or R-103757 are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the ppar agonist of formula (I), for example the Bei Tefei nobert, ammonia shellfish amine, bezafibrate, Win-35833 or gemfibrozil, for example with preferred GW 9578, GW 7647, and LY-518674 or NS-220 are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the PPAR-delta agonists of formula (I), for example take with preferred GW 501516 combinations.
In the preferred embodiment of the invention, compound and blended PPAR α/delta agonists with formula (I), for example with preferred GI-262570 (farglitazar), GW 2331, QW409544, AVE 8042, and AVE 8134, AVE 0847, and MK-0767 (KRP-297) or AZ-242 are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the blended PPAR α/gamma/delta agonist of formula (I), for example take with preferred MCC-555 combination.
In the preferred embodiment of the invention, have the compound of formula (I) with from the endothelial lipase inhibitor, the steapsin inhibitor, the gastric lipase inhibitor, hormone-responsive lipase inhibitor or hepatic lipase inhibitor are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the steapsin inhibitor of formula (I), preferred Lipstatin (lipstatin), for example orlistat is in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the poly-cholic acid absorption agent of formula (I), for example with preferred Colestyramine, colestipol, colesolvam, CholestaGel or colestimid are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the lipoprotein antagonist of formula (I), for example take with preferred gemcabene calcium (I-1027) or nicotinic acid combination.
In the preferred embodiment of the invention, have the compound and the niacin receptor antagonist of formula (I), for example and preferred niaspan, ASIMO this or pentaerythritol tetranicotinate are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the Antioxidans of formula (I), for example with preferred Probucol, AGI1067 or Bo653 are in conjunction with taking.
In the preferred embodiment of the invention, have the compound and the ldl receptor inductor of formula (I), for example take with preferred lifibrol combination.
In the preferred embodiment of the invention, compound with formula (I) and HMG-CoA reductase inhibitor from the statin class, for example with preferred lovastatin, simvastatin, Pravastatin, chlorine cut down his spit of fland, atorvastatin, Rosuvastatin, Cerivastatin (cerivastatin) or Pitavastatin Calcium (pitavastatin) are in conjunction with taking.
The present invention also provides the combination of compound with formula (I) and the material that reduces the expression of HMG-CoA reductase gene.This material can be that for example the HMG-CoA reductase enzyme is transcribed or the inhibitor of HMG-CoA reductase enzyme translation.Can for example pass through to suppress S1P (position-1) proteolytic enzyme, or reduce the inhibition that SREBP (sterol receptor binding protein) concentration affects HMG-CoA reductase gene is expressed.
The present invention also provides compound with formula (I) and the combination with material of the anti-inflammatory effect and/or the stable arteriosclerotic plague.This material can be for example from the compound of NSAID class, PAF-AH antagonist or chemokine receptor anagonists, for example IL-8 receptor antagonist or MCP-1 antagonist.
Active compound combination according to the present invention has useful pharmacology performance and can be used for prevention and the treatment disease.
Active compound combination according to the present invention is particularly suitable for treatment and elementary or intermediate coronary heart disease disease, for example myocardial infarction of preventing.In addition, they can be used for the treatment of and prevent arteriosclerosis, restenosis, apoplexy and alzheimer's disease.And the active compound combination of mentioning also can be used for the treatment of and prevent hypolipoproteinemia, dyslipidemia, hypertriglyceridemia, hyperlipidaemia, hypercholesterolemia, fat (Adipositas), obesity (Obesitas), pancreatitis, insulin-dependent and non insulin dependent diabetes, the diabetes sequela, retinopathy for example, ephrosis and neuropathy, plyability hyperlipidaemia and metabolism syndrome.Further, the active compound combination is suitable for treating hypertension, heart failure, stenocardia, ischemic and inflammation according to the present invention.
The present invention also provides and has comprised according to compound of the present invention, usually with the medicine of one or more inert non-toxic pharmacology proper auxiliary agent and they application for above-mentioned purpose.
According to compound of the present invention can whole body and/or the part work.For this purpose, they can be with suitable manner, and are for example oral, outside the intestines, and lung, nose, the hypogloeeis, tongue contains clothes, rectum, corium, through skin, conjunctiva, ear or as implanting or support (stent) is used.
For these route of administration, compound of the present invention can be taken with suitable administration form.
For oral suitable working according to prior art, transmit compound of the present invention apace and/or with improved form, this form comprises the compound of the present invention with crystallization and/or noncrystalline and/or dissolved form, sheet (the uncoated or sheet that applies for example, for example have mode dissolved or enteric coating or the clothing undissolved and release of controlling compound of the present invention) to postpone, the sheet or the film/wafer that in the oral cavity, decompose fast, film/lyophilized products (lyophylisate), capsule (for example hard or soft gel capsule), the sheet that sugar applies, particle, ball, powder, emulsion, suspension, aerosol or solution.
The outer clothes of intestines can be avoided absorption step (intravenously for example, intra-arterial is intracardiac, in the canalis spinalis or in the waist), or carry out along with absorbing (for example intramuscular is subcutaneous, and intracutaneous is through skin or intraperitoneal).Take the suitable form of taking outward particularly with solution for intestines, suspension, emulsion, the injection of lyophilized products or sterilized powder form or perfusion preparation.
For other route of administration suitable be, for example for the medicament forms that sucks (particularly powder inhalator, spraying gun), nasal drop, nose solution or sprays, hypogloeeis, tongue or contain the sheet of taking, film/wafer or capsule, suppository, the preparation that ear or eye are taken, vaginal capsule, aqueous suspension (lotion, jolting mixture), oleophylic suspension, ointment, emulsifiable paste, transdermal therapeutic system (for example plaster), milk sap, cream, foam, pouring powders is implanted or support (stent).
Preferred oral or intestines are obeyed outward, and are particularly oral.
Can change into the above-mentioned form of taking according to compound of the present invention.This can be in a manner known way undertaken by mixing with the nontoxic pharmacology proper auxiliary agent of natural instincts.These auxiliary agents are particularly including carrier (for example Microcrystalline Cellulose, lactose, N.F,USP MANNITOL), solvent (for example liquid macrogol), emulsifying agent and dispersion agent or wetting agent (for example sodium lauryl sulphate, polyoxy anhydrosorbitol oleate), tackiness agent (for example polyvinylpyrrolidone), synthetic or natural polymkeric substance (for example white protein), stablizer (for example antioxidant, for example xitix), tinting material (mineral dye for example, ferric oxide for example) and local flavor and/or smell corrigent.
Usually, have been found that the dose of preferably approximately 0.01-0.5mg/kg body weight is favourable in order to obtain under the situation that effective result obeys approximately 0.001-1mg/kg outside intestines.Under oral situation, dosage is about 0.01-100mg/kg, preferably approximately 0.01-20mg/kg and 0.1-10mg/kg body weight very particularly preferably.
Even now, it may be necessary departing from above-mentioned amount, promptly depends on body weight, route of administration, the individual is for the reaction of active compound, the type of preparation and taking the pitch time of carrying out.Therefore, take in some cases that to be less than above-mentioned minimum may be enough, yet must surpass the above-mentioned upper limit in other cases.Under the situation of taking relatively in a large number, these a plurality of each dosage that become to take in one day are divided in suggestion.
Below the embodiment that carries out has explained the present invention.This invention is not subjected to the restriction of these embodiment.
Following test and the per-cent among the embodiment are weight percents, unless specialize; Part is a weight part.Solvent ratio, the concentration of dilution ratio and liquid/liquid solution under each situation based on volume.
Test portion with compound of formula (Ia)
A. embodiment
Abbreviation and acronym
The CE cholesteryl ester
The CETP cholesteryl ester transfer protein
DAST dimethylamino sulfur trifluoride
DCI direct chemical ionization (in MS)
DDQ 2,3-two chloro-5,6-dicyano-1,4-benzoquinones
The de diastereomer is excessive
DMF N, dinethylformamide
The DMSO dimethyl sulfoxide (DMSO)
(in the productive rate) of d.Th theory
EDTA quadrol-N, N, N ', N '-tetraacethyl
The ee enantiomer is excessive
Eq. equivalent
ESI electrospray ionization (in MS)
H hour
The HDL high-density lipoprotein (HDL)
HPLC high pressure-high performance liquid chromatography
The LC/MS liquid chromatograph mass spectrography
The LDL low-density lipoprotein
Min minute
The MS mass spectrum
The MTBE methyl tertiary butyl ether
The NMR nuclear magnetic resonance spectrum
R
tRetention time (in HPLC)
The SPA scintillation proximity assay
The TBAF tetrabutyl ammonium fluoride
TBDMSOTf trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester
The TFA trifluoroacetic acid
The THF tetrahydrofuran (THF)
Parent material and intermediate
Embodiment 1A
1-ring propylidene benzylacetone
274g (1.57mol) [(1-oxyethyl group cyclopropyl) oxygen base] (trimethylammonium) silane, 650g (2.04mmol) 1-(triphenylphosphine is just being pitched base) acetone (1-(triphenylphosphoranylidene) acetone) and 29.9g (157mmol) be right-and the toluenesulphonic acids monohydrate is suspended in 1.58 liter 1, stirs 2.5h down in the 2-dichlorobenzene and at 100 ℃.After the heating, dissolving 1-(triphenyl phosphorane fork base) acetone.Reaction mixture cool to room temperature and crude product (moving phase: beginning sherwood oil, methylene dichloride afterwards) chromatographic separation on silica gel afterwards.The enriched product component and under high vacuum of short duration drying.
Productive rate: 78.5g (theoretical 46%)
1H-NMR(400MHz，CDCl
3)：δ＝6.42(t，1H)，2.31(s，3H)，1.53-1.46(m，2H)，1.36-1.29(m，2H)
MS(DCI，NH
3)：m/z＝114[M+NH
4]
+.
Embodiment 2A
Spiral shell [2.5] octane-5, the 7-diketone
Beginning adds 37.09g (687mmol) sodium methylate and along with stirring, heats under refluxing in 388ml methyl alcohol.Add 96.2g (728mmol) propanedioic acid dimethyl esters, and mixture restir 10min and cool to room temperature afterwards under refluxing.At room temperature drip 66g (687mmol) 1-ring propylidene benzylacetone afterwards, and mixture stirs 4h under refluxing subsequently from embodiment 1A.After removing heating bath, fast (z ü gig) drips the solution of 84.75g (1.51mol) potassium hydroxide in 264ml water, and under refluxing continuously stirring 1h.Use half concentrated hydrochloric acid to adjust pH afterwards to 1-2 (foaming), and mixture restir 15min.Under 55 ℃ bath temperature, on rotatory evaporator, under reduced pressure remove methyl alcohol up to the pressure that reaches 60mbar.With the content in the ethyl acetate extraction flask twice, merge organic phase, drying under reduced pressure also concentrates.Concentrate the oil that produces, in methylene dichloride, dissolve and (moving phase: methylene chloride 95: 5) chromatographic separation on silica gel.Enriched product component and remaining afterwards oil are developed with ether.The use suction filters the solid of generation and is at room temperature dry under high vacuum.
Productive rate: 37.2g (theoretical 39%)
1H-NMR(400MHz，CDCl
3)：δ＝3.49(s，2H)，2.47(s，4H)，0.58(s，4H)
MS(DCI，NH
3)：m/z＝156[M+NH
4]
+.
Embodiment 3A
4 '-cyclohexyl-2 '-cyclopentyl-3 '-[4-(trifluoromethyl) benzoyl]-4 ', 8 '-dihydro-1 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-5 ' (6 ' H)-ketone
In the 350ml Di Iso Propyl Ether, begin to add 8.0g (28.24mmol) 3-amino-3-cyclopentyl-1-(4-trifluoromethyl) acrylketone (according to WO 03/028727, embodiment 4 preparations), and add 3.63ml (47.1mmol) trifluoroacetic acid and 3.25g (23.53mmol) spiral shell [2.5] octane-5,7-diketone (embodiment 2A).After at room temperature stirring 10min, add 5.70ml (47.1mmol) hexanaphthene formaldehyde, and mixture heats 18h under refluxing afterwards.After the cooling, mixture stirs 15min under ice bath, and filters the precipitation of generation and wash with cold Di Iso Propyl Ether with suction.
Productive rate: 2.92g (theoretical 25%)
1H-NMR (400MHz, CDCl
3): δ=7.80 (d, 2H), 7.67 (d, 2H), 5.88 (s, 1H), 3.80 (d, 1H), 3.51 (quintet, 1H), 2.85 (d, 1H), 2.69 (d, 1H), 2.26-2.14 (m, 1H), 2.00 (t, 2H), 1.80-1.46 (m, 9H), 1.44-1.31 (m, 2H), 1.25-1.13 (m, 1H), 1.12-0.96 (m, 4H), and 0.93-0.75 (m, 2H), 0.62-0.43 (m, 4H)
MS(DCI)：m/z＝498[M+H]
+.
Embodiment 4A
4 '-cyclohexyl-2 '-cyclopentyl-3 '-[4-(trifluoromethyl) benzoyl]-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-5 ' (8 ' H)-ketone
In the 60ml methylene dichloride, dissolve the compound of 1.90g (3.82mmol), and at room temperature use 950mg (4.20mmol) 2 from embodiment 3A, 3-two chloro-5,6-dicyano-1,4-benzoquinones (DDQ) stirs.Enriched mixture and resistates are by chromatogram purification (silica gel, moving phase: cyclohexane/ethyl acetate 20: 1 → 10: 1) on rotatory evaporator.
Productive rate: 1.3g (theoretical 69%)
1H-NMR(400MHz，CDCl
3)：δ＝8.10-7.82(br.s，2H)，7.74(d，2H)，3.41-3.14(br.s，1H)，3.05(dd，2H)，2.71-2.50(m，3H)，1.98-1.36(m，16H)，1.24-1.05(m，2H)，0.62-0.50(m，4H)
MS(ESIpos)：m/z＝496[M+H]
+.
Embodiment 5A
[(5 ' S)-4 '-cyclohexyl-2 '-cyclopentyl-5 '-hydroxyl-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-3 '-yl] [4-(trifluoromethyl) phenyl] ketone
(1R 2S)-1-aminoidan-2-alcohol, and at room temperature adds 5.40g (33.1mmol) borine-N, N-diethylbenzene amine complex to begin to add 190mg (1.24mmol) in 150ml THF.After the release of gas stopped, mixture was cooled to 0 ℃, and added the 4.10g (8.27mmol) be dissolved among the 150ml THF compound from embodiment 4A.Follow stirring, make mixture warm up room temperature through several hrs.After reaction finishes, add methyl alcohol, concentrated reaction mixture and absorption of residual excess in ethyl acetate.Mixture 1N hydrochloric acid under each situation, saturated sodium hydrogen carbonate solution and saturated sodium chloride solution washed twice.Organic phase is filtered and is concentrated by dried over sodium sulfate.By column chromatography purifying crude product (silica gel, moving phase: begin hexanaphthene, cyclohexane/ethyl acetate is 10: 1 afterwards).
Productive rate: 3.4g (theoretical 83%)
The excessive 71%ee that is determined as of enantiomer.
Go up the chromatographic separation [post: Chiralpak AD, 500mm * 40mm of corresponding isomer subsequently mutually in chirality; Moving phase: Virahol/isohexane 2.5: 97.5; Flow velocity: 50ml/min; Temperature: 25 ℃; Detect: 254nm] title compound of 2.83g enantiomeric pure is provided.
R
t=4.96min[Chiralpak AD, 250mm * 4.6mm; Moving phase: Virahol/isohexane 2.5: 97.5; Flow velocity: 1.0ml/min; Detect: 254nm]
1H-NMR(400MHz，CDCl
3)：δ＝8.50-7.35(m，4H)，5.48-5.02(m，1H)，3.43-3.14(m，2H)，2.71-2.27(m，3H)，2.18-0.93(m，19H)，0.83-0.73(m，1H)，0.72-0.56(m，1H)，0.52-0.43(m，2H)
MS(ESIpos)：m/z＝498[M+H]
+.
Embodiment 6A
[(5 ' S)-5 '-{ [tert-butyl (dimethyl) silyl] oxygen base }-4 '-cyclohexyl-2 '-cyclopentyl-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-3 '-yl) [4-(trifluoromethyl) phenyl] ketone
Under argon, in the 0.75ml dry toluene, dissolve compound and the 86mg (0.80mmol) 2 of 100mg (0.20mmol) from embodiment 5A, the 6-lutidine also is cooled to-20 ℃.Under this temperature, drip the solution of 106mg (0.40mmol) trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester in the 0.25ml dry toluene, and mixture stirs 15min down at-20 ℃ subsequently, warm afterwards to 0 ℃ and under this temperature restir 1h.In mixture, add 3ml 0.1N hydrochloric acid, repeat to extract with ethyl acetate after it.The organic phase that merges once and with saturated sodium chloride solution is washed once with saturated sodium hydrogen carbonate solution and saturated sodium chloride solution mixture washing in 1: 1, by dried over sodium sulfate, filters and concentrates.Resistates is chromatogram purification (moving phase: begin hexanaphthene, cyclohexane/ethyl acetate is 15: 1 afterwards) on silica gel.
Productive rate: 115mg (theoretical 94%)
1H-NMR(400MHz，CDCl
3)：δ＝8.02-7.48(m，4H)，5.51-5.18(br.s，1H)，3.25-2.68(m，2H)，2.65-2.45(m，1H)，2.13-1.03(m，21H)，0.93-0.83(m，9H)，0.81-0.70(m，1H)，0.68-0.58(m，1H)，0.44-0.39(m，1H)，0.38-0.28(m，1H)，0.25-0.14(m，6H)
MS(ESIpos)：m/z＝612[M+H]
+.
Embodiment 7A
(S) ((5 ' S)-5 '-{ [tert-butyl (dimethyl) silyl] oxygen base }-4 '-cyclohexyl-2 '-cyclopentyl-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-3 '-yl) [4-(trifluoromethyl) phenyl] methyl alcohol
Under argon, in the 50ml dry toluene, begin to add 3.10g (5.07mmol) from the compound of embodiment 6A and be cooled to-50 ℃.Under this temperature, slowly drip the 1M solution of 25.4mg (25.4mmol) diisobutyl aluminium hydride in toluene.Mixture-50 ℃ of following restir 10min and after through the warm room temperature that arrives of 1h.Follow ice-coldly, in mixture, add the sodium tartrate potassium solution of 20% concentration, repeat to extract with ethyl acetate after it.The organic phase that merges with saturated sodium chloride solution washing once by dried over sodium sulfate, is filtered and is concentrated.This has produced the 3.2g crude product.
