CN101291676B - Amidines derivative and use of medicine - Google Patents

Amidines derivative and use of medicine Download PDF

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Publication number
CN101291676B
CN101291676B CN2006800388504A CN200680038850A CN101291676B CN 101291676 B CN101291676 B CN 101291676B CN 2006800388504 A CN2006800388504 A CN 2006800388504A CN 200680038850 A CN200680038850 A CN 200680038850A CN 101291676 B CN101291676 B CN 101291676B
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phenothiazine
oxolane
methyl
group
salt
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CN101291676A (en
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S·欧文
D·比戈
P-E·查布里德拉索尼尔
B·彼格诺尔
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Ipsen Pharma SAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D279/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one sulfur atom as the only ring hetero atoms
    • C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
    • C07D279/14—1,4-Thiazines; Hydrogenated 1,4-thiazines condensed with carbocyclic rings or ring systems
    • C07D279/18—[b, e]-condensed with two six-membered rings
    • C07D279/22—[b, e]-condensed with two six-membered rings with carbon atoms directly attached to the ring nitrogen atom

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  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention concerns amidine derivatives of general formula (I) exhibiting an inhibitory activity of calpains and/or a trapping activity of reactive forms of oxygen.

Description

Amidine derivative with and as the application of medicine
Theme of the present invention is calpain to be had suppress active and/or reactive oxygen species (ROS) had to catch active amidine derivative.The invention still further relates to described derivant preparation method, comprise it pharmaceutical preparation with and be used for the treatment of purpose, particularly as the purposes of inhibitor and the selectivity or the non-selective ROS trapping agent of calpain.
In view of calpain and ROS possible effect in pathophysiology, new derivatives of the present invention can produce useful or favourable effect in the treatment of the pathological changes that relates to these enzymes and/or these free radical materials, and what described pathological changes was concrete is:
-inflammatory and immune disease, for example rheumatoid arthritis, pancreatitis, multiple sclerosis, gastrointestinal inflammation (for example ulcer or non-ulcerative colitis, Crohn disease),
-cardiovascular and/or cerebrovascular disease comprise that for example arterial pressure is too high, septic shock, ischemia or the hemorrhage heart that causes or cerebral infarction, ischemia and the disease relevant with platelet aggregation,
-central or peripheral nervous system disease, neurodegenerative disease for example, the particularly brain that wherein can mention or spinal cord injury, subarachnoid hemorrhage, epilepsy, aging, alzheimer disease comprise Alzheimer, Huntington chorea, parkinson disease, peripheral neurophaty
-cachexia,
-skeletal muscle reduces disease (sarcopenia),
-hearing disability, particularly by presbyacusis, acoustic trauma or owing to use for example gentamycin, anticarcinogen cisplatin, NSAID (non-steroidal anti-inflammatory drug) salicylic acid or ibuprofen derivative, diuretic furosemide, antiulcerative cimetidine or omeprazole, the anticonvulsant hearing disability that causes of carbamazepine or valproic acid for example for example for example for example for example of medicine such as antibiotic
-osteoporosis,
-duchenne muscular dystrophy, for example, particularly Duchenne's muscular dystrophy, becker's dystrophy, myotonia type muscular dystrophy or muscular dystrophy myotonia, congenital muscular dystrophy, erb syndrome and FSHD
The non-Cancerous disease of-proliferative is atherosclerosis or restenosis for example,
-cataract,
-organ transplantation,
-autoimmune or virus disease be lupus, AIDS, parasite or viral infection, diabetes and complication thereof for example,
-cancer and carcinous proliferative disease,
-be characterized as excessive ROS to produce and/or activatory all pathological changes of calpain.
There is the experimental evidence proof in all these pathological changes, all to relate to ROS (Free Radie.Biol.Med. (1996) 20,675-705; Antioxid.Health.Dis. (1997) 4 (synthetic antioxidant handbooks (Handbook of Synthetic Antioxidants)), 1-52) and calpain (TrendsPharmacol.Sci. (1994) 15,412419; Drug News Perspect (1999) 12,73-82).For example, antioxidant (Acta.Physiol.Scand. (1994) 152,349-350; J.Cereb.BloodFlow Metabol. (1995) 15,948-952; J Pharmacol Exp Ther (1997) 2,895-904) and calpain inhibitor (Proe Natl Acad Sci U S A (1996) 93,3428-33; Stroke, (1998) 29,152-158; Stroke (1994) 25,2265-2270) reduced and cerebral infarction or the relevant brain injury of experimental cranium wound.
In order to satisfy industrial requirement, need to find calpain had and suppress active and/or reactive oxygen species had to catch active other chemical compound.
Therefore, problem to be solved by this invention provides calpain had and suppresses active and/or reactive oxygen species had to catch active noval chemical compound.
The present inventor finds that unexpectedly the compound or its salt of the general formula (I) of all combining forms of racemic form hereinafter described, diastereo-isomerism form or these forms has the activity that suppresses the active of calpain and/or catch reactive oxygen species.
The invention has the advantages that it can implement in all industries, particularly pharmacy, veterinary, cosmetics and food industries and agriculture field.
Compound or its salt of the present invention particularly has the dissolubility of increase in water-bearing media in biological media.
When description below reading and embodiment, other advantage of the present invention and feature will become apparent, and given description and embodiment only are in order to describe, rather than in order to limit.
Therefore, theme of the present invention is the compound or its salt of the general formula (I) of all combining forms of racemic form, diastereomer or these forms,
Wherein
R 1, R 2, R 4, R 5And R 6Represent hydrogen atom, halogen atom, OH group, alkyl, alkoxyl, cyano group, nitro, amino, alkyl amino or carboxylic acid independently;
R 3Expression hydrogen atom, alkyl or-COR 10Group;
R 10Expression hydrogen atom or alkyl, alkoxyl, aryl or heterocyclic group;
W represent oxygen atom or sulphur atom or-W-represents key;
R 7Expression hydrogen atom or alkyl;
R 8Expression hydrogen atom, haloalkyl or alkenyl, cycloalkyl, straight or branched alkyl, it is substituted or is not substituted, when being substituted, it carries chemical functional group such as carboxylic acid, amino, alcohol, guanidine, amidine, mercaptan, thioether, thioester, alkoxyl, heterocycle or Methanamide;
R 9Expression hydrogen atom, alkyl, aryl, aryl alkyl, biaryl alkyl, heterocyclic group, heterocyclic radical alkyl or-COR 10Group;
Should be understood that:
Figure S2006800388504D00032
Be meant
Figure S2006800388504D00033
Preferably, the R in the The compounds of this invention 1Group is a hydrogen atom.
Preferably, the R in the The compounds of this invention 2Group is a hydrogen atom.
Preferably, the R in the The compounds of this invention 3Group is a hydrogen atom.
Preferably, the R in the The compounds of this invention 4Group is a hydrogen atom.
Preferably, the R in the The compounds of this invention 5Group is a hydrogen atom.
