CN1053448C - Compounds useful as antiproliferative agents and GARFT inhibitors - Google Patents
Compounds useful as antiproliferative agents and GARFT inhibitors Download PDFInfo
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- CN1053448C CN1053448C CN94195172A CN94195172A CN1053448C CN 1053448 C CN1053448 C CN 1053448C CN 94195172 A CN94195172 A CN 94195172A CN 94195172 A CN94195172 A CN 94195172A CN 1053448 C CN1053448 C CN 1053448C
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Abstract
The present relates to compounds, such as a compound (1) which is equilibrium with a 4-hydroxy tautomer thereof, and pharmaceutically acceptable salts thereof. The compounds can be used as inhibitors of glycinamide ribonucleotide formyl transferase (GARFT) or anti-proliferation medicines. The present invention also relates to medical compositions using the compounds as the GARFT inhibitors or anti-proliferation medicines, a method thereof, and intermediate compounds used for preparing the compounds.
Description
Background of invention
The present invention relates to the compound in structural formula I as giving a definition, this compound can suppress phosphoribosyl glycinamide formyl transferase (GARFT).The invention still further relates to the intermediate of these compounds of preparation, the pharmaceutical composition that contains these compounds, purposes that they suppress GARFT and their suppress higher organism cell or the growth of microorganism (falling as bacterium, yeast and fungi) and the purposes of propagation.That these compounds have is antitumor, anti-inflammatory, treatment psoriasis and/or immunosuppressive activity.The invention still further relates to the preparation of described these compounds.
In the big class of anti-proliferative drugs, comprise the antimetabolic compound.The special antimetabolite of a class that is called as the antifolic thing is the antagonist of VITAMIN folic acid.Usually, the structure of antifolic thing and folic acid is closely similar and can mix the distinctive to benzoyl L-glutamic acid part of folic acid.The L-glutamic acid part of folic acid has two negative charges under physiological pH condition.Therefore, this compound and analogue thereof have activated energy drives movement system to pass cytolemma and to produce metabolism.
GARFT is the folic acid dependent enzyme in the from the beginning biosynthetic pathway of purine.The division of this approach pair cell and breed extremely important.This approach of known cut-out has antiproliferative effect, is antineoplastic effect specifically.Therefore, synthesized a large amount of folacin and its ability that suppresses GARFT studied.It is reported, the GARFT inhibitor 5 of typical specificity mortise, the assorted tetrahydrofolic acid (THFA) of 10-two denitrifications has anti-tumor activity.Referring to F.M.Muggia, " the antifolate inhibitor of purine de novo synthesis ", the new drug in the cancer chemotherapy, notion and result, KluwerAcademic Publishers, Boston (1992), 65-87.
Recently, at the annelated heterocycles shape glutamic acid derivatives that discloses in the International Application PCT/US94/00418 that submitted on January 18th, 1994 by the applicant as anti-proliferative drugs or GARFT inhibitor.The application continues progress in this field by the development to more useful anti-proliferative drugs and GARFT inhibitor.
Summary of the invention
The present invention relates to compound in structural formula I as giving a definition.These compounds are effective to growth and the propagation that suppresses phosphoribosyl glycinamide formyl transferase (GARFT) and higher organism cell or microorganism (for example bacterium, yeast and fungi).The invention further relates to the pharmaceutical composition that contains these compounds or its suitable salt and these compounds purposes as the GARFT enzyme inhibitors.
As mentioned above, compound of the present invention has antiproliferative activity, and this characteristic can show as anti-tumor activity.Compound of the present invention can be an activeconstituents itself, or as the precursor that can change active compound in vivo into.Preferred compound of the present invention is effective especially to suppressing the GARFT enzyme.Particularly preferred compound is effective to suppress L1210 clone (a kind of L-1210 that can grow) in tissue culture medium.Compound of the present invention is also effective to the growth that suppresses bacterium (the Gram-negative intestinal bacteria that for example can grow) in nutrient solution.
Compound of the present invention and pharmaceutically useful salt thereof can be made formulation easily, for example capsule, tablet and injectable formulation.Can also use pharmaceutically acceptable carrier, thinner or the excipient of solid or liquid.
Solid carrier comprises starch, lactose, terra alba, terra alba, sucrose, talcum, gelatin, agar, pectin, gum arabic, Magnesium Stearate and stearic acid.Liquid vehicle comprises syrup, peanut oil, sweet oil, salts solution and water.
Carrier and thinner comprise the material that any prolongation discharges, and for example Zerol or Stearic diglyceride use separately or use with wax.When using liquid vehicle, preparation can be syrup, elixir, emulsion, soft gelatin capsule, aseptic parenteral solution (for example solution) or form non-aqueous or waterborne liquid suspension.
Pharmaceutical preparation is according to pharmacist's routine techniques preparation, when the needs Tabules, for example comprise mix, steps such as granulation and compressing tablet, perhaps comprise mixing, dress irritate and be dissolved into grade that step is used in oral, parenteral, part, intravaginal, the nose, in the segmental bronchus to make, the desired product of intraocular, Er Nei or drop rectum with drug.
Composition of the present invention can also further contain one or more other pharmaceutical active compounds.For example, can in composition, contain a kind of following antitumor drug: mitotic inhibitor (for example vincaleucoblastine); Alkylating agent; Dihydrofolate reductase inhibitor or TS inhibitor; Metabolic antagonist (for example 5 FU 5 fluorouracil, cytosine arabinoside); Embedded type microbiotic (for example Zorubicin, bleomycin); Enzyme (for example asparaginase); Topoisomerase enzyme inhibitor (for example Zuyeyidal); And biological response modifier (for example Interferon, rabbit).Composition of the present invention can also contain other GARFT inhibitor or anti-proliferative drugs, for example the compound of being put down in writing among international publication number WO 94/13295 (on June 23rd, 1994 is open) or the international publication number WO 92/05153 (on April 2nd, 1992 is open) is incorporated herein its disclosed content as a reference.Composition of the present invention can also contain that one or more are antibiotic, antimycotic, parasiticide, antiviral, psoriasis or anticoccidial drug.The example of antiviral comprises: sulfanilamide (SN), for example sulfamethoxazole, Sulphadiazine Sodium, Sulfametoxydiazine and fanasil; Dihydrofolate reductase inhibitor, for example trimethoprim, bromodiaprim and trimetrexate; Penicillin; Cynnematin; Quinolone carboxylic acid and condensed isothiazole analogue thereof.
Another aspect of the present invention relates to the methods of treatment that suppresses higher organism cell or microbial growth or propagation, and this method comprises compound of the present invention from significant quantity to the host that use.Compound of the present invention is particularly useful to treatment mammalian hosts (for example human host) and birds host.A particularly preferred methods of treatment comprises compound of the present invention from the dosage that can effectively suppress GARFT to the host that use.
Many anti-proliferative compounds and pharmaceutically useful salt thereof in this record can be used for methods of treatment of the present invention.These compounds can carry out administration with the form of the pharmaceutically acceptable composition that contains above-mentioned diluent or carrier.
The dosage of composition contains the active compound of significant quantity at least, and preferably is made up of one or more pharmaceutical dosage units." significant quantity " is the amount that is enough to suppress the pathways metabolism of folic acid and produces beneficial effect thus, for example by using one or more pharmaceutical dosage units.
Exemplary every day of the dosage that is used for vertebrate host comprises the per kilogram host dosage of 1 gram active compound at the most, preferred per kilogram host body weight 0.5 gram, more preferred 100 milligrams, 50 milligrams of preferably approximatelies or still less the most.Selected dosage can comprise by any suitable medication: topical, for example cream or frost; Oral; Rectal administration, for example suppository; Parenteral admin by injection; Or by in intravaginal, the nose, in the segmental bronchus, continuing medication of leaching of Er Nei or intraocular, carry out administration to warm-blooded animal or Mammals (for example needs) by suppressing the patient that the folic acid metabolism approach is treated.