Go up the chromatographic separation [post: Chiralpak AD, 500mm * 40mm, 20 μ m of diastereomer subsequently mutually in chirality; Moving phase: Virahol/isohexane 2.5: 97.5; Flow velocity: 50ml/min; Room temperature; Detect: 254nm] produced the diastereoisomeric cis-isomeride of the title compound (trans-isomer) of 1.4g (theoretical 45%) diastereisomericallypure pure and 1.3g (theory 42%).
Trans-diastereomer
R
t=8.09min[post: Chiralpak IA, 250mm * 4.6mm; Moving phase: Virahol/isohexane 3: 97; Flow velocity: 1.0ml/min; Detect: 254nm]
1H-NMR (300MHz, CDCl
3): δ=7.58 (d, 2H), 7.42 (d, 2H), 6.68 and 6.49 (2br.s, 1H together), 5.58 and 5.21 (2br.s, 1H together), 3.45-3.22 (m, 1H), 2.99-2.78 (m, 2H), 2.33-2.18 (m, 1H), 2.14-1.05 (m, 20H), 0.95-0.82 (m, 9H), 0.80-0.70 (m, 1H), 0.68-0.43 (m, 2H), 0.42-0.26 (m, 1H), 0.25-0.02 (m, 6H)
MS(ESIpos)：m/z＝614[M+H]
+.
Cis-diastereomer
R
t=5.70min[post: Chiralpak IA, 250mm * 4.6mm; Moving phase: Virahol/isohexane 3: 97; Flow velocity: 1.0ml/min; Detect: 254nm]
1H-NMR (300MHz, CDCl
3): δ=7.59-7.51 (m, 2H), 7.48-7.28 (m, 2H), 6.64 and 6.49 (2br.s, 1H together), 5.58 and 5.22 (2br.s, 1H together), 3.45-3.22 (m, 1H), 3.10-2.76 (m, 2H), 2.33-2.18 (m, 1H), 2.12-1.05 (m, 20H), 0.94-0.82 (m, 9H), 0.80-0.70 (m, 1H), 0.68-0.43 (m, 2H), 0.42-0.27 (m, 1H), 0.26-0.01 (m, 6H)
MS(ESIpos)：m/z＝614[M+H]
+.
Embodiment 8A
(5 ' S)-5 '-{ [tert-butyl (dimethyl) silyl] oxygen base }-4 '-cyclohexyl-2 '-cyclopentyl-3 '-(S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]
Under argon, in 17ml toluene, dissolve 813mg (1.32mmol) from the compound of embodiment 7A and be cooled to-20 ℃.Under this temperature, drip 0.29ml (2.19mmol) diethylamino sulfur trifluoride.Remove cooling, and mixture restir 2h afterwards.In mixture, add water, repeat to extract with methylene dichloride after it.The organic phase that merges is washed once with saturated sodium hydrogen carbonate solution and with saturated sodium chloride solution washed twice, by dried over sodium sulfate, is filtered and concentrate.Crude product is dry under high vacuum not to be further purified with reaction.
Productive rate: 770mg (theoretical 94%)
1H-NMR (400MHz, CDCl
3): δ=7.60 (d, 2H), 7.46-6.98 (m, 3H), 5.59 with 5.22 (2br.s, 1H together), and 3.42-3.20 (m, 1H), 3.02-2.67 (m, 3H), 2.20-0.99 (m, 20H), 0.90 (s, 9H), 0.82-0.68 (m, 1H), 0.67-0.48 (m, 2H), 0.44-0.27 (m, 1H), and 0.25-0.01 (m, 6H)
MS(ESIpos)：m/z＝616[M+H]
+.
The embodiment that implements
Embodiment 1
(5 ' S)-4 '-cyclohexyl-2 '-cyclopentyl-3 '-(S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-5 '-alcohol
Under argon, in 1ml THF, dissolve the compound of 770mg (1.25mmol) from embodiment 8A, add 1M solution and the mixture of 6.25ml (6.25mmol) TBAF in THF and at room temperature stir 2h.In mixture, add 50ml 0.2N hydrochloric acid, repeat to extract with ethyl acetate after it.The organic phase that merges, is filtered and is concentrated by dried over sodium sulfate with saturated sodium chloride solution washed twice.Resistates is chromatogram purification (moving phase: begin hexanaphthene, cyclohexane/ethyl acetate is 10: 1 afterwards) on silica gel.
Productive rate: 594mg (theoretical 95%)
Chirality use on mutually the further separation of chromatogram still be present in diastereomer in the product [post: KBD 5945,400mm * 30mm is based on poly-(the N-methacryloyl-L-leucine-tert-butyl acid amides of chiral selector; Moving phase: MTBE/ isohexane 20: 80; Flow velocity: 50ml/min; Room temperature; Detect: the title compound that the 540mg diastereisomericallypure pure 254nm) is provided:
R
t=3.76min[post: KBD 5945,250mm * 4.6mm; Moving phase: MTBE/ isohexane 3: 7; Flow velocity: 1.0ml/min; Detect: 265nm]
1H-NMR (400MHz, CDCl
3): δ=7.61 (d, 2H), 7.48-7.29 (m, 3H), 5.47-5.39 and 5.18-5.07 (2m, 1H together), 3.60-3.46 (m, 1H), 3.31-3.11 (m, 1H), 2.96-2.68 (m, 1H), 2.47-2.20 (m, 2H), 2.10-1.10 (m, 19H), 0.84-0.70 (m, 2H), 0.66-0.58 (m, 1H), and 0.50-0.38 (m, 2H)
MS(ESIpos)：m/z＝502[M+H]
+.
B. the evaluation of pharmacological activity
The B-I.CETP-inhibition test
The acquisition of B-I.1.CETP
CETP is obtained with partially purified form by differential centrifugation and column chromatography by people's blood plasma and is used for this test.For this reason, use NaBr with human plasma adjust to every ml 1.21g density and under 4 ℃ under 50000rmp centrifugal 18h.(d＞1.21g/ml) is used for Sephadex to the bottom component
-Phenyl-Sepharose 4B (Pharmacia company) post washs and uses afterwards the distilled water wash-out with 0.15MNaCl/0.001M TrisHCl pH 7.4.Collect the CETP active ingredient, with 50mM sodium acetate pH 4.5 dialysis and put on CM-Sepharose
Post (Pharmacia company).Mixture uses linear gradient (0-1M NaCl) wash-out afterwards.The CETP component of collecting with 10mM TrisHCl pH 7.4 dialysis and afterwards further by chromatogram at Mono Q
Post (Pharmacia) is gone up purifying.
The B-I.2.CETP fluorescent test
The mensuration of fluorescence cholesteryl ester between the catalytic liposome of CETP transhipment [according to Bisgaier etc., J.Lipid Res.
34, 1625 (1993) step is improved]:
In order to prepare the donor liposome, along with gently the dissolving 1mg cholesteryl-4 of heating in the two alkane of 600 μ l in ultrasonic bath, 4-two fluoro-5,7-dimethyl-4-boron is mixed-3a, 4a-diaza-s-indacene (indacene)-3-dodecylate (cholesteryl BODIPY
FLC
12, Molecular Probes company), 15.35mg triolein and 6.67mg phosphatidylcholine and at room temperature in 63ml 50mM TrisHCl/150mMNaCl/2mM edta buffer liquid pH 7.3, add this solution very lentamente with the ultrasonic wave effect.Afterwards suspension in the Branson ultra sonic bath under about 50 watts at N
2Ultrasonication 30min under the atmosphere, temperature remains on about 20 ℃.
By the 86mg Oleoylcholesterol that is dissolved in 1.2ml two alkane and the above damping fluid of 114ml, 20mg triolein and 100mg phosphatidylcholine obtain the acceptor liposome similarly by the ultrasonication under 30 minutes 50 watts (20 ℃).
B-I.2.1. the CETP fluorescent test of enrichment CETP
In order to test, use by damping fluid more than 1 part 1 part of donor liposome and 2 parts of test mixtures that the acceptor liposome constitutes.
With the solution-treated 50 μ l test mixtures of material in DMSO that the CETP component (1-3 μ g) and the 2 μ l of 48 μ l enrichments will check, this CETP component obtains by the blood plasma of hydrophobic chromatography by the people, and cultivates 4 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 485/535nm changes, with the inhibition of the control batch comparative measurement transhipment that does not have material.
| Embodiment number | IC 50[nM] fluorescent test |
| 1 | 17 |
B-I.2.2. the CETP fluorescent test of human plasma
In 42 μ l (86%v/v) human plasmas (Sigma P9523), add the solution of material in DMSO that 6 μ l (12%v/v) donor liposomes and 1 μ l (2%v/v) will check, and mixture was cultivated 24 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 510/520nm changes (stitching wide 2.5nm), with the inhibition of the control batch comparative measurement transhipment that does not have material.
| Embodiment number | IC 50Fluorescent test in [nM] human plasma |
| 1 | 80 |
B-I.2.3. the CETP fluorescent test in vitro exsomatizes
In 80 μ l test mixtures, add 10 μ l damping fluids and 2 μ l serum, and mixture was cultivated 4 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 485/535nm changes, with the inhibition of the control batch comparative measurement transhipment that does not have material.
B-I.3. the acquisition of radiolabeled HDL
The fresh people's edta plasma of 50ml uses NaBr to adjust to 1.12 density and centrifugal 18h in 65 rotors of the Ty under 50000rpm under 4 ℃.The upper strata is used to obtain cold LDL mutually.Lower floor uses 3 * 4 liters of PDB damping fluids mutually, and (10mM TrisHCl, pH 7.4,0.15mM NaCl, 1mM EDTA, 0.02%NaN
3) dialysis.Every afterwards 10ml retentate volume adds 20 μ l
3H-cholesterol (Dupont NET-725; In ethanol the dissolving 1 μ C/ μ l) and mixture under 37 ℃ at N
2Under cultivated 72 hours.
This batch of material uses NaBr to adjust to density 1.21 and centrifugal 18h in 65 rotors of the Ty under 50000rpm under 20 ℃ afterwards.Reclaim the upper strata phase and pass through the gradient centrifugation purification lipoprotein component.For this reason, lipoprotein isolating, mark uses NaBr to adjust to 1.26 density.In centrifuge tube (SW 40 rotors) be that 1.21 solution and 4.5ml density are that 1.063 solution covers this solution (the density solution of PDB damping fluid and NaBr) of each part of 4ml and centrifugal at SW 40 rotors under 38000rpm and 20 ℃ afterwards with 4ml density.Middle layer between density 1.063 and 1.21, it comprises the HDL of mark, 4 ℃ of following PDB damping fluid dialysis with 3 * 100 volumes.
Retentate comprises radiolabeled
3H-CE-HDL, it adjusts to about 5 * 106cmp/ml, is used for this test.
The R-I.4.CETP-SPA test
In order to test the activity of CETP, measure
3The H-cholesteryl ester is by the transhipment to the LD lipoprotein of biotin function of people's HD lipoprotein, and reaction is by adding streptavidin-SPA
Pearl (Amersham company) finishes and the radioactivity of transfer is directly measured in liquid scintillation counter.
In test batch, 10 μ l HDL-
3H-cholesteryl ester (about 50000cpm) is comprising 50mM Hepes/0.15M NaCl/0.1% bovine serum albumin/0.05%NaN of 10 μ l CETP (1mg/ml) with 10 μ l vitamin H-LDL (Amersham company) down at 37 ℃
3Cultivated 18 hours in the material (being dissolved in the solution of 10%DMSO/1%RSA) that pH 7.4 and 3 μ l will test.Add 200 μ l SPA-streptavidin pearl solution (TRKQ 7005) afterwards, further cultivate 1h and in scintillometer, measure afterwards along with jolting.With 10 μ l damping fluids, cultivate in contrast in the correspondence of 10 μ l CETP under 4 ℃ and 10 μ l CETP under 37 ℃.
Regarding 100% as 37 ℃ of activity that shift down in the contrast batch of material that uses CETP shifts.This transfer reduce to a half material concentration be defined as IC
50Value.
| Embodiment number | IC 50[nM] SPA test |
| 1 | 32 |
B-II.1. the mensuration of isolated activity in vitro on transgenosis hCETP mouse
Suppress active in order to test CETP, use stomach tube to give inner oral this material of transgenosis hCETP mouse [Dinchuk etc., BBA 1295-301 (1995)] of feeding.For this reason, arbitrarily be arranged to and have the equal number animal beginning to test the day before yesterday buck, usually the group of n=4.Before using this material, be used for measuring its basic active blood of CETP (time point T1) of serum by the puncture collection of vena orbitalis posterior clump (retroorbitalenVenenplexus) by each mouse.Use stomach tube to take this substances afterwards to animal.Specified time after substances is used, by puncture for the second time (time point T2) from animal, get blood, usually behind application of substances 16 or 24h, but as suitable this also can carry out in another time.
In order to assess the inhibition activity of material,, promptly 16 or 18 hours, use animal only to accept not have the corresponding control group of the preparation of material to each time.In control animal, for can measure through corresponding test period at interval (16 or 24h) do not have the active change of CETP of inhibitor, in the animal of mass treatment, carry out the secondary blood specimen collection of each animal.
After solidifying end, blood samples is centrifugal and remove serum deprivation with suction pipe.In order to measure the CETP activity, measure transportation through the cholesteryl ester (Cholesterylester) of 4h.For this reason, the carrying out of in test batch, using 2 μ l serum and test as under B-I.2.3., describing usually.
Calculate for each animal the cholesteryl ester transportation difference [pM CE/h (T2)-pMCE/h (T1)] and in group, average.In one of time point, reduced＞material of the transportation of 20% cholesteryl ester is considered to active.
| Embodiment number | Inhibition % at 3mg/kg | |
| 16h | 24h | |
| 1 | 74 | 66 |
B-II.2. the mensuration of activity in vivo in Syria's cricetulus auratus
The inner female Syria cricetulus auratus of feeding and having 150-200g weight (planting BAY:DSN) is used to measure the oral effect of CETP inhibitor for serum lipoprotein and serum triglyceride.Six animals of each cage be divided into one group and feed arbitrarily and intake the domestication two weeks.
Just before on-test and after material takes, get blood and at room temperature cultivate 30min and after under the 30000g centrifugal 20 minutes, be used to obtain serum by puncture behind the socket of the eye of venous plexus.This substance dissolves is in 20%Solutol/80% water and oral by stomach tube, the solvent of control animals received equal volume and do not have substances.
Operational analysis instrument COBAS INTEGRA 400 plus (from Roche Diagnostics company) measure triglyceride level, cholesterol total amount, HDL cholesterol and LDL cholesterol according to manufacturer's specification sheets.From measured value, for each parameter, the per-cent that causes with mass treatment of change and each group calculate by to(for) each animal are defined as the mean value (n=6 or n=12) with standard deviation.If with the group of solvent treatment relatively, the influence of material is significant, add p-value by the t-test determination (
*P≤0.05;
*P≤0.01;
* *P≤0.005).
B-II.3. the mensuration of activity in vivo in transgenosis hCETP mouse
In order to measure oral effect, use stomach tube to take substances for transgenic mice [Dinchuk etc., BBA, 1295-1301 (1995)] for lipoprotein and triglyceride level.Before on-test, after measuring cholesterol in the serum and the socket of the eye of triglyceride level, draw blood by mouse.The serum that obtains as described above for hamster is in 4 ℃ of following overnight incubation and centrifugal under 6000g subsequently.After three days, in order to measure lipoprotein and triglyceride level is got blood by mouse again.The change of the parameter of measuring is expressed as the change with starting value per-cent relatively.
| Embodiment number | % (the dosage: 3 * 3mg/kg) that HDL increases after 3 days |
| 1 | 61 |
C. the embodiment of the enforcement of pharmaceutical composition
Compound of the present invention can change into pharmaceutical preparation with following method:
Sheet:
Composition:
100mg compound of the present invention, 50mg lactose (monohydrate), 50mg W-Gum (homemade), 10mg polyvinylpyrrolidone (PVP 25) (from BASF, Ludwigshafen, Germany) and 2mg Magnesium Stearate.
Sheet weight 212mg.Diameter 8mm, bending radius 12mm.
Produce:
The mixture of compound of the present invention, lactose and starch is in water and PVP solution (m/m) granulation of 5% concentration.Particle mixed with Magnesium Stearate 5 minutes after dry.This mixture use conventional tabletting machine (for the form of sheet referring to more than) compressing tablet.The pressure of 15kN is as the standard of compressing tablet.
Suspension that can be oral:
Composition:
1000mg compound of the present invention, 1000mg ethanol (96%), 400mg Rhodigel
(from the xanthan gum of FMC, Pennsylvania, the U.S.) and 99g water.
The 10ml oral suspension is corresponding to the single dose with 100mg compound of the present invention.
Produce:
Rhodigel is suspended in the ethanol and adds compound of the present invention in suspension.Add water along with stirring.Mixture stirs about 6h and finishes up to the Rhodigel swelling.
Solution that can be oral:
Composition:
500mg compound of the present invention, 2.5g polysorbate and 97g poly(oxyethylene glycol) 400.The 20g oral liquid is corresponding to the single dose with 100mg compound of the present invention.
Produce:
Along with stirring, compound of the present invention is suspended in the mixture of polyoxyethylene glycol and polysorbate, continues to stir to dissolve fully up to compound of the present invention.
Solution in the body:
Being lower than saturated deliquescent concentration, compound dissolution of the present invention is in physiology acceptable solvent (for example isotonic saline solution, glucose solution 5% and/or PEG 400 solution 30%).Solution is through in sterile filtration and pack into aseptic and the pyrogen-free injection vessel.
Test portion with compound of formula (Ib)
A. embodiment
Abbreviation and acronym
The CE cholesteryl ester
The CETP cholesteryl ester transfer protein
DAST dimethylamino sulfur trifluoride
DCI direct chemical ionization (in MS)
DDQ 2,3-two chloro-5,6-dicyano-1,4-benzoquinones
The de diastereomer is excessive
DMF N, dinethylformamide
The DMSO dimethyl sulfoxide (DMSO)
(in the productive rate) of d.Th theory
EDTA quadrol-N, N, N ', N '-tetraacethyl
The ee enantiomer is excessive
Eq. equivalent
ESI electrospray ionization (in MS)
H hour
The HDL high-density lipoprotein (HDL)
HPLC high pressure-high performance liquid chromatography
The LC/MS liquid chromatograph mass spectrography
The LDL low-density lipoprotein
Min minute
The MS mass spectrum
The MTBE methyl tertiary butyl ether
The NMR nuclear magnetic resonance spectrum
R
tRetention time (in HPLC)
The SPA scintillation proximity assay
The TBAF tetrabutyl ammonium fluoride
TBDMSOTf trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester
The TFA trifluoroacetic acid
The THF tetrahydrofuran (THF)
Parent material and intermediate
Embodiment 1A
1-ring propylidene benzylacetone
274g (1.57mol) [(1-oxyethyl group cyclopropyl) oxygen base] (trimethylammonium) silane, 650g (2.04mol) 1-(triphenyl phosphorane fork base) acetone (1-(triphenylphosphoranylidene) acetone) and 29.9g (157mmol) be right-and the toluenesulphonic acids monohydrate is suspended in 1.58 liter 1, in the 2-dichlorobenzene and at 100 ℃ of stirring 2.5h down.After the heating, dissolving 1-(triphenyl phosphorane fork base) acetone.Reaction mixture cool to room temperature and crude product (moving phase: beginning sherwood oil, methylene dichloride afterwards) chromatographic separation on silica gel afterwards.The enriched product component and under high vacuum of short duration drying.