Preferably, the R in the The compounds of this invention 6Group is a hydrogen atom.
Preferably, the R in the The compounds of this invention 7Group is a hydrogen atom.
Preferably, the R in the The compounds of this invention 8Group is an isobutyl group.
Preferably, the W in the The compounds of this invention is a sulphur atom.
Preferably, the R in the The compounds of this invention 9Group is a hydrogen atom.
Preferably, the R in the The compounds of this invention 9Group is an acetyl group.
Preferably, the R in the The compounds of this invention 9Group is a methyl.
Preferably, the R in the The compounds of this invention 9Group is a benzyl.
Preferably, the R in the The compounds of this invention 9Group is a naphthyl methyl.
Unless definition is arranged clearly in addition, otherwise alkyl is meant and comprises 1 to 12 carbon atom and preferred 1 to 6 carbon atom, more preferably the straight or branched alkyl of 1 to 4 carbon atom.Straight or branched alkyl with 1 to 6 carbon atom is fingernail base, ethyl, propyl group, isopropyl, butyl, isobutyl group, the second month in a season-butyl and tert-butyl, amyl group, neopentyl, isopentyl, hexyl, isohesyl particularly.
Haloalkyl is meant that wherein at least one hydrogen atom is by the displaced alkyl of halogen atom.Haloalkyl for example is meant-CF 3,-CHF 2Or-CH 2The Cl group.
Unless clearly definition is arranged in addition, otherwise alkenyl is meant to have at least one degree of unsaturation and comprise 2 to 12 carbon atoms, the straight or branched alkenyl of preferred 2 to 6 carbon atoms.
Unless clearly definition is arranged in addition, otherwise alkoxyl is meant the R-O-group that contains carbochain R that comprises straight or branched and have 1 to 6 carbon atom.
Unless clearly definition is arranged in addition, otherwise cycloalkyl is meant the saturated carbon cyclic group that comprises 3 to 7 carbon atoms.The cycloalkyl that comprises 3 to 7 carbon atoms is the finger ring hexyl particularly.
Unless clearly definition is arranged in addition, otherwise aryl is meant the aromatic carbocyclic group that preferably has 1 to 3 fused rings.Aryl is meant phenyl, naphthyl and phenanthryl especially, preferably phenyl and naphthyl and more preferably phenyl.
Aryl alkyl is meant that its ingredient alkyl and aryl have the aryl alkyl of top given implication respectively, should be understood that aryl by alkyl and (I) or (SI) molecule link to each other.
The biaryl alkyl is meant in the present invention and comprises at least 2 rings, wherein at least one ring be aromatics and comprise maximum 14 carbon atoms, the aromatic carbocyclyl groups of preferred maximum 10 carbon atoms, should be understood that this biaryl alkyl by alkyl and (I) or (SI) molecule link to each other.
Heterocycle is meant cyclic group aromatics or non-aromatics, that comprise 1 to 14 atom in the present invention, and these atoms are selected from one of carbon, nitrogen, oxygen or sulfur or its chemical compound.Should be understood that described heterocyclic group can be that part is undersaturated.Heterocycle for example is meant heteroaryl or Heterocyclylalkyl.
The heterocycle that the heterocyclic radical alkyl is meant wherein to be comprised in the present invention with above alkyl has shown in implication and its heterocyclic group by alkyl and (I) or (SI) the heterocyclic radical alkyl that links to each other of molecule.
Halogen atom is meant the atom that is selected from fluorine, chlorine, bromine or iodine atom.
Amino is meant-NH in the present invention 2Group.
Alkyl amino is meant in the present invention-NRH or-N (R) 2Group, R are alkyl as defined above.
Following example table is understood can protect R 8The blocking group of entrained functional group:
-methyl, ethyl, tert-butyl or benzyl ester can be protected acid functional group;
-benzyl or fluorenyl methyl-tert-butyl carbamate can be protected amine functional group;
-acetamide can be protected amine functional group;
-tert-butyl, benzyl, Pentamethylene oxide. or silyl ether can be protected alcohol functional group;
-acetyl group can be protected alcohol functional group; With
-methyl thioether or methyl thioester can be protected thiol functionalities.
In particular, the present invention relates to be selected from the chemical compound of the general formula (I) of following compounds or its salt:
N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-methoxyl group oxolane-3-yl]-the L-leucyl amine;
N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-N 2-[imino group (10H-phenothiazine-2-yl) methyl]-L-leucyl amine;
N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-the L-leucyl amine;
Acetic acid (3S)-3-(N-[imino group (10H-phenothiazine-2-yl) methyl]-the L-leucyl-} amino) oxolane-2-base ester;
N 1-[(3S)-2-hydroxyl tetrahydrofuran-3-yl]-N 2-[imino group (10H-phenothiazine-2-yl) methyl]-L-leucyl amine.
The used nomenclature of the name of top chemical compound is an English IUPAC nomenclature.
Theme of the present invention also comprises as the compound or its salt of top defined general formula (I) purposes as therapeutic active substance.
More particularly, compound or its salt of the present invention can be used as and is used for the treatment of the therapeutic active substance that is characterised in that excessive ROS generation and/or the activatory pathological changes of calpain.
More particularly, compound or its salt of the present invention can be used as to be used for the treatment of and is selected from following disease and treatment of conditions active substance: inflammatory or immune disease, cardiovascular disease, cerebrovascular disease, central or peripheral nervous system disease, cachexia, skeletal muscle reduce rejection, autoimmune disease, virus disease or cancer after disease, hearing disability, osteoporosis, duchenne muscular dystrophy, carcinous or non-carcinous proliferative disease, cataract, the organ transplantation.
Theme of the present invention preferably is used for the treatment of the purposes of the therapeutic active substance of hearing disability or duchenne muscular dystrophy as the compound or its salt of top defined general formula (I).
Theme of the present invention also relates to the medicine of one of compound or its salt of comprising at least a general formula as defined above (I).These salt are the pharmaceutically useful salt of this compounds preferably.
Theme of the present invention also relates to as the chemical compound of the general formula as defined above (I) of medicine or its pharmaceutically useful salt.
The invention still further relates to the pharmaceutical composition of the officinal salt of the chemical compound that comprises at least a general formula as defined above (I) or at least a this compounds.Described pharmaceutical composition preferably comprises at least a pharmaceutically useful excipient.The compound or its salt of the defined general formula in front (I) preferably is contained in the pharmaceutical active compositions as active component.
Pharmaceutically useful salt is meant mineral acid or organic acid addition salts especially, the addition salts of described mineral acid is for example hydrochlorate, hydrobromate, hydriodate, sulfate, phosphate, diphosphate or nitrate, and described organic acid addition salts is for example acetate, maleate, fumarate, tartrate, succinate, citrate, lactate, mesylate, right-toluene fulfonate, benzene sulfonate, pamoate or stearate.For other example of officinal salt, can be referring to " Salt selection forbasic drugs ", Int.J.Pharm. (1986), 33,201-217.