Compound of the present invention can produce one or more effects in antiproliferative, antibiotic, parasiticide, psoriasis, protozoacide, anticoccidial, anti-inflammatory, immunosuppression and the anti-mycotic efficiency.It is particularly useful that this compound produces antitumous effect for the vertebrates that suffers from tumour.
Detailed Description Of The Invention
Specifically, the present invention relates to the anti-proliferative compounds of structural formula I:
Wherein:
A is O, S or Se;
X replaces or unsubstituted C
1-C
3Alkyl, replacement or unsubstituted C
2-C
3Thiazolinyl, replacement or unsubstituted C
2-C
3Alkynyl, replacement or unsubstituted amino, sulphur or oxygen;
Y is O, S or NH;
B is hydrogen or halogen (F, Cl, Br or I);
C is hydrogen, halogen or replacement or unsubstituted C
1-C
6Alkyl; And
R
1And R
2Independent of each other be hydrogen or with the CO that links to each other
2Form the group of the ester group that is easy to hydrolysis together, for example C
1-C
6Alkyl.The invention further relates to the pharmaceutically useful salt of compound in structural formula I.The invention still further relates to the intermediate of these compounds of preparation.
Although compound in structural formula I is to represent with the form of 4-oxo, and also be to mention this compound with this form in whole specification sheets, this carbonyl group exists with tautomeric balance with corresponding 4-oh group.Therefore, be to be understood that term " compound in structural formula I " means 4-oxo and the tautomeric 4-OH-form with structrual description.Therefore, the invention still further relates to the pharmaceutically useful salt of the described 4-hydroxyl of structural formula I tautomer.
When X or C were the group that replaces, preferred substituted comprised C
1-C
6Alkyl, C
2-C
6Thiazolinyl, C
2-C
6Alkynyl, aryl, halogen, amino, hydroxyl, nitro, sulfydryl, monocyclic carbocyclic ring, monocyclic heterocycle, condense and many rings carbocyclic ring of non-condensed, condense and many rings heterocycle, the hydroxyl C of non-condensed
1-C
6Alkyl and C
1-C
6Alkoxy C
1-C
6Alkyl.
Preferred X is CH
2, CH
2CH
2, NH, oxygen, sulphur, CH (CH
2OH) or NCH
3Preferred Y is S or O.Preferred B is a hydrogen.Preferred C is hydrogen or CH
3Preferred R
1And R
2Be to be independently from each other hydrogen, C
1-C
6Alkyl, hydroxyalkyl, alkaryl and aralkyl most preferably are selected from hydrogen and C
1-C
2Alkyl.When A and Y all are that sulphur and X are CH
2The time preferred especially.
Compound in structural formula I can be used as the GARFT inhibitor.R wherein
1And R
2The compound in structural formula I that all is hydrogen is special effective antitumour or anti-proliferative drugs.R wherein
1And R
2All be and the CO that links to each other
2Formation is easy to the compound in structural formula I of the group (being preferably ethyl) of the ester group of hydrolysis, is the useful as intermediates that can form the free glutamic acid form of this compound, thereby the effect of prodrug is played in hydrolysis in vivo.
Pharmaceutically useful salt of the present invention comprises, for example the ammonium salt of an alkali metal salt of glutamic acid compounds of the present invention, alkaline earth salt, other nontoxic metal-salt and ammonium salt and replacement.The example of salt comprises, the sodium of free acid compound, potassium, lithium, calcium, magnesium, pyridine and substituted pyridines salt.
Compound of the present invention can followingly be prepared.(wherein X is CH to the preparation compound in structural formula I
2) a compound that useful starting raw material is a structural formula II:
Wherein B, C and X as above structural formula I define; D is Cl, Br or I; R
6Be hydrogen or with the CO that links to each other
2Form the group of the ester group that is easy to hydrolysis together.Preferred D is a bromine or iodine.Preferred R
6Be hydrogen, C
1-C
6Alkyl, hydroxyalkyl, alkaryl or aralkyl, more preferred hydrogen or C
1-C
2Alkyl.
The compound of structural formula II and the compound of structural formula II I are reacted:
R wherein
3Be to replace or unsubstituted C
1-C
6Alkyl or trisubstituted silyl.Preferred R
3Be CH
2OH or trimethyl silyl.
Under the existence that is reflected at suitable transition-metal catalyst (preferred palladium or nickel) and non-nucleophilicity auxiliary alkali (amine of preferred replacement) of the compound of structural formula II and III, in solvent, carry out, in described solvent, at least a reactant is partly soluble at least under reaction conditions, is enough to the compound of generating structure formula IV:
Wherein B, C and Y as above structural formula I define; R
6As above structural formula II defines; R
3As above structural formula II I defines.
Work as R
3When being trisubstituted silyl, preferably use the alkali (for example methyl alcohol of salt of wormwood or ethanolic soln) or the fluoride salt (for example Potassium monofluoride, cesium fluoride or tetrabutyl ammonium fluoride) of nucleophilicity, be that (for example methyl alcohol, dimethyl formamide, ethanol, N,N-DIMETHYLACETAMIDE, dimethyl sulfoxide (DMSO) or Virahol) removes silylation in the partly soluble at least solvent at least a reactant, the compound of generating structure formula V:
Wherein B, C and Y as above structural formula I define; R
6As above structural formula II defines.
Compound and the nucleophilic reagent of structural formula V (are preferably the glycine (glycinal) of N-protected; more preferred N-tertiary butyloxycarbonyl acyl group glycine or two-N-tertiary butyloxycarbonyl acyl group glycine); under alkaline condition with alkali (preferred two-trimethyl silyl Lithamide of non-nucleophilicity; two-trimethyl silyl potassium amide; two-trimethyl silyl sodium amide or lithium diisopropylamine) (preferably from-90 ℃ to 25 ℃) at low temperatures; to at least a reactant be partly soluble at least appropriate solvent (preferred tetrahydrofuran (THF); ether Huo diox) reaction in, the compound of generating structure formula VI:
Wherein B, C and Y as above structural formula I define; R
6As above structural formula II defines; R
4And R
5Independent of each other is hydrogen or nitrogen-protecting group of being easy to remove.Preferred R
4And R
5Be hydrogen, tertbutyloxycarbonyl, carbobenzoxy-(Cbz) or benzyl.
With the compound reduction of structural formula VI, preferably in the presence of the proper metal catalyzer, reduce then, make the compound of structural formula VII with hydrogen:
Wherein B, C and Y as above structural formula I define; R
6As above structural formula II defines; R
4And R
5As above structural formula VI defines.
Then with the compound of structural formula VII and acidylate or sulfonylation agent (preferred methylsulfonyl chloride or Tosyl chloride) in the presence of non-nucleophilicity alkali (preferred triethylamine or diisopropyl ethyl amine), be to react in the partly soluble at least appropriate solvent at least a reactant, produce the activatory hydroxyl.This activatory hydroxyl is replaced the compound of generating structure formula VIII with suitable nucleophilic reagent (preferred thioacetate, more preferred thioacetic acid potassium):
Wherein A, B, C and Y as above structural formula I define; R
6As above structural formula II defines; R
4And R
5As above structural formula VI defines; Ac is an acyl group, preferred ethanoyl.
Perhaps, with compound triphenyl phosphine, azepine diethyl dicarboxylate or dimethyl ester and the acid nucleophilic reagent (preferred thioacetic acid) of structural formula VII, in appropriate solvent, be transformed into the compound of structural formula VIII through a step chemical reaction.
Use the alkali (preferred salt of wormwood, yellow soda ash, sodium hydroxide or potassium hydroxide) of nucleophilicity in alcohol solvent (particular methanol, ethanol or Virahol) compound of structural formula VIII, in the presence of alkylating reagent (preferred chloromalonic acid dimethyl ester or diethyl ester), handle, make the compound of structural formula IX:
Wherein A, B, C and Y as above structural formula I define; R
6As above structural formula II defines; R
4And R
5As above structural formula VI defines.