Productive rate: 78.5g (theoretical 46%)
1H-NMR(400MHz，CDCl
3)：δ＝6.42(t，1H)，2.31(s，3H)，1.53-1.46(m，2H)，1.36-1.29(m，2H)MS(DCI，NH
3)：m/z＝114[M+NH
4]
+.
Embodiment 2A
Spiral shell [2.5] octane-5, the 7-diketone
Beginning adds 37.09g (687mmol) sodium methylate and along with stirring in 388ml methyl alcohol, heating under refluxing. add 96.2g (728mmol) propanedioic acid dimethyl esters, and mixture restir 10min and cool to room temperature afterwards under refluxing.At room temperature drip 66g (687mmol) 1-ring propylidene benzylacetone afterwards, and mixture stirs 4h under refluxing subsequently from embodiment 1A.After removing heating bath, drip the solution of 84.75g (1.51mol) potassium hydroxide in 264ml water fast, and under refluxing continuously stirring 1h.Use half concentrated hydrochloric acid to adjust pH afterwards to 1-2 (foaming), and mixture restir 15min.Under 55 ℃ bath temperature, on rotatory evaporator, under reduced pressure remove methyl alcohol up to the pressure that reaches 60mbar.With the content in the ethyl acetate extraction flask twice, merge organic phase, drying under reduced pressure also concentrates.Concentrate the oil that produces, in methylene dichloride, dissolve and (moving phase: methylene chloride 95: 5) chromatographic separation on silica gel.Enriched product component and remaining afterwards oil are developed with ether.The use suction filters the solid of generation and is at room temperature dry under high vacuum.
Productive rate: 37.2g (theoretical 39%)
1H-NMR(400MHz，CDCl
3)：δ＝3.49(s，2H)，2.47(s，4H)，0.58(s，4H)
MS(DCI，NH
3)：m/z＝156[M+NH
4]
+.
Embodiment 3A
2 ', 4 '-two cyclopentyl-3 '-[4-(trifluoromethyl) benzoyl]-4 ', 8 '-dihydro-1 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-5 ' (6 ' H)-ketone
In the 350ml Di Iso Propyl Ether, begin to add 9.0g (31.77mmol) 3-amino-3-cyclopentyl-1-(4-trifluoromethyl) acrylketone (according to WO 03/028727, embodiment 4 preparations), and add 4.08ml (52.95mmol) trifluoroacetic acid and 3.66g (26.47mmol) spiral shell [2.5] octane-5,7-diketone (embodiment 2A).After at room temperature stirring 10min, add 5.20g (52.95mmol) pentamethylene formaldehyde, and mixture stirs 18h under ice bath.After the cooling, mixture stirs 15min under ice bath, and filters the precipitation of generation and wash with cold Di Iso Propyl Ether with suction.
Productive rate: 1.9g (theoretical 15%)
1H-NMR (400MHz, CDCl
3): δ=7.81 (d, 2H), 7.67 (d, 2H), 5.91 (s, 1H), 3.88 (d, 1H), 3.52 (quintet, 1H), 2.88 (d, 1H), 2.70 (d, 1H), 2.26-2.14 (m, 1H), 1.94 (dd, 2H), 1.80-1.24 (m, 14H), 1.16-0.88 (m, 2H), 0.62-0.40 (m, 4H)
MS(ESIpos)：m/z＝484[M+H]
+.
Embodiment 4A
2 ', 4 '-two cyclopentyl-3 '-[4-(trifluoromethyl) benzoyl]-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-5 ' (8 ' H)-ketone
In the 150ml methylene dichloride, dissolve the compound of 4.80g (9.93mmol), and at room temperature use 2.48mg (10.92mmol) 2 from embodiment 3A, 3-two chloro-5,6-dicyano-1,4-benzoquinones (DDQ) stirs.Enriched mixture and resistates are by chromatogram purification (silica gel, moving phase: cyclohexane/ethyl acetate 20: 1 → 10: 1) on rotatory evaporator.
Productive rate: 2.7g (theoretical 56%)
1H-NMR(300MHz，CDCl
3)：δ＝7.98(d，2H)，7.76(d，2H)；3.18-2.97(m，3H)，2.72-2.55(m，3H)，1.98-1.64(m，10H)，1.60-1.36(m，6H)，0.64-0.49(m，4H)
MS(DCI)：m/z＝482[M+H]
+.
Embodiment 5A
[(5 ' S)-2 ', 4 '-two cyclopentyl-5 '-hydroxyl-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-3 '-yl] [4-(trifluoromethyl) phenyl] ketone
(1R 2S)-1-aminoidan-2-alcohol, and at room temperature adds 3.66g (22.43mmol) borine-N, N-diethylbenzene amine complex to begin to add 130mg (0.84mmol) in 100ml THF.After the release of gas stopped, mixture was cooled to 0 ℃, and added the 2.70g (5.61mmol) be dissolved among the 150ml THF compound from embodiment 4A.Follow stirring, make mixture warm up room temperature through several hrs.Reaction is added methyl alcohol, concentrated reaction mixture and absorption of residual excess in ethyl acetate after finishing in reaction mixture.Mixture 1N hydrochloric acid under each situation, saturated sodium hydrogen carbonate solution and saturated sodium chloride solution washed twice.Organic phase is filtered and is concentrated by dried over sodium sulfate.By column chromatography purifying crude product (silica gel, moving phase: begin hexanaphthene, cyclohexane/ethyl acetate is 20: 1 afterwards).
(chemical purity: about 83%, enantiomer is excessive: 91%ee) for productive rate: 2.8g.
Go up chromatographic separation [post: Chiralpak AD, 500mm * 40mm mutually in chirality subsequently to enantiomer; Moving phase: Virahol/isohexane 2.5: 97.5; Flow velocity: 50ml/min; Temperature: 24 ℃; Detect: 254nm] title compound of 2.4g enantiomeric pure is provided by the 2.65g product of above acquisition.
R
t=6.78min[Chiralpak AD, 250mm * 4.6mm; Moving phase: Virahol/isohexane 2.5: 97.5; Flow velocity: 1.0ml/min; Detect: 254nm]
1H-NMR(300MHz，CDCl
3)：δ＝8.00-7.89(m，2H)，7.72(d，2H)，5.68-5.59(m，1H)，3.36-3.14(m，2H)，2.65-2.50(m，2H)，2.34(d，1H)，2.20-2.04(m，2H)，1.94-1.30(m，16H)，0.83-0.73(m，1H)，0.72-0.59(m，1H)，0.53-0.41(m，2H)
MS(DCI)：m/z＝484[M+H]
+.
Embodiment 6A
((5 ' S)-5 '-{ [tert-butyl (dimethyl) silyl] oxygen base }-2 ', 4 '-two cyclopentyl-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-3 '-yl) [4-(trifluoromethyl) phenyl] ketone
Under argon, in the 20ml dry toluene, dissolve compound and the 1.99g (18.61mmol) 2 of 2.25mg (4.65mmol) from embodiment 5A, the 6-lutidine also is cooled to-20 ℃.Under this temperature, drip the solution of 2.46g (9.31mmol) trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester in the 5ml dry toluene, and mixture stirs 15min down at-20 ℃ subsequently, warm afterwards to 0 ℃ and under this temperature restir 1h.In mixture, add 75ml0.1N hydrochloric acid, repeat to extract with ethyl acetate after it.The organic phase that merges once and with saturated sodium chloride solution is washed once with saturated sodium hydrogen carbonate solution and saturated sodium chloride solution mixture washing in 1: 1, by dried over sodium sulfate, filters and concentrates.Resistates is chromatogram purification (moving phase: begin hexanaphthene, cyclohexane/ethyl acetate is 15: 1 afterwards) on silica gel.
Productive rate: 2.18g (theoretical 78%)
1H-NMR(300MHz，CDCl
3)：δ＝8.00-7.87(m，2H)，7.71(d，2H)，5.28-5.18(m，1H)，3.38-3.11(m，1H)，2.96(d，1H)，2.80(d，1H)，2.65-2.43(m，1H)，2.08-1.23(m，18H)，0.87(s，9H)，0.76-0.58(m，2H)，0.46-0.38(m，1H)，0.37-0.25(m，1H)，0.18(s，3H)，0.10(s，3H).
MS(ESIpos)：m/z＝598[M+H]
+.
Embodiment 7A
(S) ((5 ' S)-5 '-{ [tert-butyl (dimethyl) silyl] oxygen base }-2 ', 4 '-two cyclopentyl-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-3 '-yl) [4-(trifluoromethyl) phenyl] methyl alcohol
Under argon, in the 35ml dry toluene, begin to add 2.10g (3.51mmol) from the compound of embodiment 6A and be cooled to-50 ℃.Under this temperature, slowly drip the 1M solution of 17.56mg (17.56mmol) diisobutyl aluminium hydride in toluene.Mixture-50 ℃ stir down 10min and after through the warm room temperature that arrives of 1h.Follow ice-coldly, in mixture, add the sodium tartrate potassium solution of 20% concentration, repeat to extract with ethyl acetate after it.The organic phase that merges, is filtered and is concentrated by dried over sodium sulfate with saturated sodium chloride solution washing.This has produced the 2.4g crude product.
Go up chromatographic separation [post: Chiralpak AD, 500mm * 40mm, 20 μ m mutually in chirality subsequently to diastereomer; Moving phase: Virahol/isohexane 2.5: 97.5; Flow velocity: 50ml/min; Temperature: 24 ℃; Detect: 254nm] produced the diastereoisomeric cis-isomeride of the title compound (trans-isomer) of 1.2g (theoretical 56%) diastereisomericallypure pure and 0.9g (theory 42%).
Trans-diastereomer
R
t=5.25min[post: Chiralpak AD, 250mm * 4.6mm; Moving phase: Virahol/isohexane 2.5: 97.5; Flow velocity: 1.5ml/min; Detect: 250nm]
1H-NMR(400MHz，CDCl
3)：δ＝7.58(d，2H)，7.41(d，2H)，6.23(br.s，1H)，5.20(t，1H)，3.68-3.55(m，1H)，3.08-2.70(m，3H)，2.29-2.18(m，1H)，2.12-1.94(m，2H)，1.90-1.22(m，15H)，0.89(s，9H)，0.76-0.68(m，1H)，0.62-0.55(m，1H)，0.43-0.36(m，1H)，0.33-0.25(m，1H)，0.13(s，3H)，0.11(s，3H)
MS(ESIpos)：m/z＝600[M+H]
+.
Cis-diastereomer
R
t=4.36min[post: Chiralpak AD, 250mm * 4.6mm; Moving phase: Virahol/isohexane 2.5: 97.5; Flow velocity: 1.5ml/min; Detect: 250m]
1H-NMR(400MHz，CDCl
3)：δ＝7.56(d，2H)，7.34(d，2H)，6.23(br.s，1H)，5.20(t，1H)，3.68-3.55(m，1H)，3.13-2.60(m，3H)，2.29-2.11(m，2H)，2.10-1.98(m，1H)，1.90-1.22(m，15H)，0.89(s，9H)，0.76-0.68(m，1H)，0.62-0.55(m，1H)，0.43-0.36(m，1H)，0.33-0.25(m，1H)，0.13(s，3H)，0.11(s，3H)
MS(ESIpos)：m/z＝600[M+H]
+.
Embodiment 8A
(5 ' S)-5 '-{ [tert-butyl (dimethyl) silyl] oxygen base }-2 ', 4 '-two cyclopentyl-3 '-(S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]
Under argon, in 10ml toluene, dissolve 500mg (0.83mmol) from the compound of embodiment 7A and be cooled to-20 ℃.Under this temperature, drip 0.18ml (1.38mmol) diethylamino sulfur trifluoride.Remove cooling, and mixture restir 2h afterwards.In mixture, add water, repeat to extract with methylene dichloride after it.The organic phase that merges is washed once with saturated sodium hydrogen carbonate solution and with saturated sodium chloride solution washed twice, by dried over sodium sulfate, is filtered and concentrate.Crude product is dry under high vacuum not to be further purified with reaction.
Productive rate: 485mg (theoretical 96%)
1H-NMR(400MHz，CDCl
3)：δ＝7.61(d，2H)，7.38(d，2H)，6.91(d，1H)，5.21(t，1H)，3.66-3.55(m，1H)，2.97-2.80(m，3H)，2.16-2.00(m，2H)，1.98-1.60(m，12H)，1.50-1.22(m，3H)，1.20-1.05(m，1H)，0.90(s，9H)，0.78-0.70(m，1H)，0.63-0.57(m，1H)，0.44-0.37(m，1H)，0.35-0.28(m，1H)，0.13(s，1H)，0.11(s，3H)
MS(ESIpos)：m/z＝602[M+H]
+.
The embodiment that implements
Embodiment 1
(5 ' S)-2 ', 4 '-two cyclopentyl-3 '-(S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-5 ', 8 '-dihydro-6 ' H-spiral shell [cyclopropane-1,7 '-quinoline]-5 '-alcohol
Under argon, in 1ml THF, dissolve the compound of 475mg (0.79mmol) from embodiment 8A, add 1M solution and the mixture of 3.95ml (3.95mmol) TBAF in THF and at room temperature stir 2h.In mixture, add 50ml 0.2N hydrochloric acid, repeat to extract with ethyl acetate after it.The organic phase that merges, is filtered and is concentrated by dried over sodium sulfate with saturated sodium chloride solution washed twice.Resistates is chromatogram purification (moving phase: begin hexanaphthene, cyclohexane/ethyl acetate is 10: 1 afterwards) on silica gel.
Productive rate: 293mg (theoretical 76%), it is excessive to have a diastereomer of 90%.
Chirality use on mutually the further separation of chromatogram still be present in diastereomer in the product [post: KBD 5945,400mm * 30mm is based on poly-(the N-methacryloyl-L-leucine-tert-butyl acid amides of chiral selector; Moving phase: MTBE/ isohexane 20: 80; Flow velocity: 50ml/min; Temperature: 24 ℃; Detect: the title compound that the 251mg diastereisomericallypure pure 254nm) is provided:
R
t=4.67min[post: KBD 5945,250 * 4.6mm; Moving phase: MTBE/ isohexane 3: 7; Flow velocity: 1.0ml/min; Detect: 280nm]
1H-NMR (400MHz, CDCl
3): δ=7.62 (d, 2H), 7.36 (d, 2H), 6.97 (d, 1H), 5.12 (br.s, 1H), 3.84 (quintet, 1H), 3.24 (dd, 1H), 2.98-2.86 (m, 1H), 2.48 (d, 1H), 2.36 (d, 1H), 2.22-1.52 (m, 14H), 1.49-1.20 (m, 3H), 1.06-0.90 (m, 1H), 0.80-0.72 (m, 1H), and 0.67-0.58 (m, 1H), 0.51-0.40 (m, 2H)
MS(ESIpos)：m/z＝488[M+H]
+.
B. the evaluation of pharmacological activity
The B-I.CETP-inhibition test
The acquisition of B-I.1.CETP
CETP is obtained with partially purified form by differential centrifugation and column chromatography by people's blood plasma and is used for this test.For this reason, use NaBr with human plasma adjust to every ml 1.21g density and under 4 ℃ under 50000rmp centrifugal 18h.(d＞1.21g/ml) is used for Sephadex to the bottom component
-Phenyl-Sepharose 4B (Pharmacia company) post washs and uses afterwards the distilled water wash-out with 0.15MNaCl/0.001M TrisHCl pH 7.4.Collect the CETP active ingredient, with 50mM sodium acetate pH 4.5 dialysis and put on CM-Sepharose
Post (Pharmacia company).Mixture uses linear gradient (0-1M NaCl) wash-out afterwards.The CETP component of collecting with 10mM TrisHCl pH 7.4 dialysis and afterwards further by chromatogram at Mono Q
Post (Pharmacia) is gone up purifying.
The B-I.2.CETP fluorescent test
The mensuration of fluorescence cholesteryl ester between the catalytic liposome of CETP transhipment [according to Bisgaier etc., J.Lipid Res.
34, 1625 (1993) step is improved]:
In order to prepare the donor liposome, along with gently the dissolving 1mg cholesteryl-4 of heating in the two alkane of 600 μ l in ultrasonic bath, 4-two fluoro-5,7-dimethyl-4-boron is mixed-3a, 4a-diaza-s-indacene (indacene)-3-dodecylate (cholesteryl BODIPY
FLC
12, Molecular Probes company), 15.35mg triolein and 6.67mg phosphatidylcholine and at room temperature in 63ml 50mM TrisHCl/150mMNaCl/2mM edta buffer liquid pH 7.3, add this solution very lentamente with the ultrasonic wave effect.Afterwards suspension in the Branson ultra sonic bath under about 50 watts at N
2Ultrasonication 30min under the atmosphere, temperature remains on about 20 ℃.
By the 86mg Oleoylcholesterol that is dissolved in 1.2ml two alkane and the above damping fluid of 114ml, 20mg triolein and 100mg phosphatidylcholine obtain the acceptor liposome similarly by the ultrasonication under 30 minutes 50 watts (20 ℃).
B-I.2.1. the CETP fluorescent test of enrichment CETP
In order to test, use by damping fluid more than 1 part 1 part of donor liposome and 2 parts of test mixtures that the acceptor liposome constitutes.
With the solution-treated 50 μ l test mixtures of material in DMSO that the CETP component (1-3 μ g) and the 2 μ l of 48 μ l enrichments will check, this CETP component obtains by the blood plasma of hydrophobic chromatography by the people, and cultivates 4 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 485/535nm changes, with the inhibition of the control batch comparative measurement transhipment that does not have material.
| Embodiment number | IC 50[nM] fluorescent test |
| 1 | 15 |
B-I.2.2. the CETP fluorescent test of human plasma
In 42 μ l (86%v/v) human plasmas (Sigma P9523), add the solution of material in DMSO that 6 μ l (12%v/v) donor liposomes and 1 μ l (2%v/v) will check, and mixture was cultivated 24 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 510/520nm changes (stitching wide 2.5nm), with the inhibition of the control batch comparative measurement transhipment that does not have material.
| Embodiment number | IC 50Fluorescent test in [nM] human plasma |
| 1 | 40 |
B-I.2.3. the CETP fluorescent test in vitro exsomatizes
In 80 μ l test mixtures, add 10 μ l damping fluids and 2 μ l serum, and mixture was cultivated 4 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 485/535nm changes, with the inhibition of the control batch comparative measurement transhipment that does not have material.