The compound or its salt of the general formula (I) that the present invention is used can be a solid form, for example powder, granule, tablet, gelatine capsule, liposome or suppository.Suitable solid carrier can be for example calcium phosphate, magnesium stearate, Pulvis Talci, sugar, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine or wax.
The compound or its salt of general formula of the present invention (I) can also exist with liquid form, and for example solution, Emulsion or in a broad sense can be gel, suspension, spraying or syrup.The appropriate liquid carrier can be for example water, organic solvent such as glycerol or glycols, with and the mixture in water of various ratios.
The officinal salt that the invention still further relates to the chemical compound of general formula as defined above (I) or this compounds preparation be used for the treatment of be characterised in that excessive ROS produces and/or the medicine of activatory all pathological changes of calpain in application, and described pathological changes particularly is selected from inflammatory or immune disease, cardiovascular disease, cerebrovascular disease, the central or peripheral nervous system disease, cachexia, skeletal muscle reduces disease, hearing disability, osteoporosis, duchenne muscular dystrophy, carcinous or non-carcinous proliferative disease, cataract, rejection after the organ transplantation, autoimmune disease, virus disease or cancer.
The compound or its salt of general formula of the present invention (I) can carry out administration by local, oral, parenteral approach (by intramuscular, subcutaneous injection etc.).
The dosage that is used for the treatment of the product of the present invention of above-mentioned disease or disease changes according to medication, patient's age and body weight and patient's state, and will finally be determined by attending doctor or veterinary.Such quantity that attending doctor or veterinary determine is called as " treatment effective dose " here.
Theme of the present invention also relates to the compound or its salt of the general formula (SI) of stereoisomer or its combining form,
Figure S2006800388504D00071
Wherein:
W, R 1, R 2, R 3, R 4, R 5, R 6And R 7Have general formula I) implication in the chemical compound, and
R 11Expression alkyl, aryl, aryl alkyl, biaryl alkyl, heterocyclic group or heterocyclic radical alkyl.
Theme of the present invention also relates to the synthetic intermediate compound or its salt that obtains in the building-up process of general formula (I) chemical compound, and it is selected from following chemical compound:
10H-phenothiazine-2-imino group bamic acid (carbimidothioate) methyl ester;
N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-methoxyl group oxolane-3-yl]-the L-leucyl amine;
N 2-[(9H-fluorenes-9-ylmethoxy) carbonyl]-N 1-[(3S)-2-oxo-tetrahydrofuran-3-yl]-the L-leucyl amine;
N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-N 2-[(9H-fluorenes-9-ylmethoxy) carbonyl]-L-leucyl amine;
N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-the L-leucyl amine;
N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-N 2-[imino group (10H-phenothiazine-2-yl) methyl]-L-leucyl amine;
N 2-[(9H-fluorenes-9-ylmethoxy) carbonyl]-N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-the L-leucyl amine;
N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-the L-leucyl amine;
N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-the L-leucyl amine;
Acetic acid (3S)-3-(L-leucyl-amino) oxolane-2-base ester;
Acetic acid (3S)-3-(N-[imino group (10H-phenothiazine-2-yl) methyl]-the L-leucyl-} amino) oxolane-2-base ester;
N-ethyl-10H-phenothiazine-2-Methanamide;
N-ethyl-10H-phenothiazine-2-thioformamide;
N-ethyl-10H-phenothiazine-2-imino group bamic acid methyl ester.
The used nomenclature of the name of top chemical compound is an Englishi IUPAC nomenclature.
Theme of the present invention also relates to as therapeutic active substance and is the compound or its salt of one of above-mentioned synthetic intermediate (SI).These compound or its salts of the present invention especially can be used as and are used for the treatment of the therapeutic active substance that is characterised in that excessive ROS generation and/or the activatory pathological changes of calpain.
Specifically, be that these compound or its salts of the present invention of above-mentioned synthetic intermediate (SI) can be used as to be used for the treatment of and are selected from following disease and treatment of conditions active substance: inflammatory or immune disease, cardiovascular disease, cerebrovascular disease, central or peripheral nervous system disease, cachexia, skeletal muscle reduce rejection, autoimmune disease, virus disease or cancer after disease, hearing disability, osteoporosis, duchenne muscular dystrophy, carcinous or non-carcinous proliferative disease, cataract, the organ transplantation.
Theme of the present invention relates to also that to comprise at least a be the medicine of the compound or its salt of one of above-mentioned synthetic intermediate (SI).These salt are the pharmaceutically useful salt of this compounds preferably.
It is chemical compound or its pharmaceutically useful salt of one of above-mentioned synthetic intermediate (SI) that theme of the present invention also relates to as at least a of medicine.
The invention still further relates to that to comprise at least a be the pharmaceutical composition of the officinal salt of the compound or its salt of one of above-mentioned synthetic intermediate (SI) or at least a this compounds.Described pharmaceutical composition preferably comprises at least a pharmaceutically useful excipient.The compound or its salt that is one of above-mentioned synthetic intermediate (SI) preferably is contained in the pharmaceutical composition as active component.
Theme of the present invention also relates to chemical compound or product or a kind of synthetic intermediate (SI) or the application of its salt in pharmacy, veterinary, chemistry, cosmetics, food industries and agriculture field of at least a general formula of the present invention (I).
The preparation of general formula (I) chemical compound:
The chemical compound of general formula of the present invention (I) can be prepared according to the route of synthesis shown in following Fig. 1.R wherein 9Expression alkyl, aryl, aryl alkyl, biaryl alkyl, heterocyclic group, heterocyclic radical alkyl or-COR 10Group, R 10The chemical compound of the general formula (I) of expression hydrogen atom or alkyl, alkoxyl, aryl or heterocyclic group is called as general formula (I) 1Chemical compound.In the remainder of description, R wherein 9The chemical compound of the general formula (I) of expression hydrogen atom is called as general formula (I) 2Chemical compound.Among below Fig. 1 and Fig. 2, general formula (II), (III), (I) 1(I) 2Middle W, R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R 9, R 10And R 11Implication described in description before:
Figure S2006800388504D00101
Fig. 1
General formula (I) 1(I) 2Chemical compound be according to Fig. 1, by with the thioimido ester derivant of general formula (II) and the amino-lactonaphthol condensation of general formula (III), preferably 25 to 60 ℃ of heating down, preferably polar solvent for example isopropyl alcohol, DMF or more preferably react among the THF obtain, the response time is 4 to 20 hours.Then, can be with general formula (I) 1The hemiacetal functional group of chemical compound goes protection, thereby makes general formula (I) 2Chemical compound, for example in acid medium, with mineral acid for example HCl or HBr or organic acid for example benzenesulfonic acid for example go protection in acetone, THF, dioxanes, acetonitrile or the ethanol at solvent.That this reaction is normally carried out under about 20 ℃ and according to R 9Character, its response time changed between 4 to 20 hours.
The preparation of the intermediate of general formula (II):
Wherein W, R 1, R 2, R 3, R 4, R 5, R 6And R 7The thioimido ester of the non-commercialization of the general formula (II) described in top description can be prepared according to the route of synthesis that describes in detail among Fig. 2.