The compound of structural formula IX is handled under suitable condition partly or entirely to remove R
4And R
5Protecting group makes the compound of structural formula X:
Wherein A, B, C and Y as above structural formula I define; R
6As above structural formula II defines.When protecting group was tertbutyloxycarbonyl (t-BOC), the condition optimization that removes this group was to handle with trifluoroacetic acid, neutralized then with the compound of preparation structural formula X.
Compound and alkylating reagent (preferred oxygen Tetrafluoroboric acid trimethyl or triethyl) reaction in appropriate solvent (preferred methylene dichloride) with structural formula X generate lactim ether intermediate.This lactim ether intermediate and guanidine are reacted the compound of generating structure formula XI in alcohol solvent (particular methanol, ethanol or Virahol):
Wherein A, B, C and Y as above structural formula I define; R
6As above structural formula II defines.
Perhaps, by compound and mercaptan reagent (preferred P with structural formula X
2S
5Or 2,4-two (4-methoxyphenyl)-1,3-dithio-2,4-diphosphine (phosphetane)-2, the 4-disulfide) reaction generates the thio lactam intermediate, use alkylating reagent (preferred methyl iodide or oxygen Tetrafluoroboric acid trimethyl or triethyl), in alcohol solvent (particular methanol, ethanol or Virahol), to react the compound of generating structure formula XI with guanidine then then with its alkylation.
With compound hydrolysis under alkaline condition of structural formula XI, the compound of generating structure formula XII:
Wherein A, B, C and Y as above structural formula I define.R in the compound of structural formula XI
6When being hydrogen, then need not the reaction that is hydrolyzed, the coupling of carrying out peptide as described below of the compound of structural formula XI.
The compound of structural formula XII (or the compound of structural formula XI, wherein R
6Be hydrogen) be the free carboxylic acid form, can carry out the coupling of peptide by the hydrochloride that well known to a person skilled in the art method and glutamic acid diester, the diester of generating structure formula XIII:
Wherein A, B, C and Y as above structural formula I define; R
1And R
2Independent of each otherly be and the CO that links to each other
2Formation is easy to the group of the ester group of hydrolysis, for example C
1-C
6Alkyl, hydroxyalkyl, alkaryl or aralkyl.
At last, as needs, be the free L-glutamic acid form (R of structural formula I with the compound hydrolysis of structural formula XIII
1And R
2All be hydrogen).
(wherein X removes CH to compound in structural formula I
2Outside group) can be with the preparation of the alkene of structural formula XIV:
Wherein B, C and Y as above structural formula I define, X is the as above defined CH that removes of structural formula I
2Outside group, R
6As above structural formula II defines.
When X is sulphur, oxygen, replacement or unsubstituted when amino, the compound of structural formula XIV prepares by the alkylation with structural formula XV:
Wherein B, C and Y such as general formula I define; X is sulphur, oxygen or replacement or substituted-amino not; R
6Define as general formula I I.Available allyl halide is finished alkylated reaction, and preferred allyl bromide 98 is in the presence of non-nucleophilicity alkali (preferred triethylamine or diisopropylethylamine) and must general formula X IV compound.
When X replaces or unsubstituted C
1-C
2Alkyl (CH
2Except), replacement or unsubstituted C
2-C
3Alkenyl or replacement or unsubstituted C
2-C
3During alkynyl, general formula X IV compound is that the olefination by following formula XVI aldehyde prepares:
Wherein B, C and Y such as general formula I define; X replaces or unsubstituted C
1-C
2Alkyl (CH
2Except), replacement or unsubstituted C
2-C
3Alkenyl or replacement or unsubstituted C
2-C
3Alkynyl.At pharmaceutical chemistry magazine the 35th volume (1992), the similar approach of describing among the 1109-1116 prepares general formula X VI aldehyde by ChuanShih etc.Finish the olefination of this aldehyde with methylene radical transfer agent (preferred methylene radical-triphenyl phosphorane).
General formula X IV compound and dihydroxy reagent (preferred perosmic anhydride) reacted under suitable oxidizing agent (preferable methyl morpholine-N-oxide compound) and must general formula X VII compound:
Wherein B, C and Y such as general formula I define; X such as general formula I define, and are not CH just
2R
6Define as general formula I I.
The prepared in reaction intermediate list sulfonyl compound in the presence of non-nucleophilic base (preferred triethylamine or diisopropylethylamine) with general formula X VII compound and sulfonylation agent (preferably to benzene methylsulfonyl chloride or methylsulfonyl chloride).With (preferred sodium hydride) reaction of this intermediate and highly basic and general formula X VIII compound:
Wherein B, C, X, Y and R
6Define as general formula X VII.
With the epoxide of general formula X VIII with contain nitrogen nucleophile (preferred sodiumazide) and react down and get intermediate azide alcohol at moderate Lewis acid catalyst (preferred lithium perchlorate or magnesium perchlorate).Preferably this intermediate and hydrogen are reduced in the presence of metal catalyst, use suitable nitrogen-protecting group (preferred tertiary butoxy carbonyl, carbobenzoxy-(Cbz) or benzyl) protection then and general formula VII ' compound:
Wherein B, C, X, Y and R
6Define as general formula X VII; R
4And R
5Define as general formula VI.
Then general formula VII ' compound and acylating agent or sulfonyl agent (preferred methylsulfonyl chloride or Tosyl chloride) are reacted in appropriate solvent (at least a reactant is partly to dissolve in wherein at least) down and must activate hydroxyl at non-nucleophilic base (preferred triethylamine or diisopropylethylamine).Replace the activation hydroxyl and get general formula VIII ' compound with suitable nucleophilic reagent (preferred thiosalt, more preferably thioacetate):
Wherein A such as general formula I define; B, C, X, Y and R
6Define as general formula X VII; Ac is an acyl group, preferred ethanoyl.
In addition, available triphenyl phosphine, azepine diethyl dicarboxylate or dimethyl ester and acid nucleophilic reagent (preferred thioacetic acid) are converted into general formula VIII ' compound with one step of general formula VII ' compound in appropriate solvent.
Suitably in the presence of alkylating reagent (preferred chloromalonic acid dimethyl ester or diethyl ester), handling general formula VIII ' change thing and getting general formula I X ' compound in the alcoholic solvent (particular methanol, ethanol or Virahol) with nucleophilic alkali (preferably salt of wormwood, yellow soda ash, sodium hydroxide or potassium hydroxide):
Wherein A such as general formula I define; B, C, X, Y and R
6Define as general formula X VII; R
4And R
5Define as general formula VI.
Be suitable for removing R
4And R
5Handle general formula I X ' compound under protecting group one or both conditions and
Wherein A such as general formula I define; B, C, X, Y and R
6Define as general formula X VII.When protecting group was tertbutyloxycarbonyl, the condition optimization of removing this group was to handle with trifluoroacetic acid earlier, neutralize then and X ' compound.
With general formula X ' compound and alkylating agent (preferred Tetrafluoroboric acid trimethylammonium oxygen father-in-law or Tetrafluoroboric acid triethyl oxygen) reaction and formation intermediate lactim ether in appropriate solvent (preferred methylene dichloride).This intermediate lactim ether and guanidine reacted in alcoholic solvent (particular methanol, ethanol or Virahol) and form XI ' compound:
Wherein A such as general formula I define; B, C, X, Y and R
6Define as general formula X VII.
In addition, with general formula X ' compound and thiol reagent (preferred P
2S
5Or 2,4-two (4-p-methoxy-phenyl)-1,3-two sulphur-2,4-diphosphine alkane-2,4-disulphide) reaction forms sulphur lactan intermediate and makes general formula X ' compound is converted into general formula X I ' compound.Then it is earlier used guanidine alkanisation and must general formula X I ' compound in alcoholic solvent (particular methanol, ethanol or Virahol) then with alkylating agent (preferable methyl iodine or Tetrafluoroboric acid trimethylammonium oxygen father-in-law or Tetrafluoroboric acid triethyl oxygen).