B-I.3. the acquisition of radiolabeled HDL
The fresh people's edta plasma of 50ml uses NaBr to adjust to 1.12 density and centrifugal 18h in 65 rotors of the Ty under 50000rpm under 4 ℃.The upper strata is used to obtain cold LDL mutually.Lower floor uses 3 * 4 liters of PDB damping fluids mutually, and (10mM TrisHCl, pH 7.4,0.15mM NaCl, 1mM EDTA, 0.02%NaN
3) dialysis.Every afterwards 10ml retentate volume adds 20 μ l
3H-cholesterol (Dupont NET-725; In ethanol the dissolving 1 μ C/ μ l) and mixture under 37 ℃ at N
2Under cultivated 72 hours.
This batch of material uses NaBr to adjust to density 1.21 and centrifugal 18h in 65 rotors of the Ty under 50000rpm under 20 ℃ afterwards.Reclaim the upper strata phase and pass through the gradient centrifugation purification lipoprotein component.For this reason, lipoprotein isolating, mark uses NaBr to adjust to 1.26 density.In centrifuge tube (SW 40 rotors) be that 1.21 solution and 4.5ml density are that 1.063 solution covers this solution (the density solution of PDB damping fluid and NaBr) of each part of 4ml and centrifugal at SW 40 rotors under 38000rpm and 20 ℃ afterwards with 4ml density.Middle layer between density 1.063 and 1.21, it comprises the HDL of mark, 4 ℃ of following PDB damping fluid dialysis with 3 * 100 volumes.
Retentate comprises radiolabeled
3H-CE-HDL, it adjusts to about 5 * 106cmp/ml, is used for this test.
The B-I.4.CETP-SPA test
In order to test the activity of CETP, measure
3The H-cholesteryl ester is by the transhipment to the LD lipoprotein of biotin function of people's HD lipoprotein, and reaction is by adding streptavidin-SPA
Pearl (Amersham company) finishes and the radioactivity of transfer is directly measured in liquid scintillation counter.
In test batch, 10 μ l HDL-
3H-cholesteryl ester (about 50000cpm) is comprising 50mM Hepes/0.15M NaCl/0.1% bovine serum albumin/0.05%NaN of 10 μ l CETP (1mg/ml) with 10 μ l vitamin H-LDL (Amersham company) down at 37 ℃
3Cultivated 18 hours in the material (being dissolved in the solution of 10%DMSO/1%RSA) that pH 7.4 and 3 μ l will test.Add 200 μ l SPA-streptavidin pearl solution (TRKQ 7005) afterwards, further cultivate 1h and in scintillometer, measure afterwards along with jolting.With 10 μ l damping fluids, cultivate in contrast in the correspondence of 10 μ l CETP under 4 ℃ and 10 μ l CETP under 37 ℃.
Regarding 100% as 37 ℃ of activity that shift down in the contrast batch of material that uses CETP shifts.This transfer reduce to a half material concentration be defined as IC
50Value.
| Embodiment number | IC 50[nM] SPA test |
| 1 | 24 |
B-II.1. changeing on the solid hCETP mouse of base the in vitro mensuration of isolated activity
Suppress active in order to test CETP, use stomach tube to give inner oral this material of transgenosis hCETP mouse [Dinchuk etc., BBA 1295-301 (1995)] of feeding.For this reason, arbitrarily be arranged to and have the equal number animal beginning to test the day before yesterday buck, usually the group of n=4.Before using this material, be used for measuring its basic active blood of CETP (time point T1) of serum by the puncture collection of vena orbitalis posterior clump (retroorbitalenVenenplexus) by each mouse.Use stomach tube to take this substances afterwards to animal.Specified time after substances is used, by puncture for the second time (time point T2) from animal, get blood, usually behind application of substances 16 or 24h, but as suitable this also can carry out in another time.
In order to assess the inhibition activity of material,, promptly 16 or 18 hours, use animal only to accept not have the corresponding control group of the preparation of material to each time.In control animal, for can measure through corresponding test period at interval (16 or 24h) do not have the active change of CETP of inhibitor, in the animal of mass treatment, carry out the secondary blood specimen collection of each animal.
After solidifying end, blood samples is centrifugal and remove serum deprivation with suction pipe.In order to measure the CETP activity, measure transportation through the cholesteryl ester (Cholesterylester) of 4h.For this reason, the carrying out of in test batch, using 2 μ l serum and test as under B-I.2.3., describing usually.
Calculate for each animal the cholesteryl ester transportation difference [pM CE/h (T2)-pMCE/h (T1)] and in group, average.In one of time point, reduced＞material of the transportation of 20% cholesteryl ester is considered to active.
| Embodiment number | Inhibition % at 3mg/kg | |
| 16h | 24h | |
| 1 | 64 | 58 |
B-II.2. the mensuration of activity in vivo in Syria's cricetulus auratus
The inner female Syria cricetulus auratus of feeding and having 150-200g weight (planting BAY:DSN) is used to measure the oral effect of CETP inhibitor for serum lipoprotein and serum triglyceride.Six animals of each cage be divided into one group and feed arbitrarily and intake the domestication two weeks.
Just before on-test and after material takes, get blood and at room temperature cultivate 30min and after under the 30000g centrifugal 20 minutes, be used to obtain serum by puncture behind the socket of the eye of venous plexus.This substance dissolves is in 20%Solutol/80% water and oral by stomach tube, the solvent of control animals received equal volume and do not have substances.
Operational analysis instrument COBAS INTEGRA 400 plus (from Roche Diagnostics company) measure triglyceride level, cholesterol total amount, HDL cholesterol and LDL cholesterol according to manufacturer's specification sheets.From measured value, for each parameter, the per-cent that causes with mass treatment of change and each group calculate by to(for) each animal are defined as the mean value (n=6 or n=12) with standard deviation.If with the group of solvent treatment relatively, the influence of material is significant, add p-value by the t-test determination (
*P≤0.05;
*P≤0.01;
* *P≤0.005).
B-II.3. the mensuration of activity in vivo in transgenosis hCETP mouse
In order to measure oral effect, use stomach tube to take substances for transgenic mice [Dinchuk etc., BBA, 1295-1301 (1995)] for lipoprotein and triglyceride level.Before on-test, after measuring cholesterol in the serum and the socket of the eye of triglyceride level, draw blood by mouse.The serum that obtains as described above for hamster is in 4 ℃ of following overnight incubation and centrifugal under 6000g subsequently.After three days, in order to measure lipoprotein and triglyceride level is got blood by mouse again.The change of the parameter of measuring is expressed as the change with starting value per-cent relatively.
| Embodiment number | % (the dosage: 3 * 3mg/kg) that HDL increases after 3 days |
| 1 | 56 |
C. the embodiment that implements of pharmaceutical composition
Compound of the present invention can change into pharmaceutical preparation with following method:
Sheet:
Composition:
100mg compound of the present invention, 50mg lactose (monohydrate), 50mg W-Gum (homemade), 10mg polyvinylpyrrolidone (PVP 25) (from BASF, Ludwigshafen, Germany) and 2mg Magnesium Stearate.
Sheet weight 212mg.Diameter 8mm, bending radius 12mm.
Produce:
Compound of the present invention, the mixture of lactose and starch are in water and PVP solution (m/m) granulation of 5% concentration.Particle mixed with Magnesium Stearate 5 minutes after dry.This mixture use conventional tabletting machine (for the form of sheet referring to more than) compressing tablet.The pressure of 15kN is as the standard of compressing tablet.
Suspension that can be oral:
Composition:
1000mg compound of the present invention, 1000mg ethanol (96%), 400mg Rhodigel
(from the xanthan gum of FMC, Pennsylvania, the U.S.) and 99g water.
The 10ml oral suspension is corresponding to the single dose with 100mg compound of the present invention.
Produce:
Rhodigel is suspended in the ethanol and adds compound of the present invention in suspension.Add water along with stirring.Mixture stirs about 6h and finishes up to the Rhodigel swelling.
Solution that can be oral:
Composition:
500mg compound of the present invention, 2.5g polysorbate and 97g poly(oxyethylene glycol) 400.The 20g oral liquid is corresponding to the single dose with 100mg compound of the present invention.
Produce:
Along with stirring, compound of the present invention is suspended in the mixture of polyoxyethylene glycol and polysorbate, continues to stir to dissolve fully up to compound of the present invention.
(i.v.) solution in the body:
Being lower than saturated deliquescent concentration, compound dissolution of the present invention is in physiology acceptable solvent (for example isotonic saline solution, glucose solution 5% and/or PEG 400 solution 30%).Solution is through in sterile filtration and pack into aseptic and the pyrogen-free injection vessel.
Test portion with compound of formula (Ic)
A. embodiment
Abbreviation and acronym
The CE cholesteryl ester
The CETP cholesteryl ester transfer protein
DAST dimethylamino sulfur trifluoride
DCI direct chemical ionization (in MS)
DDQ 2,3-two chloro-5,6-dicyano-1,4-benzoquinones
The de diastereomer is excessive
DMF N, dinethylformamide
The DMSO dimethyl sulfoxide (DMSO)
(in the productive rate) of d.Th theory
EDTA quadrol-N, N, N ', N '-tetraacethyl
The ee enantiomer is excessive
Eq. equivalent
ESI electrospray ionization (in MS)
H hour
The HDL high-density lipoprotein (HDL)
HPLC high pressure-high performance liquid chromatography
The LC/MS liquid chromatograph mass spectrography
The LDL low-density lipoprotein
Min minute
The MS mass spectrum
The NMR nuclear magnetic resonance spectrum
R
fRetention index (in thin-layer chromatography)
R
tRetention time (in HPLC)
The SPA scintillation proximity assay
The TBAF tetrabutyl ammonium fluoride
TBDMSOTf trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester
The TFA trifluoroacetic acid
The THF tetrahydrofuran (THF)
HPLC and LC/MS method
Method 1: post: Chiralpak IA, 250mm * 4.6mm; Moving phase: isohexane/1-propyl alcohol 97: 3; Flow velocity: 1.0ml/min; UV-detects: 254nm.
Method 2: have the HP 1100 that DAD detects; Post: Kromasil RP-18,60mm * 2mm, 3.5 μ m; Mobile phase A: 5ml HClO
4/ l water, Mobile phase B: acetonitrile; Gradient: 0min 2%B → 0.5min 2%B → 4.5min 90%B → 9min 90%B; Flow velocity: 0.75ml/min; Temperature: 30 ℃; UV-detects: 210nm.
Method 3 (LC/MS): MS instrument type: Micromass ZQ; HPLC instrument type: HP 1100 series; UV DAD; Post: Phenomenex Synergi 2 μ Hydro-RP Mercury 20mm * 4mm; Mobile phase A: the formic acid of 1l water+0.5ml 50% concentration, Mobile phase B: the formic acid of 1l acetonitrile+0.5ml 50% concentration; Gradient: 0.0min 90%A → 2.5min 30%A → 3.0min 5%A → 4.5min 5%A; Flow velocity: 0.0min 1ml/min → 2.5min/3.0min/4.5min 2ml/min; Stove: 50 ℃; UV-detects: 210nm.
Parent material and intermediate
Embodiment 1A
4 '-cyclopentyl-2 '-sec.-propyl-3 '-[4-(trifluoromethyl) benzoyl]-4 ', 8 '-dihydro-1 ' H-spiral shell [tetramethylene-1,7 '-quinoline]-5 ' (6 ' H)-ketone
In the 188ml Di Iso Propyl Ether, begin to add 6.3g (24.5mmol, 1.2eq.) 3-amino-3-sec.-propyl-1-(4-trifluoromethyl) acrylketone is (according to WO 03/028727, embodiment 2 preparations), and add 3.14ml (40.8mmol, 2.0eq.) trifluoroacetic acid and 3.1g (20.4mmol, 1eq.) spiral shell [3.5] nonane-6,8-diketone (according to WO 03/028727, embodiment 5 preparations).After at room temperature stirring 10min, add 3.0g (30.6mmol, 1.5eq.) pentamethylene formaldehyde.Mixture heats 18h on water separator under refluxing afterwards.After the cooling, mixture stirs 30min under ice bath, and filters the precipitation of generation and wash and remove the solvent residues thing with cold Di Iso Propyl Ether under high vacuum with suction.
Productive rate: 4.37g (theoretical 45.5%)
1H-NMR (CDCl
3, 400MHz): δ=0.91 (m, 2H), 1.05 (d, 3H), 1.28 (d, 3H), 1.21-2.07 (m, 13H), 2.43 and 2.70 (2d, 2H), 2.63 (s, 2H), 3.47 (septet, 1H), 3.80 (d, 1H), 6.03 (s, 1H), 7.66 (d, 2H), 7.77 (d, 2H) ppm.
MS(ESIpos)：m/z＝472[M+H]
+.
Embodiment 2A
4 '-cyclopentyl-2 '-sec.-propyl-3 '-[4-(trifluoromethyl) benzoyl]-6 ' H-spiral shell [tetramethylene-1,7 '-quinoline]-5 ' (8 ' H)-ketone
In the 180ml methylene dichloride, dissolve the compound of 5.19g (11.0mmol) from embodiment 1A, and portion-wise addition 2.75g (12.1mmol, 1.1eq.) 2,3-two chloro-5,6-dicyano-1,4-benzoquinones (DDQ) stirring at room temperature.Mixture at room temperature stirs 1h.Enriched mixture and resistates are by chromatogram purification (silica gel, moving phase: isohexane/ethyl acetate 100: 0 → 50: 50) on rotatory evaporator.
Productive rate: 4.74g (theoretical 91.6%)
1H-NMR (CDCl
3, 300MHz): δ=1.10 (d, 3H), 1.19 (d, 3H), 1.33-2.10 (m, 14H), 2.59 (septet, 1H), 2.82 (s, 2H), 2.99 (septet, 1H), 3.30 (s, 2H), 7.75 (d, 2H), 7.94 (m, 2H) ppm.
MS(ESIpos)：m/z＝470[M+H]
+.
Embodiment 3A
[(5 ' S)-4 '-cyclopentyl-5 '-hydroxyl-2 '-sec.-propyl-5 ', 8 '-dihydro-6 ' H-spiral shell [tetramethylene-1,7 '-quinoline]-3 '-yl] [4-(trifluoromethyl) phenyl] ketone
(0.81mmol, 0.08eq.) (1R 2S)-1-aminoidan-2-alcohol, and at room temperature adds 6.6g (40.4mmol, 4.0eq.) borine-N, N-diethylbenzene amine complex to begin to add 120mg in 250ml THF.After the release of gas stopped, mixture was cooled to 0 ℃, and (10.1mmol is 1eq.) from the compound of embodiment 2A to add the 4.74g be dissolved among the 250ml THF.Follow stirring, make mixture warm up room temperature through 18h.After reaction finishes, in reaction mixture, add 20ml methyl alcohol, be evaporated to dried after it.By chromatogram purification crude product (silica gel, moving phase: isohexane/ethyl acetate mixture).
Productive rate: 4.33g (theoretical 91.1%)
Measuring enantiomer excessive according to method 1 is 94.0%ee.
1H-NMR (CDCl
3, 300MHz): δ=0.99-1.19 (m, 6H), 1.29-2.41 (m, 14H), 2.53 (septet, 1H), 2.93 (d, 1H), 3.15-3.54 (m, 2H), 5.13 (m, 1H), 7.73 (d, 2H), 7.94 (m, 2H) ppm.
MS(ESIpos)：m/z＝472[M+H]
+.
Embodiment 4A
((5 ' S)-5 '-{ [tert-butyl (dimethyl) silyl] oxygen base }-4 '-cyclopentyl-2 '-sec.-propyl-5 ', 8 '-dihydro-6 ' H-spiral shell [tetramethylene-1,7 '-quinoline]-3 '-yl) [4-(trifluoromethyl) phenyl] ketone
Under argon, in the 30ml dry toluene, dissolve the compound of 4.00g (8.48mmol) from embodiment 3A.At room temperature, add 3.64g (33.9mmol, 4eq.) 2,6-lutidine, and mixture is cooled to-16 ℃ afterwards.In this solution, drip 3.90ml (17.0mmol, 2eq.) the trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester that is dissolved in the 10ml toluene.Behind the 15min, reaction mixture warm to 0 ℃ and under this temperature restir 80min.For aftertreatment, add 124ml 0.1N hydrochloric acid, and mixture is warm uses ethyl acetate extraction after room temperature.Organic phase is washed with saturated sodium chloride solution and saturated 1: 1 mixture of sodium bicarbonate.The water that contains that merges is brought up again with ethyl acetate and is got twice.The organic phase that merges is filtered and is under reduced pressure concentrated by dried over sodium sulfate.Resistates is by chromatogram purification (silica gel, moving phase: isohexane/ethyl acetate 9: 1).
Productive rate: 4.61g (theoretical 92.7%)
1H-NMR(CDCl
3，400MHz)：δ＝0.12(s，3H)，0.23(s，3H)，0.86(s，9H)，1.06(d，3H)，1.12(d，3H)，1.24-2.12(m，14H)，2.19-2.35(m，2H)，2.51(m，1H)，2.89-3.44(m，3H)，5.17(m，1H)，7.73(d，2H)，7.84-8.02(m，2H)ppm.
MS(ESIpos)：m/z＝586[M+H]
+.
Embodiment 5A
(S) ((5 ' S)-5 '-{ [tert-butyl (dimethyl) silyl] oxygen base }-4 '-cyclopentyl-2 '-sec.-propyl-5 ', 8 '-dihydro-6 ' H-spiral shell [tetramethylene-1,7 '-quinoline]-3 '-yl) [4-(trifluoromethyl) phenyl] methyl alcohol
Under 0 ℃, in the solution of the anhydrous THF of 46ml, add 5.1ml1M lithium aluminum hydride (5.1mmol, 1.1eq.) solution in THF from the compound of embodiment 4A to 2.71g (4.6mmol).Follow stirring, mixture is warm to room temperature through 3.5h.For aftertreatment, carefully add the saturated potassium sodium tartrate solution of 120ml.After the release of gas stops, mixture ethyl acetate extraction four times, and the organic phase that merges by dried over sodium sulfate, is filtered also under reduced pressure concentrated with saturated sodium chloride solution washing.The resistates chromatogram purification has caused separation (silica gel, the moving phase: isohexane/ethyl acetate 95: 5) of product diastereomer.
Productive rate: 1.39g (theoretical 51.2%)
1H-NMR(CDCl
3，400MHz)：δ＝0.16(s，3H)，0.25(s，3H)，0.67-0.98(m，18H)，1.02-2.34(m，14H)，2.80-3.76(m，4H)，5.19(m，1H)，6.21(br.s，1H)，7.43(d，2H)，7.59(d，2H)ppm.