Figure S2006800388504D00111
Fig. 2
The thioimido ester of general formula (II), phenothiazine derivative (W=S), phenoxazine derivant (W=O) or carbazole derivates (W-is a key) can be begun 3 stages of branch by the carboxylic acid of corresponding general formula (II.1) and obtain.These carboxylic acids can be by the method described in the document, and for example Pharmazie 1984,39 (1), 22-3; Bull.Soc.Chim.1968, (7), 2832-42, Pharmazie 1966,21 (11), 645-9, Synthesis 1988, (3), 215-17, J.Med.Chem.1992,35 (4), 716-24, J.Org.Chem. (1960), 25,747-53, Heterocycles (1994), 39 (2), 833-45; J.IndianChem.Soc. (1985), 62 (7), 534-6; J.Chem.Soc.Chem.Comm. (1985), (2), the method described in the 86-7 obtains.The formation of the Methanamide of general formula (II.2) is to have ammonia (R 7=H) fortified aqueous or more preferably amine (R 7=alkyl) with peptide coupling reaction thing for example DCC or HBTU, for example carries out among the DMF under the situation at solvent.The thioformamide of general formula (II.3) can be by the Lawesson reaction reagent 1, and being used in the 4-dioxanes solution obtains.The alkylation of thioformamide that is used to produce the thioimido ester of general formula (II) can be used R 11-X carries out, and X is for example halogen atom, sulfuric ester or a triflate of a kind of leaving group.This reactant mixture was for example stirred 15 hours in acetone.If use iodomethane (R 11-X), then thioimido ester (II) is with salt, for example the form of hydriodate obtain and can be randomly with alkali for example sodium carbonate make its desalinization (desalified).
The preparation of the intermediate of general formula (III):
R wherein 8And R 9As mentioned above, Gp is that the amino-lactonaphthol derivant of the general formula (III) of blocking group (preferred carbamates blocking group) can obtain by the preparation approach of use-case as shown in following Fig. 3.
Figure S2006800388504D00121
Fig. 3
Amino-the butyrolactone derivative of general formula (III.2) can be by with the protected aminoacid of general formula (III.1) (R wherein 8Before being in the general formula (I) defined amino acid group and Gp be a kind of blocking group; for example benzyl, tert-butyl or fluorenyl methyl carbamate) with (S)-the alpha-amido butyrolactone reacts under the standard peptide synthesis condition and obtains, thereby make the Methanamide intermediate of general formula (III.2).Then, with lactone (III.2) with Reducing agent for example diisobutyl aluminum (DlBAL) at atent solvent for example THF or CH 2Cl 2In preferably be lower than-50 ℃, for example approximately reduce under-78 ℃ the temperature.Then; for example for example trifluoroacetic acid or camphorsulfonic acid are protected with strong acid in methanol or the benzylalcohol at the alcohol medium with the hemiacetal functional group of the lactonaphthol derivant of general formula (III.3); perhaps exist carboxylic acid anhydrides for example under the situation of acetic anhydride, exist under the situation of 4-monomethyl aminopyridine and in atent solvent such as dichloromethane, protecting, thereby make the acetal of general formula (III.4).
Perhaps, the amino-lactonaphthol of general formula (III) can be begun by business-like protected (S)-alpha-amido butyrolactone, divides 5 stages to be prepared.The protection step that being used to of carrying out continuously prepares the reduction of the intermediate (III.5) and the lactone of (III.6) and hemiacetal with produce intermediate (III.3) and (III.4) time described step identical.The preparation of intermediate (III.7) is preferably by carrying out to the main benzyloxycarbonyl of using in this strategy that hydrogenolysis carries out existing under the situation of Pd/C.Then, can by above-mentioned about (III.2) described condition under, the aminoacid of intermediate (III.7) and general formula (III.1) is carried out the intermediate that the peptide condensation obtains general formula (III.4).Then; according to the method described in the document (T.W.Greene and P.G.M.Wuts; blocking group in the organic synthesis (Protective Groups inOrganic Synthesis); the 2nd edition (Wiley-lnterscience, 1991)) amine functional group of intermediate of mutual-through type (III.4) goes protection.
Unless otherwise defined, otherwise all used here technology and scientific terminology have the common implication of understanding of one skilled in the art of the present invention.Similarly, mentioned here all publications, patent application, patent and other reference material all are introduced into as a reference.
With the following examples top method is described, should not regard described embodiment as limitation of the scope of the invention.
Embodiment
The used nomenclature of the name of following embodiment is an English IUPAC nomenclature.
In the following embodiments, fusing point all is by capillary tube, and is by name with commodity The device of B-545 type is measured.
Embodiment 1: N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-methoxyl group oxolane-3-yl]-L-leucyl amine hydriodate
1.1) N 2-[(benzyloxy) carbonyl]-N 1-[(3S)-2-oxo-tetrahydrofuran-3-yl]-the L-leucyl amine:
With 3.51g (13.25mmol) Cbz-L-leucine, 2.41g (1 equivalent) (S)-2-amino-4-butyrolactone hydrobromate, 1.97g HOBT (1.1 equivalent) and 5.59g (2.2 equivalent) 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) be dissolved in the 60ml dry DMF, add 7.64ml (3.3 equivalent) N then, the N-diisopropyl ethyl amine.This reactant mixture was stirred 15 hours down at 20 ℃, it is poured in ethyl acetate/aqueous mixtures of 200ml 1/1 then.After stirring and coming down in torrents, organic solution is used the saturated NaHCO of 100ml successively 3Solution, 50ml water, 100ml 1M citric acid solution wash and wash with the 100ml saline solution at last.Organic facies is carried out drying with sodium sulfate, filter and it is concentrated into drying under vacuum.The grease that obtains is washed with isopentane, use dichloromethane/isopentane crystalline mixture then.Yield with 68% obtains a kind of white solid.Fusing point: 130-131 ℃.
1.2) N 2-[(benzyloxy) carbonyl]-N 1-[(3S)-2-hydroxyl tetrahydrofuran-3-yl]-the L-leucyl amine:
Under argon, in a three-neck flask that comprises the 60ml anhydrous methylene chloride, make 1.1 dissolvings of 1.24g (3.56mmol) intermediate.This mixture is cooled to-60 ℃, then to the solution of DIBAL in dichloromethane that wherein drips 10.7ml (3 equivalent) 1M.When add finishing, remove cooling bath and it was continued to stir 15 minutes again.Then, this reaction medium carefully is poured in the Rochelle saline solution of 100ml 20%.In vigorous stirring after 2 hours, add the 100ml dichloromethane and it all is poured in the separatory funnel.Reclaim organic facies and it is washed with 50ml water and 50ml saline.After carrying out drying with sodium sulfate and filtering, organic solution is concentrated into drying under vacuum.Evaporation residue is carried out purification (eluant: heptane/AcOEt:1/1 to 2/8) with silicagel column.Yield with 72% obtains a kind of white solid.Fusing point: 48-49 ℃.