Under alkaline condition, general formula X I ' compound hydrolysis is formed general formula X II ' compound:
Wherein A such as general formula I define; B, C, X, Y and R
6Define as general formula X VII.R in general formula X I ' compound
6When being hydrogen, the reaction that then needn't be hydrolyzed connects the following peptide that carries out of general formula X I ' compound then.
The method of knowing by this area professional with the glutamic acid diester hydrochloride with the general formula X II ' compound of free carboxy acid's form (perhaps R wherein
6Be the general formula X I ' compound of hydrogen) carry out the peptide connection and formation general formula X III ' diester:
Wherein A such as general formula I define; B, C, X and Y such as general formula X VII definition; R
1And R
2Independently of one another with the CO that is connected
2Formation is easy to the ester group of hydrolysis, and it can be as C
1-C
6Alkyl, hydroxyalkyl, alkylaryl or arylalkyl.
At last, free acid form can get general formula X III ' compound hydrolysis R if desired
1And R
2Compound of Formula I for H.
The preferred compound of general formula I comprises: (4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6] [1,4] thiazine-6-yl)-ethyl]-2,5-thienyl amino-L-L-glutamic acid) diethyl ester; 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6] [1,4] thiazine-6-yl)-ethyl]-2,5-thienyl amino-L-L-glutamic acid; 4-[3-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6] [1,4] thiazine-6-yl)-propyl group]-2,5-thienyl amino-L-L-glutamic acid; 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6] [1,4] thiazine-6-yl)-ethyl]-3-methyl-2,5-thienyl amino-L-L-glutamic acid; And 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6] [1,4] thiazine-6-yl)-ethyl]-2,5-furyl amino-L-L-glutamic acid.
The compounds of this invention contains one or more chiral centres.Present invention includes racemic mixture and diastereomer and optically active compound, for example do not conform to the compound of other optical isomer basically, described optically active compound can obtain by the known method of this area professional.
The following embodiment of detailed synthetic method of general formula I preferred compound is described.Embodiment: 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6] [1,4] thiazine-6-yl)-ethyl]-2,5-thienyl amino-L-L-glutamic acid (compound 1)
The preparation of 5-bromo-2-thiophenecarboxylate 2:
According to S.Gronowitz at Ark.Kemi 8,1995,87 and S.O.Lawesson at Ark.Kemi 11,1957, the method for describing in 337 prepares compound 2 (CAS Reg.No.[62224-19-5]).
The preparation of 5-ethynyl-2-methyl-formiate base thiophene 3:
Add 4.8 milliliters of (34 mmole) trimethyl silyl acetylene in 5 gram (22.7 mmole) 3,160 milligrams of (0.23 mmole) chlorinated triphenyl phosphine palladiums of bromide (II) under agitation and the 75 milliliters of diethylamine solutions of 65 milligrams of (0.34 mmole) cuprous iodides.Stir gained solution in 25 ℃.After 18 hours, removal of solvent under reduced pressure is dissolved in thick resistates in 300 milliliters of ether, extracts with 100 milliliters of 1N hydrochloric acid and 100 milliliters of saturated bicarbonate solutions.Dry (sodium sulfate) organic layer, removal of solvent under reduced pressure.Thick resistates is carried out flash chromatography on silica gel, get the product of the yellow solid shape silyls protection of 4.4 grams (81% productive rate) with 5% ether/hexane wash-out.This product is directly used in next step.
In 50 milliliters of dimethyl formamides of acetylene (DMF) solution of the above-mentioned silyl protection of 4.4 grams (18.5 mmole), add 3.3 gram (35.2 mmole) water Potassium monofluorides.At 25 ℃ after 15 minutes, mixture is poured in 500 milliliters of ether, extract four times (4 * 100 ml water) with 100 ml waters.Dry (sodium sulfate) organic solution, removal of solvent under reduced pressure.Thick resistates is carried out flash chromatography on silica gel, get the required product 3 of orange solids shape of 2.65 grams (86% productive rate) with 10% ether/hexane wash-out.NMR(CDCl
3)δ：3.45(s，1H)，3.88(s，3H)，7.22(d，1H，J＝3.5Hz)，7.65(d，1H，J＝3.5Hz).
Ultimate analysis: C
8H
6SO
2:
Theoretical value: C, 57.81; H, 3.64; S, 19.29
Measured value: C, 57.91; H, 3.71; S, 19.18.
The following silyl of removing of also available other method.The 59.7 gram silyl acetylene solids that add as above preparation in the 700 ml methanol suspension of 3.7 gram (0.02 mole) Anhydrous potassium carbonates under agitation.Stir these suspension 2 hours in 35 ℃, evaporate methyl alcohol then.Resistates is added in 50 ml waters, with 3 * 150 milliliters of ether extraction, organic moiety that dry (sodium sulfate) merges and evaporation.Thick resistates is carried out flash chromatography on silica gel, get the required product 3 of 98% productive rate (38.72 gram) with 7% ether/hexane wash-out.
The preparation of N-(tertbutyloxycarbonyl)-glycine 4:
In 80 milliliters of dichloromethane solutions of-78 ℃ of 2 gram (12.4 mmole) N-(tertbutyloxycarbonyl)-2-hydroxyl-ethamine (obtaining) under agitation, add 1.3 milliliters of (18.6 mmole) dimethyl sulfoxide (DMSO) (DMSO) and 1.2 milliliters of (13.7 mmole) oxalyl chlorides from Sigma Chemicals.After 5 minutes, add 5.4 milliliters of (38.4 mmole) triethylamines, make solution be warming up to 25 ℃.This mixture is poured in 200 milliliters of ether, extracted with 100 ml waters, 100 milliliters of 0.5N HCl and 100 milliliters of saturated bicarbonate solutions.Dry (sodium sulfate) organic moiety, removal of solvent under reduced pressure.Thick aldehyde is dissolved in 100 milliliters of benzene, removes once more down in decompression and desolvate.This unsettled crude product is used for next step immediately.
The preparation of 4-N-(tertbutyloxycarbonyl)-3-hydroxyl-1-(2-methoxycarbonyl-5-thiophene)-butine 5:
In the time of-78 ℃, in 10 milliliters of anhydrous tetrahydro furans (THF) solution of 1.7 gram (10.2 mmole) acetylene 3, add the THF solution of two-trimethyl silyl acid amides of 14.3 milliliters of (14.0 mmole) 1M.After 10 minutes, be added dropwise to 5 milliliters of anhydrous THF solutions of above-mentioned thick aldehyde 4.With-78 ℃ of stirring reactions 10 minutes, be warming up to 0 ℃ through 20 minutes.To wherein adding 5 milliliters of saturated aqueous ammonium chlorides.This mixture is poured in 200 milliliters of ether, with 100 ml waters and 50 milliliters of saturated nacl aqueous solution washings.Dry (sodium sulfate) organic layer, removal of solvent under reduced pressure.Thick resistates is carried out flash chromatography on silica gel, get 0.96 gram (29%) faint yellow oily required pure 5 with 10-40% ethyl acetate/hexane wash-out.