MS(ESIpos)：m/z＝588[M+H]
+
LC/MS (method 3): R
t=3.03min.
Cis-diastereomer:
(R) ((5 ' S)-5 '-{ [tert-butyl (dimethyl) silyl] oxygen base }-4 '-cyclopentyl-2 '-sec.-propyl-5 ', 8 '-dihydro-6 ' H-spiral shell [tetramethylene-1,7 '-quinoline]-3 '-yl) [4-(trifluoromethyl) phenyl] methyl alcohol
Productive rate: 1.04g (theoretical 38.1%)
Embodiment 6A
(5 ' S)-5 '-{ [tert-butyl (dimethyl) silyl] oxygen base }-4 '-cyclopentyl-3 '-(S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-2 '-sec.-propyl-5 ', 8 '-dihydro-6 ' H-spiral shell [tetramethylene-1,7 '-quinoline]
-15 ℃ down and under argon, to 0.94g (1.6mmol) drip in from the solution of compound in the 15ml dry toluene of embodiment 5A 0.42ml diethylamino sulfur trifluoride (3.2mmol, 1.1eq.).Mixture stirs 5h, and is warm to 0 ℃.For aftertreatment, follow ice-cooled careful interpolation 40ml saturated sodium bicarbonate solution.Mixture is used ethyl acetate extraction three times altogether.Latter incorporated organic phase with the washing of saturated sodium chloride solution, by dried over sodium sulfate, filter and under reduced pressure concentrate.Crude product is by filtering by silica gel purification (moving phase: cyclohexane/ethyl acetate 9: 1).
Productive rate: 0.65g (theoretical 69.0%)
R
f=0.72 (isohexane/ethyl acetate 9: 1)
The embodiment that implements
Embodiment 1
(5 ' S)-4 '-cyclopentyl-3 '-(S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-2 '-sec.-propyl-5 ', 8 '-dihydro-6 ' H-spiral shell [tetramethylene-1,7 '-quinoline]-5 '-alcohol
Under 0 ℃, to 0.65g (1.1mmol) from the compound of embodiment 6A in the solution of the anhydrous THF of 6ml, drip the 4.4ml1M tetrabutyl ammonium fluoride (4.4mmol, 4.0eq.).Mixture stirs 4h in ice bath.For aftertreatment, mixture is with the dilution of 20ml ethyl acetate and use the 20ml water washing.Containing water under each situation brings up again with the 20ml ethyl acetate and gets twice.The organic phase that merges, is filtered and is under reduced pressure concentrated by dried over sodium sulfate with the saturated sodium chloride solution washing of 50ml.Chromatogram purification crude product (silica gel, moving phase: isohexane/ethyl acetate 9: 1 → 4: 1).
Productive rate: 0.47g (theoretical 88.6%)
1H-NMR (CDCl
3, 400MHz): δ=0.75 (d, 3H), 1.11 (d, 3H), 1.30-2.33 (m, 17H), 2.89 (m, 1H), 2.89 and 3.33 (2d, 2H), 3.82 (s, 1H), 5.12 (m, 1H), 6.94 (d, 1H), 7.35 (d, 2H), 7.62 (d, 2H) ppm.
MS(ESIpos)：m/z＝476[M+H]
+
R
f=0.14 (isohexane/ethyl acetate 9: 1)
B. the evaluation of pharmacological activity
The B-I.CETP-inhibition test
The acquisition of B-I.1.CETP
CETP is obtained with partially purified form by differential centrifugation and column chromatography by people's blood plasma and is used for this test.For this reason, use NaBr with human plasma adjust to every ml 1.21g density and under 4 ℃ under 50000rmp centrifugal 18h.(d＞1.21g/ml) is used for Sephadex to the bottom component
-Phenyl-Sepharose 4B (Pharmacia company) post washs and uses afterwards the distilled water wash-out with 0.15MNaCl/0.001M TrisHCl pH 7.4.Collect the CETP active ingredient, with 50mM sodium acetate pH 4.5 dialysis and put on CM-Sepharose
Post (Pharmacia company).Mixture uses linear gradient (0-1M NaCl) wash-out afterwards.The CETP component of collecting with 10mM TrisHCl pH 7.4 dialysis and afterwards further by chromatogram at Mono Q
Post (Pharmacia) is gone up purifying.
The B-I.2.CETP fluorescent test
The mensuration of fluorescence cholesteryl ester between the catalytic liposome of CETP transhipment [according to Bisgaier etc., J.Lipid Res.
34, 1625 (1993) step is improved]:
In order to prepare the donor liposome, along with gently the dissolving 1mg cholesteryl-4 of heating in the two alkane of 600 μ l in ultrasonic bath, 4-two fluoro-5,7-dimethyl-4-boron is mixed-3a, 4a-diaza-s-indacene (indacene)-3-dodecylate (cholesteryl BODIPY
FLC
12, Molecular Probes company), 15.35mg triolein and 6.67mg phosphatidylcholine and at room temperature in 63ml 50mM TrisHCl/150mMNaCl/2mM edta buffer liquid pH 7.3, add this solution very lentamente with the ultrasonic wave effect.Afterwards suspension in the Branson ultra sonic bath under about 50 watts at N
2Ultrasonication 30min under the atmosphere, temperature remains on about 20 ℃.
By the 86mg Oleoylcholesterol that is dissolved in 1.2ml two alkane and the above damping fluid of 114ml, 20mg triolein and 100mg phosphatidylcholine obtain the acceptor liposome similarly by the ultrasonication under 30 minutes 50 watts (20 ℃).
B-I.2.1. the CETP fluorescent test of enrichment CETP
In order to test, use by damping fluid more than 1 part 1 part of donor liposome and 2 parts of test mixtures that the acceptor liposome constitutes.
With the solution-treated 50 μ l test mixtures of material in DMSO that the CETP component (1-3 μ g) and the 2 μ l of 48 μ l enrichments will check, this CETP component obtains by the blood plasma of hydrophobic chromatography by the people, and cultivates 4 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 485/535nm changes, with the inhibition of the control batch comparative measurement transhipment that does not have material.
| Embodiment number | IC 50[nM] fluorescent test |
| 1 | 20 |
B-I.2.2. the CETP fluorescent test of human plasma
In 42 μ l (86%v/v) human plasmas (Sigma P9523), add the solution of material in DMSO that 6 μ l (12%v/v) donor liposomes and 1 μ l (2%v/v) will check, and mixture was cultivated 24 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 510/520nm changes (stitching wide 2.5nm), with the inhibition of the control batch comparative measurement transhipment that does not have material.
| Embodiment number | IC 50Fluorescent test in [nM] human plasma |
| 1 | 85 |
B-I.2.3. the CETP fluorescent test in vitro exsomatizes
In 80 μ l test mixtures, add 10 μ l damping fluids and 2 μ l serum, and mixture was cultivated 4 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 485/535nm changes, with the inhibition of the control batch comparative measurement transhipment that does not have material.
B-I.3. the acquisition of radiolabeled HDL
The fresh people's edta plasma of 50ml uses NaBr to adjust to 1.12 density and centrifugal 18h in 65 rotors of the Ty under 50000rpm under 4 ℃.The upper strata is used to obtain cold LDL mutually.Lower floor uses 3 * 4 liters of PDB damping fluids mutually, and (10mM TrisHCl, pH 7.4,0.15mM NaCl, 1mM EDTA, 0.02%NaN
3) dialysis.Every afterwards 10ml retentate volume adds 20 μ l
3H-cholesterol (Dupont NET-725; In ethanol the dissolving 1 μ C/ μ l) and mixture under 37 ℃ at N
2Under cultivated 72 hours.
This batch of material uses NaBr to adjust to density 1.21 and centrifugal 18h in 65 rotors of the Ty under 50000rpm under 20 ℃ afterwards.Reclaim the upper strata phase and pass through the gradient centrifugation purification lipoprotein component.For this reason, lipoprotein isolating, mark uses NaBr to adjust to 1.26 density.In centrifuge tube (SW 40 rotors) be that 1.21 solution and 4.5ml density are that 1.063 solution covers this solution (the density solution of PDB damping fluid and NaBr) of each part of 4ml and centrifugal at SW 40 rotors under 38000rpm and 20 ℃ afterwards with 4ml density.Middle layer between density 1.063 and 1.21, it comprises the HDL of mark, 4 ℃ of following PDB damping fluid dialysis with 3 * 100 volumes.
Retentate comprises radiolabeled
3H-CE-HDL, it adjusts to about 5 * 106cmp/ml, is used for this test.
The B-I.4.CETP-SPA test
In order to test the activity of CETP, measure
3The H-cholesteryl ester is by the transhipment to the LD lipoprotein of biotin function of people's HD lipoprotein, and reaction is by adding streptavidin-SPA
Pearl (Amersham company) finishes and the radioactivity of transfer is directly measured in liquid scintillation counter.
In test batch, 10 μ l HDL-
3H-cholesteryl ester (about 50000cpm) is comprising 50mM Hepes/0.15M NsCl/0.1% bovine serum albumin/0.05%NaN of 10 μ l CETP (1mg/ml) with 10 μ l vitamin H-LDL (Amersham company) down at 37 ℃
3Cultivated 18 hours in the material (being dissolved in the solution of 10%DMSO/1%RSA) that pH 7.4 and 3 μ l will test.Add 200 μ l SPA-streptavidin pearl solution (TRKQ 7005) afterwards, further cultivate 1h and in scintillometer, measure afterwards along with jolting.With 10 μ l damping fluids, cultivate in contrast in the correspondence of 10 μ l CETP under 4 ℃ and 10 μ l CETP under 37 ℃.
Regarding 100% as 37 ℃ of activity that shift down in the contrast batch of material that uses CETP shifts.This transfer reduce to a half material concentration be defined as IC
50Value.
| Embodiment number | IC 50[nM] SPA test |
| 1 | 36 |
B-II.1. the mensuration of isolated activity in vitro on transgenosis hCETP mouse
Suppress active in order to test CETP, use stomach tube to give inner oral this material of transgenosis hCETP mouse [Dinchuk etc., BBA 1295-301 (1995)] of feeding.For this reason, arbitrarily be arranged to and have the equal number animal beginning to test the day before yesterday buck, usually the group of n=4.Before using this material, be used for measuring its basic active blood of CETP (time point T1) of serum by the puncture collection of vena orbitalis posterior clump (retroorbitalenVenenplexus) by each mouse.Use stomach tube to take this substances afterwards to animal.Specified time after substances is used, by puncture for the second time (time point T2) from animal, get blood, usually behind application of substances 16 or 24h, but as suitable this also can carry out in another time.
In order to assess the inhibition activity of material,, promptly 16 or 18 hours, use animal only to accept not have the corresponding control group of the preparation of material to each time.In control animal, for can measure through corresponding test period at interval (16 or 24h) do not have the active change of CETP of inhibitor, in the animal of mass treatment, carry out the secondary blood specimen collection of each animal.
After solidifying end, blood samples is centrifugal and remove serum deprivation with suction pipe.In order to measure the CETP activity, measure transportation through the cholesteryl ester (Cholesterylester) of 4h.For this reason, the carrying out of in test batch, using 2 μ l serum and test as under B-I.2.3., describing usually.
Calculate for each animal the cholesteryl ester transportation difference [pM CE/h (T2)-pMCE/h (T1)] and in group, average.In one of time point, reduced＞material of the transportation of 20% cholesteryl ester is considered to active.
| Embodiment number | Inhibition % at 3mg/kg | |
| 16h | 24h | |
| 1 | 43 | 35 |
B-II.2. the mensuration of activity in vivo in Syria's cricetulus auratus
The inner female Syria cricetulus auratus of feeding and having 150-200g weight (planting BAY:DSN) is used to measure the oral effect of CETP inhibitor for serum lipoprotein and serum triglyceride.Six animals of each cage be divided into one group and feed arbitrarily and intake the domestication two weeks.
Just before on-test and after material takes, get blood and at room temperature cultivate 30min and after under the 30000g centrifugal 20 minutes, be used to obtain serum by puncture behind the socket of the eye of venous plexus.This substance dissolves is in 20%Solutol/80% water and oral by stomach tube, the solvent of control animals received equal volume and do not have substances.
Operational analysis instrument COBAS INTEGRA 400 plus (from Roche Diagnostics company) measure triglyceride level, cholesterol total amount, HDL cholesterol and LDL cholesterol according to manufacturer's specification sheets.From measured value, for each parameter, the per-cent that causes with mass treatment of change and each group calculate by to(for) each animal are defined as the mean value (n=6 or n=12) with standard deviation.If with the group of solvent treatment relatively, the influence of material is significant, add p-value by the t-test determination (
*P≤0.05;
*P≤0.01;
* *P≤0.005).
B-II.3. the mensuration of activity in vivo in transgenosis hCETP mouse
In order to measure oral effect, use stomach tube to take substances for transgenic mice [Dinchuk etc., BBA, 1295-1301 (1995)] for lipoprotein and triglyceride level.Before on-test, after measuring cholesterol in the serum and the socket of the eye of triglyceride level, draw blood by mouse.The serum that obtains as described above for hamster is in 4 ℃ of following overnight incubation and centrifugal under 6000g subsequently.After three days, in order to measure lipoprotein and triglyceride level is got blood by mouse again.The change of the parameter of measuring is expressed as the change with starting value per-cent relatively.
C. the embodiment of the enforcement of pharmaceutical composition
Compound of the present invention can change into pharmaceutical preparation with following method:
Sheet:
Composition:
100mg compound of the present invention, 50mg lactose (monohydrate), 50mg W-Gum (homemade), 10mg polyvinylpyrrolidone (PVP 25) (from BASF, Ludwigshafen, Germany) and 2mg Magnesium Stearate.
Sheet weight 212mg.Diameter 8mm, bending radius 12mm.
Produce:
Compound of the present invention, the mixture of lactose and starch are in water and PVP solution (m/m) granulation of 5% concentration.Particle mixed with Magnesium Stearate 5 minutes after dry.This mixture use conventional tabletting machine (for the form of sheet referring to more than) compressing tablet.The pressure of 15kN is as the standard of compressing tablet.
Suspension that can be oral:
Composition:
1000mg compound of the present invention, 1000mg ethanol (96%), 400mg Rhodigel
(from the xanthan gum of FMC, Pennsylvania, the U.S.) and 99g water.
The 10ml oral suspension is corresponding to the single dose with 100mg compound of the present invention.
Produce:
Rhodigel is suspended in the ethanol and adds compound of the present invention in suspension.Add water along with stirring.Mixture stirs about 6h and finishes up to the Rhodigel swelling.
Solution that can be oral:
Composition:
500mg compound of the present invention, 2.5g polysorbate and 97g poly(oxyethylene glycol) 400.The 20g oral liquid is corresponding to the single dose with 100mg compound of the present invention.
Produce:
Along with stirring, compound of the present invention is suspended in the mixture of polyoxyethylene glycol and polysorbate, continues to stir to dissolve fully up to compound of the present invention.
(i.v.) solution in the body:
Being lower than saturated deliquescent concentration, compound dissolution of the present invention is in physiology acceptable solvent (for example isotonic saline solution, glucose solution 5% and/or PEG 400 solution 30%).Solution is through in sterile filtration and pack into aseptic and the pyrogen-free injection vessel.
Test portion with compound of formula (Id)
A. embodiment
Abbreviation and acronym
The CE cholesteryl ester
The CETP cholesteryl ester transfer protein
DAST diethylamino sulfur trifluoride
DCI direct chemical ionization (in MS)
DDQ 2,3-two chloro-5,6-dicyano-1,4-benzoquinones
The de diastereomer is excessive
DMF N, dinethylformamide
The DMSO dimethyl sulfoxide (DMSO)
(in the productive rate) of d.Th theory
EDTA quadrol-N, N, N ', N '-tetraacethyl
The ee enantiomer is excessive
Eq. equivalent
ESI electrospray ionization (in MS)
H hour
The HDL high-density lipoprotein (HDL)
HPLC high pressure-high performance liquid chromatography
The LC/MS liquid chromatograph mass spectrography
The LDL low-density lipoprotein
Min minute
The MS mass spectrum
The NMR nuclear magnetic resonance spectrum
R
fRetention index (in thin-layer chromatography)
R
tRetention time (in HPLC)
The SPA scintillation proximity assay
The TBAF tetrabutyl ammonium fluoride
TBDMSOTf trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester
The TFA trifluoroacetic acid
The THF tetrahydrofuran (THF)
HPLC and LC/MS method
Method 1A: post: Chiralpak IA, 250mm * 4.6mm; Moving phase: isohexane/1-propyl alcohol 97: 3; Flow velocity: 1.0ml/min; UV-detects: 254nm.
Method 1B: post: Chiralpak AD, 250mm * 4.6mm, 10 μ m; Moving phase: isohexane/1-propyl alcohol 97.5: 2.5; Flow velocity: 1.0ml/min; UV-detects: 254nm.
Method 2: instrument: have the HP 1100 that DAD detects; Post: Kromasil RP-18,60mm * 2mm, 3.5 μ m; Mobile phase A: 5ml HClO
4/ l water, Mobile phase B: acetonitrile; Gradient: 0min 2%B → 0.5min 2%B → 4.5min 90%B → 9min 90%B; Flow velocity: 0.75ml/min; Temperature: 30 ℃; UV-detects: 210nm.
Method 3 (LC/MS): MS instrument type: Micromass ZQ; HPLC instrument type: HP 1100 series; UV DAD; Post: Phenomenex Synergi 2 μ Hydro-RP Mercury 20mm * 4mm; Mobile phase A: the formic acid of 1l water+0.5ml 50% concentration, Mobile phase B: the formic acid of 1l acetonitrile+0.5ml 50% concentration; Gradient: 0.0min 90%A → 2.5min 30%A → 3.0min 5%A → 4.5min 5%A; Flow velocity: 0.0min 1ml/min → 2.5min/3.0min/4.5min 2ml/min; Stove: 50 ℃; UV-detects: 210nm.