1.3) N 2-[(benzyloxy) carbonyl]-N 1-[(3S)-2-methoxyl group oxolane-3-yl]-the L-leucyl amine:
Under 20 ℃, excessive trifluoroacetic acid (5ml) is added drop-wise in the solution of 0.82g (2.34mmol) intermediate 1.2 in 50ml methanol.It is continued down to stir 15 hours at 20 ℃.Then, this reactant mixture is concentrated in the vacuum lower part and it is dissolved in the 50ml dichloromethane again.Organic solution is used the saturated NaHCO of 50ml successively 3Solution, 50ml water and 50ml saline wash.After with dried over sodium sulfate, filter and, evaporation residue is carried out purification (eluant: heptane/AcOEt:1/1 is paramount to 3/7) with silicagel column its vacuum concentration.Yield with 80% obtains a kind of white solid.Fusing point: 112-113 ℃.
1.4) N 1-[(3S)-2-methoxyl group oxolane-3-yl]-the L-leucyl amine:
2g (5.5mmol) intermediate 1.3 and 600mg 10%Pd/C are incorporated in the stainless steel reactor that comprises 60ml methanol.This mixture was stirred 1 hour under the 2atm hydrogen-pressure.After filtering catalyst, vaporising under vacuum falls methanol.Oily residue (1.20g with gained; 94%) is directly used in next step.
1.5) 10H-phenothiazine-2-thioformamide:
With at the 40ml 1 that has added the 20ml pyridine, comprise 3.4g (14mmol) 10H-phenothiazine-2-Methanamide (J.Org.Chem.1961 in the 4-dioxanes solution, 26,1138-1143) and the reactant mixture of 3.4g (8.4mmol) Lawesson reagent be heated to 110 ℃ the heating 1 hour 30 minutes.Then, with this brown solution vacuum concentration and with residue 200ml AcOEt and 100ml H 2O dilutes.After stirring and coming down in torrents, organic facies is washed with HCl aqueous solution and the 100ml saline of 100ml 1N successively.After with dried over sodium sulfate, filtration and vaporising under vacuum fall solvent, obtain a kind of orange powder.With this powder Et 2O washing is removed filtrate and with acetone it is extracted.Then, with acetone filtrate vacuum concentration, then evaporation residue is carried out purification (eluant: heptane/AcOEt:1/1 to 4/6) with silicagel column.Orange powder.Fusing point: 208-209 ℃.
1.6) 10H-phenothiazine-2-imino group bamic acid methyl ester hydriodate:
Under 23 ℃, 0.3ml (1.2 equivalent) iodomethane is joined in the solution of 1.05g (4.1mmol) intermediate 1.5 in 10ml acetone.This reactant mixture was stirred 15 hours.The precipitation that forms is leached and with acetone and isopentane it cleaned successively.Yield with 85% obtains a kind of brown-purple solid.Fusing point: 207-208 ℃.
1.7) N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-methoxyl group oxolane-3-yl]-L-leucyl amine hydriodate:
1.18g (1 equivalent) intermediate 1.6 is joined in the solution of 0.68g (2.95mmol) intermediate 1.4 in the 20ml isopropyl alcohol.This reactant mixture was stirred 15 hours down at 60 ℃.Successively with the methanthiol that discharges during soda solution and the potassium permanganate solution capture reaction.By filtering to isolate formed solid and using Et 2O cleans it, with silicagel column it is carried out purification (eluant: heptane/AcOEt:1/1 to 0/1) then.Yield with 70% obtains a kind of orange solids.Fusing point: 155-165 ℃.
Embodiment 2: N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-N 2-[imino group (10H-phenothiazine-2-yl) methyl]-L-leucyl amine hydriodate
2.1) N 2-[(9H-fluorenes-9-ylmethoxy) carbonyl]-N 1-[(3S)-2-oxo-tetrahydrofuran-3-yl]-the L-leucyl amine:
Used experimental program is identical with the synthetic described scheme of intermediate 1.1, with Fmoc-L-leucine in place Cbz-L-leucine.By using the AcOEt crystallization, the yield with 72% obtains the 3.15g white solid.Fusing point: 175-176 ℃
2.2) N 2-[(9H-fluorenes-9-ylmethoxy) carbonyl]-N 1-[(3S)-2-hydroxyl tetrahydrofuran-3-yl]-the L-leucyl amine:
Used experimental program is identical with the synthetic described scheme of intermediate 1.2, replaces intermediate 1.1 with intermediate 2.1.(heptane/AcOEt:1/1), the yield with 68% obtains the 2.16g white solid after carrying out purification with silicagel column.Fusing point: 155-156 ℃.
2.3) N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-N 2-[(9H-fluorenes-9-ylmethoxy) carbonyl]-L-leucyl amine:
0.41ml (1.1 equivalent) benzylalcohol and 0.11g (0.13 equivalent) camphorsulfonic acid are joined in the suspension of 1.57g (3.58mmol) intermediate 2.2 in the 7ml dichloromethane.Along with the carrying out of reaction, this reaction medium becomes homogeneous phase.After stirring 24 hours, this mixture with 25ml water and the dilution of 25ml dichloromethane, is stirred and comes down in torrents.Organic solution is carried out drying with sodium sulfate, filter and it is concentrated into drying.Residue is carried out purification (heptane/AcOEt:1/0 to 1/1) with silicagel column.After evaporation, the yield with 76% obtains the 1.43g white solid.Fusing point: 116-117 ℃.
2.4) N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-the L-leucyl amine:
0.2ml (5 equivalent) diethylamine is added drop-wise in the solution of 0.2g (0.38mmol) intermediate 2.3 in the 3.5ml dichloromethane.This reactant mixture was stirred 5 hours 30 minutes down at 23 ℃, then that its vacuum concentration is extremely dry.With residue Et 2O is partly dissolved and it is deposited a few hours under 4 ℃.White precipitate by removing by filter formation also is concentrated into drying with filtrate.Vaporized residue is directly used in next step.
2.5) N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-N 2-[imino group (10H-phenothiazine-2-yl) methyl]-L-leucyl amine hydriodate:
Used experimental program is identical with the synthetic described scheme of intermediate 1.7, with intermediate 1.6 be used to replace the intermediate 2.4 of intermediate 1.4 to react.With silicagel column the product of this reaction is carried out purification (heptane/AcOEt:1/1 to 0/1).After to the purest fraction evaporation, residue is mixed in isopentane/AcOEt to produce a kind of greenish orange color precipitation.Yield with 53% obtains the desired product of 430mg.Fusing point: 140-145 ℃.
Embodiment 3: N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-L-leucyl amine hydriodate
3.1) N 2-[(9H-fluorenes-9-ylmethoxy) carbonyl]-N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-the L-leucyl amine:
Used experimental program is identical with the synthetic described scheme of intermediate 2.3, replaces benzylalcohol by intermediate 2.2 beginnings and with 2-hydroxymethyl naphthalene.(heptane/AcOEt:7/3), the yield with 66% obtains the 1.38g white solid after carrying out purification with silicagel column.Fusing point: 79-80 ℃.