NMR (CDCl
3) δ: 1.46 (s, 9H), 3.38 and 3.58 (AB, 2H, J=3,7Hz), 3.88 (s, 3H), 4.71 (dt, 1H, J=2.9,5.2Hz), 5.05 (brs, 1H), 7.15 (d, 1H, J=3.9Hz), 7.64 (d, 1H, J=3.9Hz) .IR (neat): 2978.3,1729.5,1685,1523,1452,1368,1286,1167,1098,959,822,750.4cm
-1. high resolution mass spec, calculated value: C
15H
19NO
5S:M
+Cs
+, 458.0038. measured value: 458.0051.5-[(4-N-(tertbutyloxycarbonyl)-amino-3-hydroxyl)-butyl]-preparation of thienyl-2-methyl-formiate 6:
Add 320 milligrams of 5%Pd/C in 50 milliliters of ethyl acetate solutions of 940 milligrams of (2.89 mmole) above-mentioned acetylene 5 under agitation.This mixture is placed hydrogen under the 40psi, stirred 17 hours in 25 ℃.By diatomite filtration mixture, removal of solvent under reduced pressure.Thick resistates is carried out flash chromatography on silica gel, get 800 milligrams of required alcohol of (84% productive rate) yellow oily with 20% ethyl acetate/dichloromethane wash-out.NMR (CDCl
3) δ: 1.44 (s, 9H), 1.81 (m, 2H), 2.28 (brs, 1H), 2.99 (m, 2H), 3.10 and 3.29 (AB, 2H, J=3,6.9Hz), 3.74 (m, 1H), 3.86 (s, 3H), 4.89 (brs, 1H), 6.82 (d, 1H, J=3.7Hz), 7.63 (d, 1H, J=3.7Hz). calculated value: C
15H
23NO
5S:C, 54.69; H, 7.04; N, 4.25; S, 9.73. measured value: C, 54.79; H, 7.02; N, 4.29; S, 9.63.
Another method for preparing alcohol 6 is used following two-tertbutyloxycarbonyl allylamine 14.
The preparation of two-tertbutyloxycarbonyl allylamine 14:
In 500 milliliters of acetonitrile solutions of dimethyl aminopyridine (DMAP) of the allylamines of 57.10 grams (1.0 moles) and 1.2 grams (0.01 mole), add 220 grams (1 mole) (t-BOC)
2100 milliliters of acetonitrile solutions of O stir the gained mixture 6 hours.With toluene (100 milliliters) diluted reaction mixture, in 60 ℃ of solvent evaporated under reduced pressure.Gained oil is dissolved in the acetonitrile (40 milliliters) once more, adds 1.2 gram (0.01 mole) DMAP, in this solution, slowly add 220 grams (1 mole) (t-BOC)
2100 milliliters of acetonitrile solutions of O.In 60 ℃ of stirred reaction mixtures 12 hours,, add rare sodium bicarbonate (100 milliliters) with 60 ℃ of solvent evaporated under reduced pressure.With it with 3 * 150 milliliters of dichloromethane extraction, the salt water washing of the organic layer of merging, dry (sodium sulfate) and evaporation.Thick resistates is carried out flash chromatography on silica gel, get the required product 14 of 156.9 gram (63% productive rate) clarification crystalline solid with 5-20% ethyl acetate/hexane gradient elution.(m.p.43-44 ℃)
1H NMR (CDCl
3) δ (ppm): 1.5 (s, 18H), 4.18 (dd, 2H, J=15Hz, J=1Hz), 5.14 (ddd, 2H, J=15Hz, J=10Hz, J=1Hz), 5.85 (ddt, 2H, J=10Hz, J=5Hz, J=1Hz) .IR (KBr): 2978,2935,2860,1724,1689,1342,1130cm
-1. calculated value: C
13H
23NO
4: C, 60.68; H, 9.01; N, 5.44. measured value: C, 60.78; H, 9.04; N, the preparation of 5.50.1-(two t-butoxycarbonyl aminos)-2-acetaldehyde 15:
-78 ℃ of (40 volts of 20 milliliters of dichloromethane solution ozonize with 0.60 gram (2.34 mmole) two-tertbutyloxycarbonyl allylamine 14,500 amperes, 1.01/ minute, Yang Qi @3psi) stable until blueness, add 10 milliliters of dimethyl sulphides, stirred this mixture 14 hours in 25 ℃.This mixture is added in the weak brine, with dichloromethane extraction (3 * 50 milliliters), dry (sodium sulfate) organic moiety and evaporation.Obtain the required product 15 of clarification crystalline solid (fusing point 37-39 ℃) of 603 milligrams (99% productive rates) behind the sudden strain of a muscle formula silica gel chromatography.
1H NMR (CDCl
3) δ (ppm): 1.50 (s, 18H), 4.38 (s, 2H), 9.55 (s, 1H) .IR (KBr): 2984,2935,2724,1792,1734,1699,1362,1153cm
-1. calculated value: C
12H
21NO
5: C, 55.58; H, 8.16; N, 5.40. measured value: C, 55.20; H, 8.19; N, 5.19.
5-[(4-N-(tertbutyloxycarbonyl)-amino-3-hydroxyl)-butyl]-preparation of thienyl-2-methyl-formiate 6:
In THF (250 milliliters) solution of 9.64 gram (59 mmole) 5-ethynyl-2-methoxycarbonyl thiophene 3, add 65.6 milliliters of (60 mmole) hexamethyldisilane Lithium Azides (LiHMDS) under-78 ℃, stirred this mixture 2 hours in-78 ℃.THF (40 milliliters) solution of 15.6 gram (60 mmole) 1-(two t-butoxycarbonyl aminos)-2-acetaldehyde 15 is added in the reaction mixture, reaction mixture was stirred 8 hours in-78 ℃.Add methyl alcohol (10 milliliters) the solution quenching of 3.4 milliliters of (60 mmole) acetate, stirring the mixture was warming up to 0 ℃ in 10 minutes, added entry (60 milliliters).With ethyl acetate extraction (3 * 100 milliliters), organic extraction washs and dry (sodium sulfate) with rare sodium bicarbonate.Removal of solvent under reduced pressure must be about 50 milliliters.Add ethyl acetate (125 milliliters) in this gained resistates, this solution is added in the Parr bottle that contains 7.38 gram (the acetylene initiators of 30 weight %) 5% palladium on carbon, hydrogenation is 24 hours in 55psi hydrogen.By the thick hydrogenated products of diatomite filtration, filter cake washs with methyl alcohol (100 milliliters) and ethyl acetate (100 milliliters).Solvent evaporated under reduced pressure is by the thick resistates of filtered through silica gel, with the hexane solution wash-out of 50% ethyl acetate, evaporating solvent.Resistates is dissolved in 20 ml methanol-benzene (50 milliliters) azeotrope, is dissolved in again in the anhydrous methanol (20 milliliters).This mixture is added in the methanol solution of 60 milliliters of (2M) sodium methylates of new system.Stirred reaction mixture 45 minutes adds dilute hydrochloric acid (0.1M, 50 milliliters), with ethyl acetate extraction (3 * 75 milliliters).Merge organic moiety, with damping fluid (1M) washing of pH 7, dry (sodium sulfate) and evaporation.Thick resistates is dodged the enterprising circumstances in which people get things ready for a trip spectrum of formula silica gel, getting the required product 6 of 9.32 grams (48% productive rate) with 40% ethyl acetate/hexane wash-out.
5-[(4-N-(tertbutyloxycarbonyl)-amino-3-acetyl thio)-butyl]-preparation of 2-thiophenecarboxylate 7:
Add 5.9 milliliters of (42 mmole) triethylamines (TEA) and 2.4 milliliters of (31 mmole) methylsulfonyl chlorides in 100 milliliters of THF solution of 0 ℃ of 9.26 gram (28.1 mmole) alcohol 6 under agitation.After 20 minutes, add 100 milliliters of DMF and 12.8 gram (110 mmole) thioacetic acid potassiums, gained solution is warming up to 25 ℃.After 3 days, mixture is poured in 500 ml waters, with 800 milliliters of ether extraction.Organic layer washs with 200 ml waters, 200 milliliters of 1N hydrochloric acid, 200 milliliters of saturated bicarbonate solutions and 100 milliliters of saturated nacl aqueous solutions.Dry (sal epsom) organic layer, removal of solvent under reduced pressure.Thick resistates is carried out flash chromatography on silica gel, with behind the 25% ethyl acetate/hexane wash-out and 10.3 gram (95% productive rate) yellow oilies required thioacetates 7.