Parent material and intermediate
Embodiment 1A
2,4-two cyclopentyl-7,7-dimethyl-3-(4-trifluoromethyl benzoyl)-4,6,7,8-tetrahydrochysene-1H-quinoline-5-ketone
In the 600ml Di Iso Propyl Ether, begin to add 16.8g (59.4mmol, 1.0eq.) 3-amino-3-cyclopentyl-1-(4-trifluoromethyl) acrylketone is (according to WO 03/028727, embodiment 4 preparations), and add 9.16ml (118.9mmol, 2.0eq.) trifluoroacetic acid and 8.3g (59.4mmol, 1eq.) 5,5-dimethyl cyclohexane-1,3-diketone.After at room temperature stirring 30min, and interpolation 7.0g (71.3mmol, 1.2eq.) pentamethylene formaldehyde, and mixture heats 15h on water separator under refluxing afterwards.Behind the cool to room temperature, (10.2mmol, 0.17eq.) pentamethylene formaldehyde, mixture heat under refluxing again and solvent volume reduces to about 500ml to add another 1.0g.Mixture under refluxing on water separator reheat 20h.For aftertreatment, be evaporated to drying after the mixture cooling, and resistates by chromatogram in silica gel (moving phase: isohexane/ethyl acetate 4: 1) go up pre--purifying.Also at room temperature stir 16h with product component absorption in a small amount of Di Iso Propyl Ether that this mode obtains.Filter the precipitation of generation with suction, with cold Di Iso Propyl Ether washing and under high vacuum, remove the solvent residues thing.
Productive rate: 3.8g (theoretical 13%)
1H-NMR (CDCl
3, 300MHz): δ=1.14 (s, 3H), 1.16 (s, 3H), 1.21-2.00 (m, 16H), 2.20 (m, 1H), 2.28 and 2.51 (2d, 2H), 2.34 (s, 2H), 3.50 (sept, 1H), 3.83 (d, 1H), 5.91 (s, 1H), 7.66 (d, 2H), 7.78 (d, 2H) ppm.
MS(ESIpos)：m/z＝486[M+H]
+.
Embodiment 2A
2,4-two cyclopentyl-7,7-dimethyl-3-(4-trifluoromethyl benzoyl)-7,8-dihydro-6H-quinoline-5-ketone
In the 78ml methylene dichloride, dissolve the compound of 3.79g (7.8mmol) from embodiment 1A, and at 0 ℃ of following portion-wise addition 1.95mg (8.6mmol, 1.1eq.) 2,3-two chloro-5,6-dicyano-1,4-benzoquinones (DDQ).Follow stirring, mixture is warm to room temperature through 3h.Mixture concentrates on rotatory evaporator and resistates passes through chromatogram purification (silica gel, moving phase: isohexane/ethyl acetate 9: 1,4: 1).
Productive rate: 3.53g (theoretical 93.6%)
1H-NMR(CDCl
3，400MHz)：δ＝1.10(d，3H)，1.17(d，3H)，1.35-1.97(m，16H)，2.51-2.71(m，3H)，2.94-3.15(m，3H)，7.75(d，2H)，7.93(m，2H)ppm.
MS(ESIpos)：m/z＝484[M+H]
+.
Embodiment 3A
[(5S)-2,4-two cyclopentyl-5-hydroxyl-7,7-dimethyl-5,6,7,8-tetrahydroquinoline-3-yl] (4-trifluoromethyl) ketone
(0.58mmol, 0.08eq.) (1R 2S)-1-aminoidan-2-alcohol, and at room temperature adds 4.76g (29.2mmol, 4.0eq.) borine-N, N-diethylbenzene amine complex to begin to add 87mg in 340ml THF.After the release of gas stops, mixture be cooled to 0 ℃ and the 3.53g that adds to be dissolved among the 25ml THF (7.3mmol is 1eq.) from the compound of embodiment 2A. follow stirring, make the mixture can be warm to room temperature through 16h.After reaction finishes, in reaction mixture, add 20ml methyl alcohol, concentrate after it.Resistates distributes between 150ml water and 150ml ethyl acetate.Contain water and under each situation, use twice of 100ml ethyl acetate extraction.The organic phase that merges is with the washing of 50ml saturated nacl aqueous solution and use dried over sodium sulfate, filters and under reduced pressure concentrates.Resistates is by chromatogram purification (silica gel, moving phase: isohexane/ethyl acetate 100: 0,4: 1 afterwards).
Productive rate: 3.13g (theoretical 88.2%)
Enantiomer is excessive to be determined as 93.5%ee according to method 1A.
1H-NMR(CDCl
3，300MHz)：δ＝1.00(m，3H)，1.23(m，3H)，1.29-2.03(m，19H)，2.56(m，1H)，2.64-3.08(m，2H)，3.29(m，1H)，5.17(m，1H)，7.73(d，2H)，7.93(m，2H)ppm.
MS(ESIpos)：m/z＝486[M+H]
+.
Embodiment 4A
((5S)-5-{[tert-butyl (dimethyl) silyl] the oxygen base }-2,4-two cyclopentyl-7,7-dimethyl-5,6,7,8-tetrahydroquinoline-3-yl) [4-(trifluoromethyl) phenyl] ketone
Under argon, in the 64ml dry toluene, add the compound of 3.12g (6.43mmol) from embodiment 3A.At room temperature, add afterwards 2.76g (25.7mmol, 4eq.) 2,6-lutidine, and mixture is cooled to-18 ℃. in this solution, drip 2.95ml (12.9mmol, 2eq.) trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester.Reaction mixture stirs 2h down at 0 ℃.For aftertreatment, add saturated ammonium chloride solution (100ml), and mixture is warm uses ethyl acetate extraction after room temperature.Contain water and use ethyl acetate extraction twice again, the organic phase of merging, is filtered and is under reduced pressure concentrated by dried over sodium sulfate with saturated sodium chloride solution washing.Resistates is by chromatogram purification (silica gel, moving phase: isohexane/ethyl acetate 9: 1).
Productive rate: 3.90g (quantitatively)
1H-NMR(CDCl
3，300MHz)：δ＝0.08(s，3H)，0.18(s，3H)，0.86(s，9H)，0.91(s，3H)，1.24(s，3H)，1.28-2.06(m，18H)，2.47-3.23(m，3H)，3.32(m，1H)，5.20(m，1H)，7.73(d，2H)，7.81-8.03(m，2H)ppm.
MS(ESIpos)：m/z＝600[M+H]
+.
Embodiment 5A
(S) ((5S)-5-{[tert-butyl (dimethyl) silyl] the oxygen base }-2,4-two cyclopentyl-7,7-dimethyl-5,6,7,8-tetrahydroquinoline-3-yl) [4-(trifluoromethyl) phenyl] methyl alcohol
Under 0 ℃, add 9.8ml 1M lithium aluminum hydride (9.8mmol, 1.5eq.) solution in from the solution of compound in the anhydrous THF of 65ml of embodiment 4A to 3.9g (6.5mmol).0 ℃ of following restir 4 hours.For aftertreatment, carefully add the saturated potassium sodium tartrate solution of 120ml.After the release of gas stopped, mixture washed with saturated ammonium chloride solution and saturated sodium chloride solution with the organic phase of ethyl acetate extraction three times and merging, by dried over sodium sulfate, filters and under reduced pressure concentrates.The resistates chromatogram purification has caused separation (silica gel, the moving phase: isohexane/ethyl acetate 95: 5) of product diastereomer.
Productive rate: 1.87g (theoretical 47.8%)
1H-NMR(CDCl
3，300MHz)：δ＝0.13(s，3H)，0.19(s，3H)，0.82-0.97(m，15H)，1.09-2.12(m，18H)，2.19(m，1H)，2.56(d，1H)，2.86-3.05(m，2H)，3.70(m，1H)，5.21(t，1H)，6.23(br.s，1H)，7.42(d，2H)，7.59(d，2H)ppm.
MS(DCI)：m/z＝602[M+H]
+
R
f=0.26 (isohexane/ethyl acetate 9: 1)
Cis-diastereomer:
(R) ((5S)-5-([tert-butyl (dimethyl) silyl] oxygen base }-2,4-two cyclopentyl-7,7-dimethyl-5,6,7,8-tetrahydroquinoline-3-yl) [4-(trifluoromethyl) phenyl] methyl alcohol
Productive rate: 2.16g (theoretical 53.9%)
R
f=0.34 (isohexane/ethyl acetate 9: 1)
Embodiment 6A
(5S)-and 5-{[tert-butyl (dimethyl) silyl] the oxygen base }-2,4-two cyclopentyl-(S)-and fluorine [4-(trifluoromethyl) phenyl] methyl }-7,7-dimethyl-5,6,7,8-tetrahydroquinoline
-17 ℃ down and under argon, to 1.78g (3.0mmol) add in from the solution of compound in the 29ml anhydrous methylene chloride of embodiment 5A 0.59ml diethylamino sulfur trifluoride (4.4mmol, 1.5eq.).Mixture be cooled to-66 ℃ and afterwards through 6h follow stir warm to 0 ℃.Reaction soln is cooled to-78 ℃ again, and add again 0.25ml diethylamino sulfur trifluoride (1.9mmol, 0.64eq.).Follow stirring, mixture is warm to 10 ℃ through 10h afterwards.For aftertreatment, follow ice-cooled careful interpolation 40ml saturated sodium bicarbonate solution.Mixture is used ethyl acetate extraction three times altogether.Latter incorporated organic phase with the washing of saturated sodium chloride solution, by dried over sodium sulfate, filter and under reduced pressure concentrate.Crude product is by filtering by chromatogram purification (moving phase: cyclohexane/ethyl acetate 9: 1).
Productive rate: 1.67g (theoretical 93.3%)
1H-NMR(CDCl
3，300MHz)：δ＝0.13(s，3H)，0.19(s，3H)，0.90(3，9H)，0.93(s，3H)，1.21(s，3H)，1.58-2.15(m，17H)，2.51-3.05(m，4H)，3.70(m，1H)，5.21(t，1H)，6.92(d，1H)，7.38(d，2H)，7.62(d，2H)ppm.
HPLC (method 2): R
t=6.30min.
MS(ESIpos)：m/z＝604[M+H]
+
R
f=0.68 (isohexane/ethyl acetate 9: 1)
The embodiment that implements
Embodiment 1
(5S)-2,4-two cyclopentyl-3-{ (S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-7,7-dimethyl-5,6,7,8-tetrahydroquinoline-5-alcohol
Under 0 ℃, in the solution of the anhydrous THF of 16ml, drip 11.0ml 1M tetrabutyl ammonium fluoride (11.0mmol, 4.0eq.) solution in THF from the compound of embodiment 6A to 1.67g (2.8mmol).Reaction mixture stirs 4h in ice bath.For aftertreatment, mixture washs with 100ml water with the saturated sodium chloride solution of 50ml with the dilution of 100ml ethyl acetate and under each situation.Organic phase is filtered and is under reduced pressure concentrated by dried over sodium sulfate.Chromatogram purification crude product (silica gel, moving phase: isohexane/ethyl acetate 9: 1 → 4: 1).
Productive rate: 0.74g (theoretical 55.1%)
1H-NMR (CDCl
3, 400MHz): δ=1.02 (s, 3H), 1.18 (s, 3H), 1.34-2.19 (m, 18H), 2.61-2.97 (m, 3H), 3.72 (m, 1H), 3.84 (septet, 1H), 5.14 (q, 1H), 6.96 (d, 1H), 7.34 (d, 2H), 7.61 (d, 2H) ppm.
MS(ESIpos)：m/z＝490[M+H]
+
R
f=0.13 (isohexane/ethyl acetate 9: 1)
Still the further separation [post: Chiralpak AD, 250mm * 20mm, 5 μ m that is present in diastereomer in the product by chromatogram; Moving phase: isohexane/Virahol 97: 3; Flow velocity: 15ml/min]
Productive rate: 0.57g (theoretical 42.4%)
HPLC (method 1B): R
t=5.60min.
B. the evaluation of pharmacological activity
The B-I.CETP-inhibition test
The acquisition of B-I.1.CETP
CETP is obtained with partially purified form by differential centrifugation and column chromatography by people's blood plasma and is used for this test.For this reason, use NaBr with human plasma adjust to every ml 1.21g density and under 4 ℃ under 50000rmp centrifugal 18h.(d＞1.21g/ml) is used for Sephadex to the bottom component
-Phenyl-Sepharose 4B (Pharmacia company) post washs and uses afterwards the distilled water wash-out with 0.15MNaCl/0.001M TrisHCl pH 7.4.Collect the CETP active ingredient, with 50mM sodium acetate pH 4.5 dialysis and put on CM-Sepharose
Post (Pharmacia company).Mixture uses linear gradient (0-1M NaCl) wash-out afterwards.The CETP component of collecting with 10mM TrisHCl pH 7.4 dialysis and afterwards further by chromatogram at Mono Q
Post (Pharmacia) is gone up purifying.
The B-I.2.CETP fluorescent test
The mensuration of fluorescence cholesteryl ester between the catalytic liposome of CETP transhipment [according to Bisgaier etc., J.Lipid Res.
34, 1625 (1993) step is improved]:
In order to prepare the donor liposome, along with gently the dissolving 1mg cholesteryl-4 of heating in the two alkane of 600 μ l in ultrasonic bath, 4-two fluoro-5,7-dimethyl-4-boron is mixed-3a, 4a-diaza-s-indacene (indacene)-3-dodecylate (cholesteryl BODIPY
FLC
12, Molecular Probes company), 15.35mg triolein and 6.67mg phosphatidylcholine and at room temperature in 63ml 50mM TrisHCl/150mMNaCl/2mM edta buffer liquid pH 7.3, add this solution very lentamente with the ultrasonic wave effect.Afterwards suspension in the Branson ultra sonic bath under about 50 watts at N
2Ultrasonication 30min under the atmosphere, temperature remains on about 20 ℃.
By the 86mg Oleoylcholesterol that is dissolved in 1.2ml two alkane and the above damping fluid of 114ml, 20mg triolein and 100mg phosphatidylcholine obtain the acceptor liposome similarly by the ultrasonication under 30 minutes 50 watts (20 ℃).
B-I.2.1. the CETP fluorescent test of enrichment CETP
In order to test, use by damping fluid more than 1 part 1 part of donor liposome and 2 parts of test mixtures that the acceptor liposome constitutes.
With the solution-treated 50 μ l test mixtures of material in DMSO that the CETP component (1-3 μ g) and the 2 μ l of 48 μ l enrichments will check, this CETP component obtains by the blood plasma of hydrophobic chromatography by the people, and cultivates 4 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 485/535nm changes, with the inhibition of the control batch comparative measurement transhipment that does not have material.
| Embodiment number | IC 50[nM] fluorescent test |
| 1 | 30 |
B-I.2.2. the CETP fluorescent test of human plasma
In 42 μ l (86%v/v) human plasmas (Sigma P9523), add the solution of material in DMSO that 6 μ l (12%v/v) donor liposomes and 1 μ l (2%v/v) will check, and mixture was cultivated 24 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 510/520nm changes (stitching wide 2.5nm), with the inhibition of the control batch comparative measurement transhipment that does not have material.
| Embodiment number | IC 50Fluorescent test in [nM] human plasma |
| 1 | 40 |
B-I.2.3. the CETP fluorescent test in vitro exsomatizes
In 80 μ l test mixtures, add 10 μ l damping fluids and 2 μ l serum, and mixture was cultivated 4 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 485/535nm changes, with the inhibition of the control batch comparative measurement transhipment that does not have material.
B-I.3. the acquisition of radiolabeled HDL
The fresh people's edta plasma of 50ml uses NaBr to adjust to 1.12 density and centrifugal 18h in 65 rotors of the Ty under 50000rpm under 4 ℃.The upper strata is used to obtain cold LDL mutually.Lower floor uses 3 * 4 liters of PDB damping fluids mutually, and (10mM TrisHCl, pH 7.4,0.15mM NaCl, 1mM EDTA, 0.02%NaN
3) dialysis.Every afterwards 10ml retentate volume adds 20 μ l
3H-cholesterol (Dupont NET-725; In ethanol the dissolving 1 μ C/ μ l) and mixture under 37 ℃ at N
2Under cultivated 72 hours.
This batch of material uses NaBr to adjust to density 1.21 and centrifugal 18h in 65 rotors of the Ty under 50000rpm under 20 ℃ afterwards.Reclaim the upper strata phase and pass through the gradient centrifugation purification lipoprotein component.For this reason, lipoprotein isolating, mark uses NaBr to adjust to 1.26 density.In centrifuge tube (SW 40 rotors) be that 1.21 solution and 4.5ml density are that 1.063 solution covers this solution (the density solution of PDB damping fluid and NaBr) of each part of 4ml and centrifugal at SW 40 rotors under 38000rpm and 20 ℃ afterwards with 4ml density.Middle layer between density 1.063 and 1.21, it comprises the HDL of mark, 4 ℃ of following PDB damping fluid dialysis with 3 * 100 volumes.
Retentate comprises radiolabeled
3H-CE-HDL, it adjusts to about 5 * 106cmp/ml, is used for this test.
The B-I.4.CETP-SPA test
In order to test the activity of CETP, measure
3The H-cholesteryl ester is by the transhipment to the LD lipoprotein of biotin function of people's HD lipoprotein, and reaction is by adding streptavidin-SPA
Pearl (Amersham company) finishes and the radioactivity of transfer is directly measured in liquid scintillation counter.
In test batch, 10 μ l HDL-
3H-cholesteryl ester (about 50000cpm) is comprising 50mM Hepes/0.15M NaCl/0.1% bovine serum albumin/0.05%NaN of 10 μ l CETP (1mg/ml) with 10 μ l vitamin H-LDL (Amersham company) down at 37 ℃
3Cultivated 18 hours in the material (being dissolved in the solution of 10%DMSO/1%RSA) that pH 7.4 and 3 μ l will test.Add 200 μ l SPA-streptavidin pearl solution (TRKQ 7005) afterwards, further cultivate 1h and in scintillometer, measure afterwards along with jolting.With 10 μ l damping fluids, cultivate in contrast in the correspondence of 10 μ l CETP under 4 ℃ and 10 μ l CETP under 37 ℃.
Regarding 100% as 37 ℃ of activity that shift down in the contrast batch of material that uses CETP shifts.This transfer reduce to a half material concentration be defined as IC
50Value.
| Embodiment number | IC 50[nM] SPA test |
| 1 | 55 |
B-II.1. the mensuration of isolated activity in vitro on transgenosis hCETP mouse
Suppress active in order to test CETP, use stomach tube to give inner oral this material of transgenosis hCETP mouse [Dinchuk etc., BBA 1295-301 (1995)] of feeding.For this reason, arbitrarily be arranged to and have the equal number animal beginning to test the day before yesterday buck, usually the group of n=4.Before using this material, be used for measuring its basic active blood of CETP (time point T1) of serum by the puncture collection of vena orbitalis posterior clump (retroorbitalenVenenplexus) by each mouse.Use stomach tube to take this substances afterwards to animal.Specified time after substances is used, by puncture for the second time (time point T2) from animal, get blood, usually behind application of substances 16 or 24h, but as suitable this also can carry out in another time.
In order to assess the inhibition activity of material,, promptly 16 or 18 hours, use animal only to accept not have the corresponding control group of the preparation of material to each time.In control animal, for can measure through corresponding test period at interval (16 or 24h) do not have the active change of CETP of inhibitor, in the animal of mass treatment, carry out the secondary blood specimen collection of each animal.