3.2) N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-the L-leucyl amine:
Used experimental program is identical with the synthetic described scheme of intermediate 2.4, replaces intermediate 2.3 with intermediate 3.1.After removing dibenzo fulvene derivant, obtain described product and it is directly used in next step.
3.3) N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-L-leucyl amine hydriodate:
Used experimental program is identical with the synthetic described scheme of intermediate 1.7, replaces intermediate 1.4 by intermediate 1.6 beginnings and with intermediate 3.2.The product of this condensation reaction is carried out purification (heptane/AcOEt:1/1 to 0/1) with silicagel column.After the purest fraction is evaporated, residue is mixed in isopentane/AcOEt to produce a kind of orange precipitation.Yield with 64% obtains the desired product of 530mg.Fusing point: 145-148 ℃.
Embodiment 4: acetic acid (3S)-3-(N-[imino group (10H-phenothiazine-2-yl) methyl]-the L-leucyl-} amino) oxolane-2-base ester hydriodate
4.1) acetic acid (3S)-3-(the N-[(benzyloxy) carbonyl]-the L-leucyl-} amino) oxolane-2-base ester:
Under argon gas atmosphere, 2g (5.73mmol) intermediate 1.2 and 0.14g (0.2 equivalent) 4-dimethylaminopyridine are dissolved in the 13ml anhydrous methylene chloride.Dropwise 5 .4ml in this solution (10 equivalent) acetic anhydride.It after stirring 5 hours under 23 ℃, is diluted this reactant mixture with 50ml dichloromethane and 50ml water.Then, organic facies is used successively the saturated NaHCO of 50ml 3Solution, 50ml water also wash with saline at last.With dichloromethane solution Na 2SO 4Drying is filtered and it is concentrated into drying under vacuum.Residue and Et with gained 2O mixes, and filters and with isopentane it is cleaned.Yield with 50% obtains the 1.14g white solid.Fusing point: 158-159 ℃.
4.2) acetic acid (3S)-3-(L-leucyl-amino) oxolane-2-base ester:
1.14g (2.89mmol) intermediate 4.1 and 227mg 10%Pd/C are incorporated in the stainless steel reactor that comprises 30ml acetic acid.This mixture was stirred under the 2atm hydrogen-pressure 4 hours 30 minutes.After filtering catalyst, vaporising under vacuum falls acetic acid.With the oily residue of gained at 50ml dichloromethane and the saturated NaHCO of 50ml 3Distribute between the solution.Stir, come down in torrents, water and saline wash organic facies then.Using Na 2SO 4After the drying, filter and be concentrated into drying, obtain a kind of colorless oil, it spontaneously forms crystallization, thereby makes the 0.45g white solid with 60% yield.Fusing point: 75-80 ℃.
4.3) acetic acid (3S)-3-(N-[imino group (10H-phenothiazine-2-yl) methyl]-the L-leucyl-} amino) oxolane-2-base ester hydriodate:
Used experimental program is identical with the synthetic described scheme of intermediate 1.7, and by intermediate 1.6 and intermediate 4.2 beginnings, just its reaction dissolvent is THF in this case, and is restricted to 4 hours heat time heating time.The top (heptane/AcOEt:3/7 to 0/1) that directly is adsorbed onto this reactant mixture on the silicon dioxide and is placed on chromatographic column is to carry out purification.After collecting and pure fraction evaporated, the yield with 18% obtains the 0.27g orange solids.Fusing point: 130-131 ℃.
Embodiment 5: N 1-[(3S)-2-hydroxyl tetrahydrofuran-3-yl]-N 2-[imino group (10H-phenothiazine-2-yl) methyl]-L-leucyl amine hydriodate
Under 23 ℃, 12mg (0.1 equivalent) benzenesulfonic acid is joined in the solution of 0.42g (0.69mmol) intermediate 4.3 in 42ml THF.Descend stirring after 5 hours 30 minutes, at 23 ℃ it to the NaHCO that wherein adds 137 μ l 0.5M 3Solution (0.1 equivalent).Continue again to stir 5 minutes, then formed precipitation is leached.Filtrate is concentrated into drying and with silicagel column residue is carried out purification (dichloromethane/EtOH:95/5 to 90/10).Collect pure fraction and it is evaporated, thereby make the 171mg orange solids with 43% yield.Fusing point: 148-150 ℃.
Embodiment 6:
6.1) N-ethyl-10H-phenothiazine-2-Methanamide:
5.8ml (3.3 equivalent) DIEA is added drop-wise in 2.43g (10mmol) 10H-phenothiazine-2-formic acid, 1.79g (2.2 equivalent) ethylamine hydrochloride and the solution of 4.17g (1.1 equivalent) HBTU in 50mlDMF that is cooled to 0 ℃.This reactant mixture was stirred 15 hours down at 23 ℃, then it is poured into the saturated NaHCO of 100ml 3In the mixture of solution and 100ml AcOEt.After it is stirred a few minutes, by removing by filter formed precipitation and filtrate being come down in torrents.Citric acid solution and salt water washing with organic facies water successively, 1M.Organic solution is carried out drying with sodium sulfate, filter and it is concentrated into drying under vacuum.The solid of gained is suspended in Et 2Among the O, grind, filter.Obtain a kind of beige solid with quantitative yield.Fusing point: 150-151 ℃.
6.2) N-ethyl-10H-phenothiazine-2-thioformamide:
Used experimental program is identical with intermediate 1.5 described schemes, replaces 10H-phenothiazine-2-Methanamide with intermediate 6.1.Yield with 60% obtains the 2.17g yellow solid.Fusing point: 155-156 ℃.
6.3) N-ethyl-10H-phenothiazine-2-imino group bamic acid methyl ester hydrochloride:
Used experimental program is identical with intermediate 1.6 described schemes, uses iodomethane and replaces intermediate 1.5 with intermediate 6.2.Then, the salifiable chemical compound that will obtain with the hydriodate form is at saturated NaHCO 3Distribute between solution and the AcOEt.After coming down in torrents,, filter with dried over sodium sulfate and to it with organic facies water and salt water washing.Then, under 0 ℃, in this refrigerative organic solution, add the HCl of 1.1 equivalent 1N at anhydrous Et 2Solution among the O.It after stirring 1 hour under 23 ℃, is concentrated into drying with this reactant mixture under vacuum.At last, evaporation residue is suspended in Et 2Filter among the O and to it.Obtain a kind of dark red solid.Fusing point: 142-143 ℃.
The pharmaceutical research of The compounds of this invention:
All percentage ratios that provide all are percents by volume.