NMR (CDCl
3) δ: 1.44 (s, 9H), 1.91 and 2.04 (ABm, 2H), 2.37 (s, 3H), 2.96 (m, 2H), 3.36 (m, 2H), 3.61 (m, 1H), 3.86 (s, 3H), 4.74 (brs, 1H), 6.79 (d, 1H, J=3.6Hz), 7.62 (d, 1H, J=3.6Hz) .IR (neat): 3366,2976.4,1713,1520,1462.1,1366,1290.5,1267.3,1169,1098,752,631cm
-1. high resolution mass spec, calculated value: C
17H
25NO
5S
2: M
+Cs
+, 520.0229. measured value: 520.0240.5-[(4-N-(tertbutyloxycarbonyl)-amino-3-(dimethyl propylene diacyl) sulfo-)-butyl]-preparation of 2-thiophenecarboxylate 8:
Add 7.2 gram (52 mmole) salt of wormwood and 3.7 milliliters of (29 mmole) chloromalonic acid dimethyl esters in 200 milliliters of absolute methanol solutions of 0 ℃ of above-mentioned thioacetate 7 under 10.2 grams (26 mmole) stir.After 3 hours, mixture is poured in 500 ml waters, with ether extraction (3 * 500 milliliters).The organic layer that merges washs dry (sal epsom) with 500 ml waters and 200 milliliters of saturated nacl aqueous solutions.Removal of solvent under reduced pressure is carried out flash chromatography with thick resistates on silica gel, get the required thioether 8 of 11.46 gram (93% productive rate) faint yellow oilies with 30% ethyl acetate/hexane wash-out.
NMR (CDCl
3) δ: 1.44 (s, 9H), 1.85 and 2.00 (ABm, 2H), 3.03 (m, 3H), 3.30 (m, 2H), 3.80 (s, 6H), 3.86 (s, 3H), 5.10 (brs, 1H), 6.82 (d, 1H, J=3.8Hz), 7.63 (d, 1H, J=3.8Hz) .IR (neat): 3442,2974,2955,1734,1713,1512,1460,1435,1366,1291,1267,1167,1098,1022,752cm
-1. high resolution mass spec,
Calculated value: C
20H
29NO
8S
2: M
+Cs
+, 608.0389. measured value: 608.0370.
6-[(5-methoxycarbonyl thiophene-2-yl)-ethyl]-2-methoxycarbonyl-3-oxo-3,4,5, the preparation of 6-tetrahydrochysene-[1,4]-thiazine 9:
Add 4 milliliters of trifluoroacetic acids (TFA) in 12 milliliters of dichloromethane solutions of 0 ℃ of 470 milligrams of (0.99 mmole) above-mentioned thioether 8 under agitation.1.5 after hour, mixture is poured in 50 milliliters of saturated sodium bicarbonate solutions also with dichloromethane extraction (4 * 100 milliliters).Removal of solvent under reduced pressure is dissolved in thick resistates in 10 ml methanol.After 1.5 hours, removal of solvent under reduced pressure is carried out flash chromatography with thick resistates on silica gel in 25 ℃ of stirrings, gets 298 milligrams of required lactan 9 of (88% productive rate) colorless oil with 40% ethyl acetate/dichloromethane wash-out.
NMR (CDCl
3) δ: 1.94 (m, 2H), 3.01 (m, 2H), 3.4-3.7 (m, 4H), 3.80 and 3.82 (s, 3H), 3.87 (s, 3H), 6.25 (brs, 1H), 6.83 (d, 1H, J=3.7Hz), 7.64 (d, 1H, J=3.7Hz) .IR (KBr): 2951,1732,1669,1462,1294,1267,1194,1157,1098,1005,752cm
-1. calculated value: C
14H
17NO
5S
2: C, 48.96; H, 4.99; N, 4.08; S, 18.67. measured value: C, 49.06; H, 4.93; N, 4.09; S, 18.60.
[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6] [1,4] thiazine-6-yl)-ethyl]-2, the preparation of 5-thiophenecarboxylate 10:
At once add 1.06 gram (7.2 mmole) Tetrafluoroboric acid trimethylammonium oxygen in 150 milliliters of anhydrous methylene chloride solution of above-mentioned lactan 9 under agitation.Stirred this solution 6 hours in 25 ℃.The gained mixture is poured in 50 milliliter of 10% wet chemical, with dichloromethane extraction (3 * 200 milliliters).The organic layer that dry (sal epsom) merges, removal of solvent under reduced pressure.Products therefrom is directly used in next step.
1.58 gram (16.6 mmole) anhydrous hydrochloric acid guanidines are placed separating flask.To wherein adding 600 milliliters of anhydrous methanols.Dry argon gas was fed in the solution 10 minutes, add 926 milligrams of (17.1 mmole) anhydrous methanol sodium then.20 ml methanol solution of the thick lactam ethers of above-mentioned preparation are added in the gained mixture through sleeve pipe.Reflux solution is 20 hours under argon gas.Regulate pH value to 4, removal of solvent under reduced pressure with 1N hydrochloric acid.Thick resistates is dissolved in 500 milliliters of chloroforms, washs with 2 * 100 ml waters.The organic layer that dry (sal epsom) merges, removal of solvent under reduced pressure.Thick resistates is carried out flash chromatography on silica gel, get 413 milligrams of pale solids with 5-10% ethanol/methylene wash-out.This product is dissolved in 25 milliliters of hot methanols then, slowly cooled to 4 ℃ through 18 hours.Solid collected by filtration and 180 milligrams of required pyrimidones of (9% productive rate) faint yellow solid shape.
NMR (d
6-DMSO) δ: 1.72 (m, 1H), 1.88 (m, 1H), 2.8-3.22 (m, 4H), 3.50 (dt, 1H, J=2,8Hz), 5.99 (brs, 2H), 6.64 (brs, 1H), 6.98 (d, 1H, J=3.8Hz), 7.62 (d, 1H, J=3.8Hz), 10.02 (brs, 1H). high resolution mass spec, calculated value: C
14H
16N
4O
3S
2: M
+Na
+, 375.0562. measured value: 375.0550.
Also can be prepared as follows pyrimidone 10.Add 648 milligrams of (1.60 mmole) Lawesson ' s reagent (2,4-two (4-p-methoxy-phenyl)-1,3-two sulphur-2,4-diphosphine-2,4-disulfide) in 20 milliliters of anhydrous THF solutions of 500 milligrams of (1.46 mmole) lactan 9 under agitation.In 25 ℃ stirred the mixture 20 hours after, be poured in 50 milliliters of saturated bicarbonate solutions, with 2 * 200 milliliters of ethyl acetate extraction.The organic layer that merges washs with 50 milliliters of saturated nacl aqueous solutions, dry (sal epsom), removal of solvent under reduced pressure.Thick sulphur lactan is carried out flash chromatography on silica gel, gets 525 milligrams of corresponding sulphur lactan 11 with 8% ethyl acetate/dichloromethane wash-out:
This product is directly used in next step.
In 10 milliliters of anhydrous THF of 525 milligrams of sulphur lactan 11 and 10 milliliters of anhydrous methanol mixtures, add 1.6 milliliters of 1N sodium hydroxide.To wherein adding 0.1 milliliter of (1.6 mmole) methyl-iodide methylated sulphur lactan, needn't purifying and use it for down the step.1.39 gram (14.6 mmole) anhydrous hydrochloric acid guanidines are placed separating flask.To wherein adding 300 milliliters of anhydrous methanols.Dry argon gas is fed solution 10 minutes, add 796 milligrams of (14.7 mmole) anhydrous methanol sodium then.In the gained mixture, add the as above sulphur lactan that slightly methylates of preparation through sleeve pipe.Reflux solution is 48 hours under argon gas.Regulate pH value to 4, removal of solvent under reduced pressure with 1N hydrochloric acid.Thick resistates is dissolved in 500 milliliters of chloroforms, washs with 2 * 100 ml waters.Dry (sal epsom) organic layer, removal of solvent under reduced pressure.Thick resistates is carried out flash chromatography on silica gel, with 5-10% ethanol/methylene wash-out.Then product is dissolved in 20 milliliters of hot methanols, was cooled to 4 ℃ through 18 hours.Filtration collect 131 milligrams of (9% productive rate) required pyrimidones 10.