After solidifying end, blood samples is centrifugal and remove serum deprivation with suction pipe.In order to measure the CETP activity, measure transportation through the cholesteryl ester (Cholesterylester) of 4h.For this reason, the carrying out of in test batch, using 2 μ l serum and test as under B-I.2.3., describing usually.
Calculate for each animal the cholesteryl ester transportation difference [pM CE/h (T2)-pMCE/h (T1)] and in group, average.In one of time point, reduced＞material of the transportation of 20% cholesteryl ester is considered to active.
| Embodiment number | Inhibition % at 3mg/kg | |
| 16h | 24h | |
| 1 | 45 | 24 |
B-II.2. the mensuration of activity in vivo in Syria's cricetulus auratus
The inner female Syria cricetulus auratus of feeding and having 150-200g weight (planting BAY:DSN) is used to measure the oral effect of CETP inhibitor for serum lipoprotein and serum triglyceride.Six animals of each cage be divided into one group and feed arbitrarily and intake the domestication two weeks.
Just before on-test and after material takes, get blood and at room temperature cultivate 30min and after under the 30000g centrifugal 20 minutes, be used to obtain serum by puncture behind the socket of the eye of venous plexus.This substance dissolves is in 20%Solutol/80% water and oral by stomach tube, the solvent of control animals received equal volume and do not have substances.
Operational analysis instrument COBAS INTEGRA 400 plus (from Roche Diagnostics company) measure triglyceride level, cholesterol total amount, HDL cholesterol and LDL cholesterol according to manufacturer's specification sheets.From measured value, for each parameter, the per-cent that causes with mass treatment of change and each group calculate by to(for) each animal are defined as the mean value (n=6 or n=12) with standard deviation.If with the group of solvent treatment relatively, the influence of material is significant, add p-value by the t-test determination (
*P≤0.05;
*P≤0.01;
* *P≤0.005).
B-II.3. the mensuration of activity in vivo in transgenosis hCETP mouse
In order to measure oral effect, use stomach tube to take substances for transgenic mice [Dinchuk etc., BBA, 1295-1301 (1995)] for lipoprotein and triglyceride level.Before on-test, after measuring cholesterol in the serum and the socket of the eye of triglyceride level, draw blood by mouse.The serum that obtains as described above for hamster is in 4 ℃ of following overnight incubation and centrifugal under 6000g subsequently.After three days, in order to measure lipoprotein and triglyceride level is got blood by mouse again.The change of the parameter of measuring is expressed as the change with starting value per-cent relatively.
| Embodiment number | % (the dosage: 3 * 3mg/kg) that HDL increases after 3 days |
| 1 | 42 |
C. the embodiment of the enforcement of pharmaceutical composition
Compound of the present invention can change into pharmaceutical preparation with following method:
Sheet:
Composition:
100mg compound of the present invention, 50mg lactose (monohydrate), 50mg W-Gum (homemade), 10mg polyvinylpyrrolidone (PVP 25) (from BASF, Ludwigshafen, Germany) and 2mg Magnesium Stearate.
Sheet weight 212mg.Diameter 8mm, bending radius 12mm.
Produce:
Compound of the present invention, the mixture of lactose and starch are in water and PVP solution (m/m) granulation of 5% concentration.Particle mixed with Magnesium Stearate 5 minutes after dry.This mixture use conventional tabletting machine (for the form of sheet referring to more than) compressing tablet.The pressure of 15kN is as the standard of compressing tablet.
Suspension that can be oral:
Composition:
1000mg compound of the present invention, 1000mg ethanol (96%), 400mg Rhodigel
(from the xanthan gum of FMC, Pennsylvania, the U.S.) and 99g water.
The 10ml oral suspension is corresponding to the single dose with 100mg compound of the present invention.
Produce:
Rhodigel is suspended in the ethanol and adds compound of the present invention in suspension.Add water along with stirring.Mixture stirs about 6h and finishes up to the Rhodigel swelling.
Solution that can be oral:
Composition:
500mg compound of the present invention, 2.5g polysorbate and 97g poly(oxyethylene glycol) 400.The 20g oral liquid is corresponding to the single dose with 100mg compound of the present invention.
Produce:
Along with stirring, compound of the present invention is suspended in the mixture of polyoxyethylene glycol and polysorbate, continues to stir to dissolve fully up to compound of the present invention.
(i.v.) solution in the body:
Being lower than saturated deliquescent concentration, compound dissolution of the present invention is in physiology acceptable solvent (for example isotonic saline solution, glucose solution 5% and/or PEG 400 solution 30%).Solution is through in sterile filtration and pack into aseptic and the pyrogen-free injection vessel.
Test portion with compound of formula (Ie)
A. embodiment
Abbreviation and acronym
The CE cholesteryl ester
The CETP cholesteryl ester transfer protein
DAST dimethylamino sulfur trifluoride
DCI direct chemical ionization (in MS)
DDQ 2,3-two chloro-5,6-dicyano-1,4-benzoquinones
The de diastereomer is excessive
DMF N, dinethylformamide
The DMSO dimethyl sulfoxide (DMSO)
(in the productive rate) of d.Th theory
EDTA quadrol-N, N, N ', N '-tetraacethyl
The ee enantiomer is excessive
Eq. equivalent
ESI electrospray ionization (in MS)
H hour
The HDL high-density lipoprotein (HDL)
HPLC high pressure-high performance liquid chromatography
The LC/MS liquid chromatograph mass spectrography
The LDL low-density lipoprotein
Min minute
The MS mass spectrum
The NMR nuclear magnetic resonance spectrum
R
fRetention index (in thin-layer chromatography)
R
tRetention time (in HPLC)
The SPA scintillation proximity assay
The TBAF tetrabutyl ammonium fluoride
TBDMSOTf trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester
The TFA trifluoroacetic acid
The THF tetrahydrofuran (THF)
HPLC and LC/MS method
Method 1A: post: Chiralpak IA, 250mm * 4.6mm; Moving phase: isohexane/1-propyl alcohol 97: 3; Flow velocity: 1.0ml/min; UV-detects: 254nm.
Method 1B: post: Chiralpak AD, 250mm * 4.6mm, 10 μ m; Moving phase: isohexane/Virahol 97.5: 2.5; Flow velocity: 1.0ml/min; UV-detects: 254nm.
Method 1C: post: Chiralpak AD, 250mm * 4.6mm, 10 μ m; Moving phase: isohexane/Virahol 97.5: 2.5; Flow velocity: 1.5ml/min; UV-detects: 254nm.
Method 2: instrument: have the HP 1100 that DAD detects; Post: Kromasil RP-18,60mm * 2mm, 3.5 μ m; Mobile phase A: 5ml HClO
4/ l water, Mobile phase B: acetonitrile; Gradient: 0min 2%B → 0.5min 2%B → 4.5min 90%B → 9min 90%B; Flow velocity: 0.75ml/min; Temperature: 30 ℃; UV-detects: 210nm.
Method 3 (LC/MS): MS instrument type: Micromass ZQ; HPLC instrument type: HP 1100 series; UV DAD; Post: Phenomenex Synergi 2 μ Hydro-RP Mercury 20mm * 4mm; Mobile phase A: the formic acid of 1l water+0.5ml 50% concentration, Mobile phase B: the formic acid of 1l acetonitrile+0.5ml 50% concentration; Gradient: 0.0min 90%A → 2.5min 30%A → 3.0min 5%A → 4.5min 5%A; Flow velocity: 0.0min 1ml/min → 2.5min/3.0min/4.5min 2ml/min; Stove: 50 ℃; UV-detects: 210nm.
Parent material and intermediate
Embodiment 1A
4-cyclohexyl-2-sec.-propyl-7,7-dimethyl-3-(4-trifluoromethyl) benzoyl]-4,6,7,8-tetrahydroquinoline-5 (1H)-ketone
In the 150ml Di Iso Propyl Ether, begin to add 5.0g (19.4mmol, 1.2eq.) 3-amino-3-sec.-propyl-1-(4-trifluoromethyl) acrylketone is (according to WO 03/028727, embodiment 2 preparations), and add 2.50ml (32.4mmol, 2.0eq.) trifluoroacetic acid and 2.3g (16.2mmol, 1eq.) 5,5-dimethyl cyclohexane-1,3-diketone.After at room temperature stirring 10min, add 2.7g (24.3mmol, 1.5eq.) hexanaphthene formaldehyde.Mixture heats 18h on water separator under refluxing afterwards.After the cooling, mixture stirs 30min in ice bath, with aspirating the precipitation that filters acquisition, washs and remove the solvent residues thing with cold Di Iso Propyl Ether under high vacuum.
Productive rate: 2.63g (theoretical 34.3%)
1H-NMR (CDCl
3, 400MHz): δ=0.78-1.36 (m, 6H), 1.04 (d, 3H), 1.16 (2s, 6H), 1.29 (d, 3H), 1.28 (d, 3H), 1.46-1.67 (m, 5H), 2.32 and 2.49 (2d, 2H), 2.34 (s, 2H), 3.46 (septet, 1H), 3.75 (d, 1H), 5.89 (s, 1H), 7.65 (d, 2H), 7.77 (d, 2H) ppm.
MS(DCI)：m/z＝474[M+H]
+.
Embodiment 2A
4-cyclohexyl-2-sec.-propyl-7,7-dimethyl-3-[4-(trifluoromethyl) benzoyl]-7,8-dihydroquinoline-5 (6H)-ketone
In the 50ml methylene dichloride, dissolve the compound of 2.30g (4.9mmol) from embodiment 1A, and at 0 ℃ of following portion-wise addition 1.10mg (4.9mmol, 1eq.) 2,3-two chloro-5,6-dicyano-1,4-benzoquinones (DDQ).After the mixture 0 ℃ stir down 1h and after 18h at room temperature.Mixture concentrates on rotatory evaporator and resistates passes through chromatogram purification (silica gel, moving phase: cyclohexane/ethyl acetate 5: 1).
Productive rate: 2.16g (theoretical 94.2%)
1H-NMR(CDCl
3，300MHz)：δ＝1.00-1.89(m，10H)，1.08(d，3H)，1.11(s，3H)，1.14(d，3H)，1.17(s，3H)，2.48-2.69(m，3H)，3.09(s，2H)，3.27(m，1H)，7.75(d，2H)，7.94(m，2H)ppm.
MS(DCI)：m/z＝472[M+H]
+.
Embodiment 3A
[(5S)-and 4-cyclohexyl-5-hydroxyl-2-sec.-propyl-7,7-dimethyl-5,6,7,8-tetrahydroquinoline-3-yl] [4-(trifluoromethyl) phenyl] ketone
(0.37mmol, 0.08eq.) (1R 2S)-1-aminoidan-2-alcohol, and at room temperature adds 2.98g (18.3mmol, 4.0eq.) borine-N, N-diethylbenzene amine complex to begin to add 55mg in 115ml THF.After the release of gas stopped, mixture is cooled to 0 ℃ and the 2.16g that adds to be dissolved among the 115ml THF, and (4.6mmol was 1eq.) from the compound of embodiment 2A.Follow stirring, make mixture warm up room temperature through 18h.After reaction finishes, in reaction mixture, add 20ml methyl alcohol, be evaporated to drying after it.Afterwards by chromatogram purification crude product (silica gel, moving phase: isohexane/ethyl acetate mixture).
Productive rate: 2.01g (theoretical 93.0%)
Enantiomer is excessive to be determined as 88%ee according to method 1A.
Enantiomer is by going up chromatographic separation (post: Chiralpak AD-H, 250mm * 20mm, 20 μ m mutually in chirality; Moving phase: isohexane/Virahol 97: 3; Flow velocity: 15ml/min).
Productive rate: 1.70 (theoretical 78.5%).
Enantiomer is excessive to be determined as＞99%ee according to method 1A; R
t(method 1A)=5.22min.
1H-NMR (CDCl
3, 300MHz): δ=0.88-2.12 (m, 26H), 2.50 (septet, 1H), 2.71 (d, 1H), 2.98 (d, 1H), 5.18 (m, 1H), 7.45-8.50 (br.m, 4H) ppm.
MS(ESIpos)：m/z＝474[M+H]
+.
Embodiment 4A
((5S)-5-{[tert-butyl (dimethyl) silyl] the oxygen base }-4-cyclohexyl-2-sec.-propyl-7,7-dimethyl-5,6,7,8-tetrahydroquinoline-3-yl) [4-(trifluoromethyl) phenyl] ketone
Under argon, in the 22ml dry toluene, begin to add the compound of 2.82g (5.95mmol) from embodiment 3A.At room temperature, add 2.55g (23.8mmol, 4eq.) 2,6-lutidine, and mixture is cooled to-16 ℃ afterwards.In this solution, drip 2.74ml (11.9mmol, 2eq.) the trifluoromethayl sulfonic acid tert-butyl dimetylsilyl ester that is dissolved in the 7ml toluene.After 15 minutes, reaction mixture warm to 0 ℃ and under this temperature restir 80min.For aftertreatment, add 124ml0.1N hydrochloric acid, and mixture is warm uses ethyl acetate extraction after room temperature.Organic phase is washed with 1: 1 mixture of saturated nacl aqueous solution and saturated sodium bicarbonate solution.The organic phase that merges is brought up again with ethyl acetate and is got twice.The organic phase that merges is filtered and is under reduced pressure concentrated by dried over sodium sulfate.Resistates is by chromatogram purification (silica gel, moving phase: isohexane/ethyl acetate 9: 1).
Productive rate: 3.17g (theoretical 90.7%)
1H-NMR(CDCl
3，400MHz)：δ＝0.14(s，3H)，0.19(s，3H)，0.86(s，9H)，0.65-1.83(m，24H)，1.96-2.07(m，1H)，2.47(m，1H)，2.63(m，1H)，3.10(m，1H)，5.25(m，1H)，7.43-8.54(br.m，4H)ppm.
MS(ESIpos)：m/z＝588[M+H]
+.
Embodiment 5A
(S) ((5S)-5-{[tert-butyl (dimethyl) silyl] the oxygen base }-4-cyclohexyl-2-sec.-propyl-7,7-dimethyl-5,6,7,8-tetrahydroquinoline-3-yl) [4-(trifluoromethyl) phenyl] methyl alcohol
Under 0 ℃, add 5.9ml 1M lithium aluminum hydride (5.9mmol, 1eq.) solution in THF in from the solution of compound in the anhydrous THF of 75ml of embodiment 4A to 3.17g (5.4mmol).Follow stirring, mixture is warm to room temperature through 5h.For aftertreatment, carefully add the saturated potassium sodium tartrate solution of 120ml.After the release of gas stops, mixture ethyl acetate extraction four times, and the organic phase that merges by dried over sodium sulfate, is filtered also under reduced pressure concentrated with saturated sodium chloride solution washing.The resistates chromatogram purification has caused the separation (post: Chiralpak AD, 500mm * 40mm, 20 μ m of product diastereomer; Moving phase: isohexane/Virahol 97.5: 2.5; Flow velocity: 50ml/min; Temperature: 24 ℃; UV-detects: 254nm).
Productive rate: 0.95g (theoretical 29.8%)
1H-NMR(CDCl
3，400MHz)：δ＝0.20(br.s，6H)，0.76-2.03(m，30H)，1.02-2.34(m，14H)，2.17(m，1H)，2.43-3.10(m，3H)，3.36(m，1H)，5.26(m，1H)，6.66(br.s，1H)，7.32-7.65(m，4H)ppm.
MS(ESIpos)：m/z＝590[M+H]
+
LC/MS (method 3): R
t=3.03min.
HPLC (method 1B): R
t=2.84min.
Cis-diastereomer:
(R) ((5S)-5-{[tert-butyl (dimethyl) silyl] the oxygen base }-4-cyclohexyl-2-sec.-propyl-7,7-dimethyl-5,6,7,8-tetrahydroquinoline-3-yl) [4-(trifluoromethyl) phenyl] methyl alcohol
Productive rate: 1.25g (theoretical 39.2%)
Embodiment 6A
(5S)-and 5-{[tert-butyl (dimethyl) silyl] the oxygen base }-4-cyclohexyl-3-{ (S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-2-sec.-propyl-7,7-dimethyl-5,6,7,8-tetrahydroquinoline
-60 ℃ down and under argon, to 1.35g (2.3mmol) drip in from the solution of compound in the 30ml dry toluene of embodiment 5A 0.45ml diethylamino sulfur trifluoride (3.4mmol, 1.5eq.).Follow and warm up-10 ℃, mixture stirs 3h.For aftertreatment, follow ice-cooled careful interpolation 40ml saturated sodium bicarbonate solution.Mixture is used ethyl acetate extraction three times altogether.Latter incorporated organic phase with the washing of saturated sodium chloride solution, by dried over sodium sulfate, filter and under reduced pressure concentrate.Crude product is by filtering by silica gel purification (moving phase: cyclohexane/ethyl acetate 9: 1).
Productive rate: 1.28g (theoretical 94.8%)
1H-NMR (CDCl
3, 400MHz): δ=0.20 (br.s, 6H), 0.71 (d, 3H), 0.91 (s, 9H), 0.77-2.06 (m, 21H), 2.60 and 3.03 (2d, 2H), 2.78 (m, 1H), 3.36 (m, 1H), 5.25 (m, 1H), 7.10-7.50 (m, 3H), 7.63 (d, 2H) ppm.
MS(ESIpos)：m/z＝592[M+H]
+.
The embodiment that implements
Embodiment 1
(5S)-4-cyclohexyl-3-{ (S)-fluorine [4-(trifluoromethyl) phenyl] methyl }-2-sec.-propyl-7,7-dimethyl-5,6,7,8-tetrahydroquinoline-5-alcohol
Under 0 ℃, to compound in the solution of 25ml anhydrous THF Dropwise 5 .4ml 1M tetrabutylammonium chloride (5.4mmol, 2.5eq.) the solution among THFs of 1.28g (2.2mmol) from embodiment 6A.Mixture stirs 4h in ice bath.For aftertreatment, mixture is with the dilution of 20ml ethyl acetate and use the 20ml water washing.Containing water brings up again with the 20ml ethyl acetate under each situation and gets twice.The organic phase that merges, is filtered and is under reduced pressure concentrated by dried over sodium sulfate with the saturated sodium chloride solution washing of 50ml.The chromatogram purification crude product has caused the still separation (post: Chiralpak AD-H, 250mm * 20mm, 5 μ m of the diastereomer of existence; Moving phase: isohexane/Virahol 97: 3; Flow velocity: 25ml/min).
Productive rate: 0.67g (theoretical 65.3%)
1H-NMR(CDCl
3，300MHz)：δ＝0.60(d，3H)，1.03(s，3H)，1.12(d，3H)，1.17(s，3H)，1.08-1.51(m，4H)，1.67-2.14(m，9H)，2.61-2.94(m，3H)，3.52(m，1H)，5.14(m，1H)，7.33(d，2H)，7.37(d，1H)，7.61(d，2H)ppm.