The compounds of this invention is to the influence of people's bone cells (SKM) calpain enzymatic activity
Test principle is as follows: thorn tail ichtyhotoxisin (maitotoxin) is a kind of toxin that causes the cell calcium channel opener.When the beginning cell death, produce calcium and in cell, flow.In process of cell death, a kind of cysteine dependence protein enzyme---calpain is activated.This test comprises cultivates cell with the activation calpain under the situation that has thorn tail ichtyhotoxisin, and adds the calpain substrate, and when this substrate of described enzymatic lysis, it fluoresces.At last, calpain inhibitor is analyzed.
Sarcoplast is inoculated into the quantity of 2500 cells in every hole added amphotericin B, people in the 96-orifice plate and recombinate in DMEM 10%FBS (peptide Ox blood serum) culture medium of epidermal growth factor and dexamethasone.After inoculation 3 days, cell adhesion is to the bottom in hole, comes inducing cell to break up to myotube by adding the DMEM F12 culture medium that 100 μ l comprise 2% horse serum.After having spent 3 days again, 100 μ l test compounds are placed on the bottom in hole.With it at 5%CO 2Descend cultivation after 1 hour at 37 ℃ under the atmosphere, and the fluorogenic substrate (Suc-Leu-Tyr-AMC) of adding calpain and thorn tail ichtyhotoxisin (MTX) (Sigma, ref:M-9159).In order to measure the gross activity of cellular enzymes, preparation does not contain the control wells (be diluted to 1% 100 μ l DMSO, wherein added 10 μ lMTX and substrates) of test compounds onboard.Other control wells by the no MTX of preparation is measured background noise.Each production concentration is all tested in triplicate.These plates are stirred, under 380/460nm, at 0 o'clock fluorescence is carried out reading with the Victor device.Cultivated 3 hours down at 30 ℃ in the dark.
Can calculate each compound concentrations-effect according to fluorescent value.Calculate IC by this concentration-effect 50(with the test compounds concentration of enzymatic activity inhibition 50%).
The chemical compound of embodiment 5 has the IC that is lower than 20 μ M in this test 50
The research of the effect of lipid peroxidation in rat cerebral cortex
By measuring the inhibitory action of the influence of level of lipid peroxidation being measured product of the present invention, level of lipid peroxidation is to measure with the concentration of malonaldehyde (MDA).The MDA that the unsaturated fatty acid peroxidating produces be lipid peroxidation good indicator (H Esterbauer and KH Cheeseman, Meth.Enzymol. (1990), 186,407-421).By broken end heavy 200 to 250g male Sprague Dawley rat (Charles River) is put to death.Take out cerebral cortex, then with Thomaspotter with it at the Tris-HCl buffer, 20mM carries out homogenize among the pH=7.4.This homogenate 50, is descended each 10 minutes centrifugal 2 times at 4 ℃ under the 000g.Deposit is kept under-80 ℃.On experiment same day, with this deposit with the concentration of 1g/15ml suspendible again and with it under 4 ℃ under 515g centrifugal 10 minutes.Directly measure lipid peroxidation with supernatant.The homogenate (500 μ l) of rat cerebral cortex was cultivated 15 minutes under the situation that has test compounds or solvent (10 μ l) under 37 ℃.By adding the FeCl of 50 μ l 1mM 2, 1mM EDTA and 4mM ascorbic acid begin lipid peroxidation.It after cultivating 30 minutes under 37 ℃, is stopped this reaction by adding 50 μ l hydroxylatings, two-t-butyltoluene (BHT, 0.2%).By with color development reagent (R)---N-methyl-2-phenylindone (650 μ l) and 200 μ l homogenate 45 ℃ down reaction came with the quantitative MDA of colorimetric determination in 1 hour.A part MDA and two molecular agents R condensations have produced a kind of stable chromophore, its maximum absorption wavelength equal 586nm (people such as Caldwell, European J.Pharmacol. (1995), 285,203-206).
The chemical compound of embodiment 1 to 5 has the IC that is lower than 5 μ M in this test 50
The compounds of this invention is to the dead protective effect of the thorn tail inductive people's bone cells of ichtyhotoxisin (SKM)
Test principle is as follows: thorn tail ichtyhotoxisin is a kind of toxin that causes the cell calcium channel opener.When the beginning cell death, produce calcium and in cell, flow.In process of cell death, a kind of cysteine dependence protein enzyme---calpain is activated and has produced a large amount of free radicals.This test comprises cultivates cell to postpone or to stop cell death and to measure protective effect thus existing under the situation of test molecule.
Sarcoplast is inoculated into the quantity of 2500 cells in every hole added amphotericin B, people in the 96-orifice plate and recombinate in DMEM 10%FBS (peptide Ox blood serum) culture medium of epidermal growth factor and dexamethasone.After inoculation 3 days, cell adhesion is to the bottom in hole, comes inducing cell to break up to myotube by adding the DMEM F12 culture medium that 100 μ l comprise 2% horse serum.After having spent 3 days again, 100 μ l test compounds are placed on the bottom in hole.With it at 5%CO 2Cultivation is after 1 hour down at 37 ℃ under the atmosphere, and (Wako ref:131-10731) assesses with the protective effect (concentration-effect) to the death of test compounds pair cell to add thorn tail ichtyhotoxisin (MTX).
After cultivating 3 hours or 96 hours, culture medium is replaced with the DMEM10%FBS culture medium of having added 10%WST-1.WST-1 (Roche, numbering 1644807) is that a kind of metabolic activity cell that dyes is the reagent of living cells.Cell was cultivated 1 hour existing under the situation of WST-1.Install by under 450nm, reading the number that absorbance is measured living cells with Perkin Elmer Wallac Envision 2101.Then, calculate the concentration-effect of described product pair cell surviving fraction.
By this concentration-calculation of effect EC 50(the substances concentration of cell death does not appear in the cell of protection 50%).
After cultivating 3 hours under the situation that has thorn tail ichtyhotoxisin, the chemical compound of embodiment 5 has the EC that is lower than 16.4 μ M in this test 50, after cultivating 96 hours under the situation that has thorn tail ichtyhotoxisin, the chemical compound of embodiment 5 has the EC that is lower than 18.3 μ M in this test 50
Compare test with alpha-methylprednisolone (it is the chemical compound that is used for the treatment of Duchenne's muscular dystrophy).Comparatively speaking, with under identical condition, after cultivating 3 hours under the situation that has thorn tail ichtyhotoxisin, alpha-methylprednisolone has the EC of 354.3 μ M with identical test 50, after cultivating 96 hours under the situation that has thorn tail ichtyhotoxisin, alpha-methylprednisolone has the EC of 195.7 μ M 50
Handle the inductive ototoxicity in back with gentamycin: the protective effect of The compounds of this invention
Prove with the protective effect of the The compounds of this invention of therapeutic agent form administration altogether by the inductive ciliated cell loss of gentamycin.
Test principle: gentamycin and other aminoglycosides have caused people's ciliated cell infringement and hearing disability.Brachydanio rerio (Zebrafish) has the sensory organ that is called as neuromast on its body surface.These neuromast ciliated cells are similar to the ciliated cell of people's in ear portion on 26S Proteasome Structure and Function.In these fishes, can use DASPEI (iodate 2,4-dimethyl-aminobenzene vinyl-N-ethylpyridine) to neuromast ciliated cell dye and this dyeing has reflected the number of functional ciliated cell.