[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6] [1,4] thiazine-6-yl)-ethyl]-2, the preparation of 5-thiophenic acid 12:
In 180 milligrams of (0.51 mmole) pyrimidones 10, add 5 milliliters of 1N aqueous sodium hydroxide solutions.Stirred gained solution 20 hours in 25 ℃.After being cooled to 0 ℃, regulate pH value extremely with 1N hydrochloric acid.The filtering brown solid washes with water, and vacuum-drying gets 111 milligrams of (64% productive rate) filbert solid state required sour 12.NMR (d
6-DMSO) δ: 1.72 (m, 1H), 1.92 (m, 1H), 2.78-3.68 (m), 6.07 (brs, 2H), 6.68 (brs, 1H), 6.92 (d, 1H, J=3.7Hz), 7.52 (d, 1H, J=3.7Hz), 10.12 (brs, 1H), 12.89 (brs, 1H). high resolution mass spec, calculated value: C
13H
14N
4O
3S
2: M
+, 338.0507. measured value: 338.0517.
4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6] [1,4] thiazine-6-yl)-ethyl]-2, the preparation of 5-thienyl amino-L-glutamate diethyl ester 13:
Add 48 milligrams of (0.36 mmole) I-hydroxybenzotriazole hydrates, 62 microlitres (0.36 mmole) diisopropylethylamine, 86 milligrams of (0.36 mmole) diethyl glutamate hydrochlorides and 106 milligrams of (0.36 mmole) 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide methyl iodide in 5 milliliters of anhydrous DMF solutions of 110 milligrams (0.33 mmoles) above-mentioned sour 12 under agitation.Stir gained solution 20 hours down in 25 ℃ of argon gas, pour 50 ml waters then into.With 2 * 150 milliliters of ethyl acetate extraction water layers.The organic layer that merges is stripped with 3 * 50 ml waters and 10 milliliters of saturated nacl aqueous solutions, dry (sal epsom) then.Removal of solvent under reduced pressure is carried out flash chromatography with thick resistates on silica gel, get the required acid amides 13 of 120 milligrams of (71% productive rate) faint yellow amorphous solid shapes with 0-10% ethanol/methylene wash-out.NMR(d
6-Acetone)δ：1.20(m，6H)，2.2(m，1H)，2.46(t，1H，J＝7.6Hz)，2.78(s，1H)，2.82(s，1H)，2.92-3.1(m，2H)，3.41(m，1H)，3.72(dt，1H，J＝3.0，12.7Hz)，4.02-4.2(m，4H)，4.58(m，1H)，5.90(brs，1H)，6.05(brs，1H)，6.90(d，1H，8。J=3.8Hz), 7.58 (d, 1H, J=3.8Hz), 7.78 (d, 1H, J=8Hz). high resolution mass spec, calculated value: C
22H
29N
5O
6S
2: M
+H
+, 524,1638. measured values: 524.1650.
4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6] [1,4] thiazine-6-yl)-ethyl]-2, the preparation of 5-thienyl amino-L-L-glutamic acid 1:
In above-mentioned 115 milligrams (0.22 mmole) diethyl esters 13, add 3 milliliters of 1N aqueous sodium hydroxide solutions.The gained mixture was stirred 14 hours in 25 ℃.After being cooled to 0 ℃, regulate pH value to 3.5 with aqueous hydrochloric acid.Cross filter solid, wash with water, vacuum-drying and the required acid of 82 milligrams of (80% productive rate) pale solid shapes.
NMR(d
6-DMSO)δ：1.62-2.02(m，4H)，2.27(m，2H)，2.78-3.00(m，3H)，4.27(dd，1H，J＝6，6.8Hz)，6.00(brs，2H)，6.63(brs，1H)，6.88(d，1H，J＝3.7Hz)，7.63(d，1H，J＝3.7Hz)，8.37(d，1H，J＝7.4Hz)，10.05(brs，1H)，12.85(brs，2H).IR(KBr)：3371，1700，1643，1543，1345cm
-1.
High resolution mass spec: C
18H
21N
5O
6S
2: theoretical value: M
+H
+, 468.1012,
Measured value: 468.1025.Ultimate analysis: C
18H
21N
5O
6S
21.5H
2O: theoretical value: C, 43.71; H, 4.89; N14.16; S, 12.97
Measured value: C, 43.50; H4.67; N14.07; S, 12.72 biology and biological chemistry evaluation
The GAR transformylase suppresses determining of constant:
In biological chemistry 23 (1984), be used for as follows after GAR-transformylase (GARFT) analytical procedure among the 3979-3986 is modified Young etc.Reaction mixture contains people GARFT catalytic domain, 0-250nM compound 1,20uM glycinamide ribonucleotide (GAR), 10 or 20uMN
10-formyl radical-5,8-two denitrogenations mix folic acid (FDDF), 50mMHEPES-KOH (PH 7.5) and 50mMKCl.It is that 11nM starts reaction that the adding enzyme makes final concentration, and monitoring 20 ℃ of absorptions at 294nm then increases (e
294=18.9mM
-1Cm
-1).
GARFT suppresses constant (Ki) and is determined by the dependency of steady state catalytic rate to inhibitor and concentration of substrate.(Ki, app) dependence to FDDF concentration has determined the inhibition type for to the FDDF competitive inhibition to apparent Ki, is described by following formula: Ki, app=Ki+ (Ki/Km) [FDDF].The Michaelis constant of FDDF, promptly Km is determined by the dependency of catalytic rate to FDDF concentration independently.The Michaelis equation of Km and Ki data fit nonlinear method, perhaps competitive inhibition Michaelis equation.The data that analysis obtains suppressing from combining closely, with data substitution nonlinear method Morrison combine closely equation (Biochem Biophys Acta 185 (1969), 269-286) and Ki.
Clone:
Clone and the origin thereof used are listed in the table 1.The upgrowth situation of each clone and substratum require as shown in table 2.All substratum are remained on 37 ℃, in the moist insulation can of 5% air-carbonic acid gas.
Growth in vitro suppresses:
Preparation inhibitor storing solution is preserved to be used for cell culture experiments with 1 ml aliquots sample in-20 ℃ in the 10mM sodium bicarbonate aqueous solution.Measure the cell growth-inhibiting by the Mosmann method (J.Immuol.Methods 65 (1983)) after modifying.