MS(ESIpos)：m/z＝478[M+H]
+
HPLC (method 1C): R
t=4.33min.
B. the evaluation of pharmacological activity
The B-I.CETP-inhibition test
The acquisition of B-I.1.CETP
CETP is obtained with partially purified form by differential centrifugation and column chromatography by people's blood plasma and is used for this test.For this reason, use NaBr with human plasma adjust to every ml 1.21g density and under 4 ℃ under 50000rmp centrifugal 18h.(d＞1.21g/ml) is used for Sephadex to the bottom component
-Phenyl-Sepharose 4B (Pharmacia company) post washs and uses afterwards the distilled water wash-out with 0.15MNaCl/0.001M TrisHCl pH 7.4.Collect the CBTP active ingredient, with 50mM sodium acetate pH 4.5 dialysis and put on CM-Sepharose
Post (Pharmacia company).Mixture uses linear gradient (0-1M NaCl) wash-out afterwards.The CETP component of collecting with 10mM TrisHCl pH 7.4 dialysis and afterwards further by chromatogram at Mono Q
Post (Pharmacia) is gone up purifying.
The B-I.2.CETP fluorescent test
The mensuration of fluorescence cholesteryl ester between the catalytic liposome of CETP transhipment [according to Bisgaier etc., J.Lipid Res.
34, 1625 (1993) step is improved]:
In order to prepare the donor liposome, along with gently the dissolving 1mg cholesteryl-4 of heating in the two alkane of 600 μ l in ultrasonic bath, 4-two fluoro-5,7-dimethyl-4-boron is mixed-3a, 4a-diaza-s-indacene (indacene)-3-dodecylate (cholesteryl BODIPY
FLC
12, Molecular Probes company), 15.35mg triolein and 6.67mg phosphatidylcholine and at room temperature in 63ml 50mM TrisHCl/150mMNaCl/2mM edta buffer liquid pH 7.3, add this solution very lentamente with the ultrasonic wave effect.Afterwards suspension in the Branson ultra sonic bath under about 50 watts at N
2Ultrasonication 30min under the atmosphere, temperature remains on about 20 ℃.
By the 86mg Oleoylcholesterol that is dissolved in 1.2ml two alkane and the above damping fluid of 114ml, 20mg triolein and 100mg phosphatidylcholine obtain the acceptor liposome similarly by the ultrasonication under 30 minutes 50 watts (20 ℃).
B-I.2.1. the CETP fluorescent test of enrichment CETP
In order to test, use by damping fluid more than 1 part 1 part of donor liposome and 2 parts of test mixtures that the acceptor liposome constitutes.
With the solution-treated 50 μ l test mixtures of material in DMSO that the CETP component (1-3 μ g) and the 2 μ l of 48 μ l enrichments will check, this CETP component obtains by the blood plasma of hydrophobic chromatography by the people, and cultivates 4 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 485/535nm changes, with the inhibition of the control batch comparative measurement transhipment that does not have material.
| Embodiment number | IC 50[nM] fluorescent test |
| 1 | 30 |
B-I.2.2. the CETP fluorescent test of human plasma
In 42 μ l (86%v/v) human plasmas (Sigma P9523), add the solution of material in DMSO that 6 μ l (12%v/v) donor liposomes and 1 μ l (2%v/v) will check, and mixture was cultivated 24 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 510/520nm changes (stitching wide 2.5nm), with the inhibition of the control batch comparative measurement transhipment that does not have material.
| Embodiment number | IC 50Fluorescent test in [nM] human plasma |
| 1 | 75 |
B-I.2.3. the CETP fluorescent test in vitro exsomatizes
In 80 μ l test mixtures, add 10 μ l damping fluids and 2 μ l serum, and mixture was cultivated 4 hours down at 37 ℃.
It is to measuring that CE transports that fluorescence under 485/535nm changes, with the inhibition of the control batch comparative measurement transhipment that does not have material.
B-I.3. the acquisition of radiolabeled HDL
The fresh people's edta plasma of 50ml uses NaBr to adjust to 1.12 density and centrifugal 18h in 65 rotors of the Ty under 50000rpm under 4 ℃.The upper strata is used to obtain cold LDL mutually.Lower floor uses 3 * 4 liters of PDB damping fluids mutually, and (10mM TrisHCl, pH 7.4,0.15mM NaCl, 1mM EDTA, 0.02%NaN
3) dialysis.Every afterwards 10ml retentate volume adds 20 μ l
3H-cholesterol (Dupont NET-725; In ethanol the dissolving 1 μ C/ μ l) and mixture under 37 ℃ at N
2Under cultivated 72 hours.
This batch of material uses NaBr to adjust to density 1.21 and centrifugal 18h in 65 rotors of the Ty under 50000rpm under 20 ℃ afterwards.Reclaim the upper strata phase and pass through the gradient centrifugation purification lipoprotein component.For this reason, lipoprotein isolating, mark uses NaBr to adjust to 1.26 density.In centrifuge tube (SW 40 rotors) be that 1.21 solution and 4.5ml density are that 1.063 solution covers this solution (the density solution of PDB damping fluid and NaBr) of each part of 4ml and centrifugal at SW 40 rotors under 38000rpm and 20 ℃ afterwards with 4ml density.Middle layer between density 1.063 and 1.21, it comprises the HDL of mark, 4 ℃ of following PDB damping fluid dialysis with 3 * 100 volumes.
Retentate comprises radiolabeled
3H-CE-HDL, it adjusts to about 5 * 106cmp/ml, is used for this test.
The B-I.4.CETP-SPA test
In order to test the activity of CETP, measure
3The H-cholesteryl ester is by the transhipment to the LD lipoprotein of biotin function of people's HD lipoprotein, and reaction is by adding streptavidin-SPA
Pearl (Amersham company) finishes and the radioactivity of transfer is directly measured in liquid scintillation counter.
In test batch, 10 μ l HDL-
3H-cholesteryl ester (about 50000cpm) is comprising 50mM Hepes/0.15M NaCl/0.1% bovine serum albumin/0.05%NaN of 10 μ l CETP (1mg/ml) with 10 μ l vitamin H-LDL (Amersham company) down at 37 ℃
3Cultivated 18 hours in the material (being dissolved in the solution of 10%DMSO/1%RSA) that pH 7.4 and 3 μ l will test.Add 200 μ l SPA-streptavidin pearl solution (TRKQ 7005) afterwards, further cultivate 1h and in scintillometer, measure afterwards along with jolting.With 10 μ l damping fluids, cultivate in contrast in the correspondence of 10 μ l CETP under 4 ℃ and 10 μ l CETP under 37 ℃.
Regarding 100% as 37 ℃ of activity that shift down in the contrast batch of material that uses CETP shifts.This transfer reduce to a half material concentration be defined as IC
50Value.
| Embodiment number | IC 50[nM] SPA test |
| 1 | 42 |
B-II.1. the mensuration of isolated activity in vitro on transgenosis hCETP mouse
Suppress active in order to test CETP, use stomach tube to give inner oral this material of transgenosis hCETP mouse [Dinchuk etc., BBA 1295-301 (1995)] of feeding.For this reason, arbitrarily be arranged to and have the equal number animal beginning to test the day before yesterday buck, usually the group of n=4.Before using this material, be used for measuring its basic active blood of CETP (time point T1) of serum by the puncture collection of vena orbitalis posterior clump (retroorbitalenVenenplexus) by each mouse.Use stomach tube to take this substances afterwards to animal.Specified time after substances is used, by puncture for the second time (time point T2) from animal, get blood, usually behind application of substances 16 or 24h, but as suitable this also can carry out in another time.
In order to assess the inhibition activity of material,, promptly 16 or 18 hours, use animal only to accept not have the corresponding control group of the preparation of material to each time.In control animal, for can measure through corresponding test period at interval (16 or 24h) do not have the active change of CETP of inhibitor, in the animal of mass treatment, carry out the secondary blood specimen collection of each animal.
After solidifying end, blood samples is centrifugal and remove serum deprivation with suction pipe.In order to measure the CETP activity, measure transportation through the cholesteryl ester (Cholesterylester) of 4h.For this reason, the carrying out of in test batch, using 2 μ l serum and test as under B-I.2.3., describing usually.
Calculate for each animal the cholesteryl ester transportation difference [pM CE/h (T2)-pMCE/h (T1)] and in group, average.In one of time point, reduced＞material of the transportation of 20% cholesteryl ester is considered to active.
| Embodiment number | Inhibition % at 3mg/kg | |
| 16h | 24h | |
| 1 | 50 | 36 |
B-II.2. the mensuration of activity in vivo in Syria's cricetulus auratus
The inner female Syria cricetulus auratus of feeding and having 150-200g weight (planting BAY:DSN) is used to measure the oral effect of CETP inhibitor for serum lipoprotein and serum triglyceride.Six animals of each cage be divided into one group and feed arbitrarily and intake the domestication two weeks.
Just before on-test and after material takes, get blood and at room temperature cultivate 30min and after under the 30000g centrifugal 20 minutes, be used to obtain serum by puncture behind the socket of the eye of venous plexus.This substance dissolves is in 20%Solutol/80% water and oral by stomach tube, the solvent of control animals received equal volume and do not have substances.
Operational analysis instrument COBAS INTEGRA 400 plus (from Roche Diagnostics company) measure triglyceride level, cholesterol total amount, HDL cholesterol and LDL cholesterol according to manufacturer's specification sheets.From measured value, for each parameter, the per-cent that causes with mass treatment of change and each group calculate by to(for) each animal are defined as the mean value (n=6 or n=12) with standard deviation.If with the group of solvent treatment relatively, the influence of material is significant, add p-value by the t-test determination (
*P≤0.05;
*P≤0.01;
* *P≤0.005).
B-II.3. the mensuration of activity in vivo in transgenosis hCETP mouse
In order to measure oral effect, use stomach tube to take substances for transgenic mice [Dinchuk etc., BBA, 1295-1301 (1995)] for lipoprotein and triglyceride level.Before on-test, after measuring cholesterol in the serum and the socket of the eye of triglyceride level, draw blood by mouse.The serum that obtains as described above for hamster is in 4 ℃ of following overnight incubation and centrifugal under 6000g subsequently.After three days, in order to measure lipoprotein and triglyceride level is got blood by mouse again.The change of the parameter of measuring is expressed as the change with starting value per-cent relatively.
| Embodiment number | % (the dosage: 3 * 3mg/kg) that HDL increases after 3 days |
| 1 | 68 |
C. the embodiment of the enforcement of pharmaceutical composition
Compound of the present invention can change into pharmaceutical preparation with following method:
Sheet:
Composition:
100mg compound of the present invention, 50mg lactose (monohydrate), 50mg W-Gum (homemade), 10mg polyvinylpyrrolidone (PVP 25) (from BASF, Ludwigshafen, Germany) and 2mg Magnesium Stearate.
Sheet weight 212mg.Diameter 8mm, bending radius 12mm.
Produce:
Compound of the present invention, the mixture of lactose and starch are in water and PVP solution (m/m) granulation of 5% concentration.Particle mixed with Magnesium Stearate 5 minutes after dry.This mixture use conventional tabletting machine (for the form of sheet referring to more than) compressing tablet.The pressure of 15kN is as the standard of compressing tablet.
Suspension that can be oral:
Composition:
1000mg compound of the present invention, 1000mg ethanol (96%), 400mg Rhodigel
(from the xanthan gum of FMC, Pennsylvania, the U.S.) and 99g water.
The 10ml oral suspension is corresponding to the single dose with 100mg compound of the present invention.
Produce:
Rhodigel is suspended in the ethanol and adds compound of the present invention in suspension.Add water along with stirring.Mixture stirs about 6h and finishes up to the Rhodigel swelling.
Solution that can be oral:
Composition:
500mg compound of the present invention, 2.5g polysorbate and 97g poly(oxyethylene glycol) 400.The 20g oral liquid is corresponding to the single dose with 100mg compound of the present invention.
Produce:
Along with stirring, compound of the present invention is suspended in the mixture of polyoxyethylene glycol and polysorbate, continues to stir to dissolve fully up to compound of the present invention.
(i.v.) solution in the body:
Being lower than saturated deliquescent concentration, compound dissolution of the present invention is in physiology acceptable solvent (for example isotonic saline solution, glucose solution 5% and/or PEG 400 solution 30%).Solution is through in sterile filtration and pack into aseptic and the pyrogen-free injection vessel.
Claims (16)
1. the compound that has following formula (I)
R wherein
1Represent cyclohexyl or cyclopentyl, R
2And R
3Represent methylidene or form tetramethylene, R together separately
4Represent cyclopentyl or sec.-propyl,
Or its salt, the solvate of solvate or salt.
2. the compound that has following formula (Ia)
Or its salt, the solvate of solvate or salt.
3. the compound that has following formula (Ib)
Or its salt, the solvate of solvate or salt.
4. the compound that has following formula (Ic)
Or its salt, the solvate of solvate or salt.
5. the compound that has following formula (Id)
Or its salt, the solvate of solvate or salt.
6. the compound that has following formula (Id)
Or its salt, the solvate of solvate or salt.
7. as the compound that is used for treatment of diseases and/or prevention of definition among one of any of the claim 1-6 with formula (I).
8. be used to prepare as the compound with formula (I) of definition among one of any of the claim 1-6 and be used for the elementary or intermediate application that prevents the medicine of coronary heart disease disease.
9. being used for preparation as the compound with formula (I) of definition among one of any of the claim 1-6 is used for the treatment of and/or prevents hypolipoproteinemia, dyslipidemia, hypertriglyceridemia, hyperlipidaemia, hypercholesterolemia, arteriosclerosis, restenosis, obesity, diabetes, the application of the medicine of apoplexy and alzheimer's disease.
10. medicine, it comprises the compound with formula (I) and the inert non-toxic pharmacology proper auxiliary agent of definition among one of any of the claim 1-6.
11. medicine, it comprises the compound with formula (I) of definition among one of any of the claim 1-6 and one or more further are selected from following active compound: antidiabetic drug, anticoagulant, antithrombotics, calcium antagonist, Angiotensin A II antagonist, ACE inhibitor, beta-blocker, phosphodiesterase inhibitor, the stimulant of soluble guanylate cyclase, cGMP toughener, hydragog(ue), the thryoid receptor agonist, HMG-CoA reductase inhibitor, squalene synthetic inhibitor, the squalene epoxidase inhibitor, the squalene oxide cyclase inhibitor, ACAT inhibitor, MTP inhibitor, the PPAR agonist, Fibrate, lipase inhibitor, cholesterol absorption inhibitor, cholic acid reuptake inhibithors, polymerization cholic acid absorption agent and lipoprotein antagonist.
12. the medicine of claim 10 or 11, it is used for elementary or intermediate prevention coronary heart disease disease.
13. the medicine of claim 10 or 11, it is used for the treatment of and/or prevents hypolipoproteinemia, dyslipidemia, hypertriglyceridemia, hyperlipidaemia, hypercholesterolemia, arteriosclerosis, restenosis, obesity, obesity, diabetes, the medicine of apoplexy and alzheimer's disease.
Elementary or the intermediate method that prevents humans and animals coronary heart disease disease of medicine of definition during one of 14. compound with formula (I) by definition among one of any of the claim 1-6 that takes significant quantity or claim 10-13 are any.
The pharmacological agent of definition and/or prevent the hypolipoproteinemia of humans and animals during one of 15. compound with formula (I) by definition among one of any of the claim 1-6 that takes significant quantity or claim 10-13 are any, dyslipidemia, hypertriglyceridemia, hyperlipidaemia, hypercholesterolemia, arteriosclerosis, restenosis, obesity, diabetes, the method for apoplexy and alzheimer's disease.
16. the method for preparation as the compound with formula (I) that defines in the claim 1 is characterised in that the have formula compound of (II), wherein R
1, R
2, R
3And R
4Separately as defined above,
At first change into the have formula compound of (III), after it by asymmetric reduction
[A] changes into the have formula compound of (IV) by introducing hydroxy-protective group,
Wherein
PG representation hydroxy blocking group preferably has formula-SiR
1R
2R
3Group, wherein
R
1, R
2And R
3Be identical or different and representative (C
1-C
4)-alkyl,
Change into compound by the cis-selectivity reduction afterwards with formula V,
Wherein PG as defined above,
Or
[B] at first produces the compound with formula (IV) by the cis-selectivity reduction,
Introducing hydroxy-protective group PG by regioselectivity after it changes into the compound with formula V,
Use the fluorizating agent reaction to produce compound after having the formula V compound with formula (VII),
Wherein PG as defined above,
Remove hydroxy-protective group PG by ordinary method afterwards and produce compound with formula (I)
With optional compound with formula (I) with suitable (i) solvent and/or (ii) alkali or the sour solvate that changes into it, the solvate of salt and/or salt.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE102004061003.7 | 2004-12-18 | ||
| DE102004061001.0 | 2004-12-18 | ||
| DE102004060998.5 | 2004-12-18 | ||
| DE102004060999.3 | 2004-12-18 | ||
| DE102004061002A DE102004061002A1 (en) | 2004-12-18 | 2004-12-18 | New (5'S)-2',4'-dicyclopentyl -3'-9(S)-fluoro(4-(trifluoromethyl) phenyl)methyl)-5',8' -dihydro-6'H-spiro(cyclopropane-1,7'-quinoline)-5'-ol is a cholesterol ester transfer protein inhibitor useful for treating e.g. coronary heart diseases |
| DE102004061002.9 | 2004-12-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101080393A true CN101080393A (en) | 2007-11-28 |
Family
ID=36571209
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2005800434885A Pending CN101080393A (en) | 2004-12-18 | 2005-12-10 | New (5's)-2',4'-dicyclopentyl -3'-9(s)-fluoro(4-(trifluoromethyl) phenyl)methyl)-5',8' -dihydro-6'h-spiro(cyclopropane-1,7'-quinoline)- 5'-ol is a cholesterol ester transfer protein inhibitor useful f |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN101080393A (en) |
| DE (1) | DE102004061002A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI570118B (en) * | 2012-01-06 | 2017-02-11 | 第一三共股份有限公司 | Acid addition salt of substituted pyridine compound |
-
2004
- 2004-12-18 DE DE102004061002A patent/DE102004061002A1/en not_active Withdrawn
-
2005
- 2005-12-10 CN CNA2005800434885A patent/CN101080393A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI570118B (en) * | 2012-01-06 | 2017-02-11 | 第一三共股份有限公司 | Acid addition salt of substituted pyridine compound |
Also Published As
| Publication number | Publication date |
|---|---|
| DE102004061002A1 (en) | 2006-06-22 |
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