Handle the infringement of induced internal ciliated cell in Brachydanio rerio with gentamycin.For of the protective effect of analytical test chemical compound to the ciliated cell of gentamycin infringement, with this chemical compound as co-therapeutic agents with the gentamycin administration.Then, inner ciliated cell is dyeed with quantitative.
This research is to carry out with 5 the biggest fishes, and this fish was hatched 24 hours under the situation that has or do not exist described chemical compound with 1 μ g/ml gentamycin.Carry out parallel control; Only use substrate (1%DMSO; Positive control).With the fish of only handling with gentamycin as negative control.
Calculate EC by this concentration-effect 50(protecting 50% cell the substances concentration of cell death not occur).
Carry out DASPEI dyeing so that ciliated cell visible (n=6/group).Come the dyeing signal of ciliated cell is carried out quantitatively with morphometric analysis.The DASPEI dyeing signal of positive control is defined as 100%.
The result of table 1 shows:
The dyeing signal of-negative control accounts for 20%+/-0.6 of control signal, and promptly after handling with gentamycin, ciliated cell loses 80%.
-account for 52%+/-10 of control signal with the dyeing signal of the animal of the compound treatment of gentamycin and 50 μ M embodiment 5, promptly handled 40% protection that has obtained highly significant of the ciliated cell of infringement by gentamycin.In the concentration range of 25 μ M to 100 μ M, EC 50(its be equivalent to make that 50% staining cell is visual valid density) is 32 μ M.
-account for 49%+/-10 of control signal with the dyeing signal of the animal of the compound treatment of gentamycin and 50 μ M embodiment 4, promptly handled 36.2% protection that has obtained highly significant of the ciliated cell of infringement by gentamycin.In the concentration range of 25 μ M to 100 μ M, EC 50(its be equivalent to make that 50% staining cell is visual valid density) is 51 μ M.
Table 1:
Figure S2006800388504D00241
The painted ciliated cell percentage ratio of table 1:DASPEI
Positive control (Brachydanio rerio-1%DMSO); The influence (Brachydanio rerio-1%DMSO-gentamycin-chemical compound) of negative control (Brachydanio rerio-1%DMSO-gentamycin 1 μ g/ml) and product.Experimentize to every group of 6 animals.

Claims (14)

1. the compound or its salt of the general formula of racemic form or diastereomeric form (I),
Figure FSB00000512033500011
Wherein:
R 1, R 2, R 3, R 4, R 5, R 6And R 7The expression hydrogen atom;
W represents sulphur atom;
R 8Expression straight or branched C 1-6Alkyl;
R 9Expression hydrogen atom, C 1-6Alkyl, phenyl C 1-6Alkyl, naphthyl C 1-6Alkyl or-COR 10Group;
R wherein 10Expression C 1-6Alkyl;
Should be understood that:
Be meant
Figure FSB00000512033500013
2. chemical compound as claimed in claim 1 is characterized in that R 8It is isobutyl group.
3. chemical compound as claimed in claim 1 is characterized in that R 9It is hydrogen atom.
4. chemical compound as claimed in claim 1 is characterized in that R 9It is acetyl group.
5. chemical compound as claimed in claim 1 is characterized in that R 9It is methyl.
6. chemical compound as claimed in claim 1 is characterized in that R 9It is benzyl.
7. chemical compound as claimed in claim 1 is characterized in that R 9It is naphthyl methyl.
8. chemical compound as claimed in claim 1 is characterized in that it is selected from following compound or its salt:
N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-methoxyl group oxolane-3-yl]-the L-leucyl amine;
N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-N 2-[imino group (10H-phenothiazine-2-yl) methyl]-L-leucyl amine;
N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-the L-leucyl amine;
Acetic acid (3S)-3-(N-[imino group (10H-phenothiazine-2-yl) methyl]-the L-leucyl-} amino) oxolane-2-base ester;
N 1-[(3S)-2-hydroxyl tetrahydrofuran-3-yl]-N 2-[imino group (10H-phenothiazine-2-yl) methyl]-L-leucyl amine.
9. the described compound or its salt of claim 8 is used for the treatment of application in the medicine of hearing disability in preparation.
10.N 1-[(3S)-2-hydroxyl tetrahydrofuran-3-yl]-N 2-[imino group (10H-phenothiazine-2-yl) methyl]-L-leucyl amine or its salt are used for the treatment of application in the medicine of duchenne muscular dystrophy in preparation.
11. comprise at least a medicine as one of defined compound or its salt of claim 1 to 8.
12. comprise the pharmaceutical composition of at least a officinal salt as the defined chemical compound of claim 1 to 8 or at least a this compounds.
13. be selected from the compound or its salt of following compounds:
10H-phenothiazine-2-imino group bamic acid methyl ester;
N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-methoxyl group oxolane-3-yl]-the L-leucyl amine;
N 2-[(9H-fluorenes-9-ylmethoxy) carbonyl]-N 1-[(3S)-2-oxo-tetrahydrofuran-3-yl]-the L-leucyl amine;
N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-N 2-[(9H-fluorenes-9-ylmethoxy) carbonyl]-L-leucyl amine;
N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-the L-leucyl amine;
N 1-[(3S)-2-(benzyloxy) oxolane-3-yl]-N 2-[imino group (10H-phenothiazine-2-yl) methyl]-L-leucyl amine;
N 2-[(9H-fluorenes-9-ylmethoxy) carbonyl]-N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-the L-leucyl amine;
N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-the L-leucyl amine;
N 2-[imino group (10H-phenothiazine-2-yl) methyl]-N 1-[(3S)-2-(2-naphthyl methoxyl group) oxolane-3-yl]-the L-leucyl amine;
Acetic acid (3S)-3-(L-leucyl-amino) oxolane-2-base ester;
Acetic acid (3S)-3-(N-[imino group (10H-phenothiazine-2-yl) methyl]-the L-leucyl-} amino) oxolane-2-base ester;
N-ethyl-10H-phenothiazine-2-Methanamide;
N-ethyl-10H-phenothiazine-2-thioformamide;
N-ethyl-10H-phenothiazine-2-imino group bamic acid methyl ester.
14. the compound or its salt of the general formula of stereoisomer form (SI),
Figure FSB00000512033500031
Wherein:
W, R 1, R 2, R 3, R 4, R 5, R 6And R 7Have in the claim 1 about the defined identical meanings of formula (I) chemical compound, and
R 11Expression C 1-6Alkyl.
CN2006800388504A 2005-10-21 2006-10-18 Amidines derivative and use of medicine Expired - Fee Related CN101291676B (en)

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FR0601999A FR2898357B1 (en) 2006-03-07 2006-03-07 AMIDINE DERIVATIVES AND THEIR APPLICATIONS AS A MEDICINAL PRODUCT
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