(the Hyclone Laboratories Inc. of calf serum after containing dialysis, Logan, UT) new system RPMI growth medium (Mediatech, Washington, DC) with mid-log phase phase cell dilution to 18500 cells/ml of each clone, be divided into 2 to 12 groups of 96 hole titer plate then in.The 1st group of acellular new system substratum that is filled with equal-volume (135 milliliters) is blank to be used as.Then plate is placed 37 ℃, the insulation can of 5% air-carbonic acid gas.After 1 to 4 hour, take out plate, in 12 to 4 groups, add compound 1 (10 * final concentration, 15 milliliters/hole, bisection dilution method) then.In reverse experiment, all contain xanthoglobulin (1.75mM) or AICA (1.75mM) in all drug solutions (final concentration 175mM).Four parts of holes of containing compound 1 each concentration of preparation on every plate.15 milliliters of substratum that do not contain compound 1 are added in the hole of 1 group of plate.Then cell is put into insulation can, until finishing incubation period.The 3rd day or the 5th day of CCRF-CEM cell of L1210 and L1210/CI920 cell will be dissolved in 50 milliliters of 0.8mg/ml MTT ((4,5-dimethylthiazole-2-yl)-2,5-phenylbenzene bromination tetrazolium in the tissue culture medium (TCM); Sigma catalog no.M2128) is added in each hole of all plates, and then is put in the insulation can.After 4 hours, take out plate, centrifugal 7 minutes at 1200rpm.Sop up substratum, 150 milliliters of DMSO are added in the hole in all plates.Then in the room temperature dark place in turbine mixer slow mixed plate.Measured by metabolic MTT with metric measurement on Molecular DevicesVmax kinetics microplate in 540nm.The required drug concentrations of 50% cell growth inhibiting by the MTT metabolic determination goes up and is lower than 50%O.D. (subtracting blank) at contrast O.D. (subtracting blank) by interpolation technique to be determined.＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝
Table 1
The tissue origin and the root clone root origin L1210 ATCC# mouse that are used for the clone of in vitro study, the white blood of lymphocyte
Sick CCRF-CEM ATCC# people, acute lymphocytoblast
Leukemia=============================#ATCC=U.S. typical case's culture center=============================
Table 2
The substratum situation, plate density and the incubation time cell line culture medium DFCS plate density incubation time that use during microtitre is analyzed
Concentration (%)
*(cells/well) (my god) L1210 RPMI-1640 5 2500 3CCRF-CEM RPMI-1640 10 2500 5DFCS concentration
*Calf serum concentration after the=dialysis.＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝
Table 3
(72 hours) contact back compound 1 growth-inhibiting datumization compound GARFT Ki IC continuously
50Cell cultures IC
50Cell cultures
(nM) L1210 (nM)
aL1210 (nM)
a1 4.5 16 4.3a: average IC
50± standard deviation=============================
The anti-tumor in vivo activity:
In the time of the 0th day, with the 2mm of 6C3HED lymphosarcoma
2The trocar fragment is in subcutaneous each group of 6C3H/He female rats that is implanted to of armpit.Beginning in first day was with the 40%Encapsin solution intraperitoneal administration once a day of test compound 9 days.The control animals received either same treatment does not just have test compound.Inhibition percentage ratio shown in the following table 4 be by relatively the 11st day control group (only accepting thinner) calculate with the tumor quality of accepting the test compounds group and get.
Table 4 compound dosage (mg/kg) suppresses percentage ratio several 1 12.5 100 6/6 1 25 100 3/6 1 50 toxicity 0/6 of surviving
At the 0th day, with the 2mm of C3H/BA mammary cancer
2The trocar fragment is through subcutaneous each group of 6C3H/He female rats that is implanted to of armpit.Since first day, with the 40%Encapsin solution intraperitoneal administration once a day Ninth Heaven of test compound.Except that use-testing compound not, the processing that control animals received either is same.Suppress percentage ratio and be by relatively the 14th day control group (only accepting thinner) calculate with the tumor weight of reception test compound group and get.
The result is summarized in table 5.
Table 5 compound dosage (mg/kg) suppresses percentage and survives several 15 16 5/6 1 10 84 6/6 1 20 100 3/6
Can make various modifications and change to method of the present invention and product, this is conspicuous for those skilled in the art.
As possibility, the chemical group that this paper enumerates can be substituted.In some cases, this possibility for example replaces or unsubstituted C by enumerating
1-C
3Alkyl and become clear and definite.
In any structure formula of this paper, enumerated more than one R
6During group, each R
6All can be independently from each other the above possible group that provides.
Claims (21)
1. a compound in structural formula I or its pharmaceutically useful salt
Wherein:
A is a sulphur;
X is CH
2
Y is a sulphur;
B is a hydrogen;
C is a hydrogen; And
R
1And R
2Independent of each other is hydrogen or low alkyl group.
2. according to the compound or the salt of claim 1, wherein X is CH
2
3. according to the compound or the salt of claim 1, wherein Y is a sulphur, and B is a hydrogen, and C is a hydrogen.
4. according to the compound or the salt of claim 3, wherein A is a sulphur, and Y is a sulphur, R
1Or R
2All be hydrogen.
5. according to the compound or the salt of claim 4, wherein X is CH
2
6. according to the compound or the salt of claim 1, R wherein
1And R
2Be independently from each other hydrogen or C
1-C
6Alkyl.
7. according to the compound or the salt of claim 6, R wherein
1And R
2Be independently from each other hydrogen or C
1-C
2Alkyl.
8. according to the compound or the salt of claim 7, wherein A is a sulphur, and Y is a sulphur, and X is CH
2
9. compound according to Claim 8 or salt, wherein B is a hydrogen, C is a hydrogen, R
1And R
2All be hydrogen.
10. according to the compound of claim 1, this compound is selected from (4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-Mi Dingbing [5,4-6]-[1,4] thiazine-6-yl)-ethyl]-2,5-thiophene amino-L-L-glutamic acid) diethyl ester; 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydrochysene-3H-pyrimidine-[5,4-6] [1,4] thiazine-6-yl)-ethyl]-2,5-thiophene amino-L-L-glutamic acid.
11. a pharmaceutical composition contains:
(I) compound in structural formula I of significant quantity or its pharmaceutical salts:
Wherein:
A is a sulphur;
X is CH
2
Y is a sulphur;
B is a hydrogen;
C is a hydrogen; And
R
1And R
2Each be hydrogen or low alkyl group; With
(ii) pharmaceutically useful carrier, excipient or attenuant.
12. according to the pharmaceutical composition of claim 11, wherein X is CH
2
13. according to the pharmaceutical composition of claim 11, wherein Y is a sulphur, B is a hydrogen, and C is a hydrogen.
14. according to the pharmaceutical composition of claim 13, wherein A is a sulphur, Y is a sulphur, and X is CH
2
15. according to the pharmaceutical composition of claim 14, wherein C is a hydrogen, R
1And R
2All be hydrogen.
16. formula I compound or pharmaceutically acceptable salt thereof is used to prepare the purposes of the medicine of the growth that is used to suppress microorganism or higher organism cell or propagation,
Wherein:
A is a sulphur;
X is CH
2
Y is a sulphur;
B is a hydrogen;
C is a hydrogen; And
R
1And R
2Each be hydrogen or low alkyl group.
17. according to the purposes of claim 16, wherein X is CH
2
18. according to the purposes of claim 16, wherein Y is a sulphur, B is a hydrogen, and C is a hydrogen.
19. according to the purposes of claim 18, wherein A is a sulphur, Y is a sulphur, and X is CH
2
20. according to the purposes of claim 19, wherein C is a hydrogen, R
1And R
2All be hydrogen.
21. the compound or its salt of claim 1, wherein said compound is a following formula: compound:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94195172A CN1053448C (en) | 1994-07-28 | 1994-07-28 | Compounds useful as antiproliferative agents and GARFT inhibitors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94195172A CN1053448C (en) | 1994-07-28 | 1994-07-28 | Compounds useful as antiproliferative agents and GARFT inhibitors |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1159193A CN1159193A (en) | 1997-09-10 |
| CN1053448C true CN1053448C (en) | 2000-06-14 |
Family
ID=5039670
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN94195172A Expired - Fee Related CN1053448C (en) | 1994-07-28 | 1994-07-28 | Compounds useful as antiproliferative agents and GARFT inhibitors |
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| Country | Link |
|---|---|
| CN (1) | CN1053448C (en) |
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| CN110156858B (en) * | 2019-06-10 | 2020-08-18 | 绍兴文理学院 | Water-soluble hydrogen sulfide fluorescent probe, preparation method thereof and application thereof in detection of water quality sulfide and cell hydrogen sulfide |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1986005181A1 (en) * | 1985-03-08 | 1986-09-12 | The Trustees Of Princeton University | PYRIDO AD2,3-d BDPYRIMIDIN DERIVATIVES |
| EP0438261A2 (en) * | 1990-01-16 | 1991-07-24 | Takeda Chemical Industries, Ltd. | Condensed heterocyclic glutamic acid derivatives, their production and use |
| WO1994013295A1 (en) * | 1992-12-16 | 1994-06-23 | Agouron Pharmaceuticals, Inc. | Antiproliferative substituted 5-thiapyrimidinone and 5-selenopyrimidinone compounds |
-
1994
- 1994-07-28 CN CN94195172A patent/CN1053448C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1986005181A1 (en) * | 1985-03-08 | 1986-09-12 | The Trustees Of Princeton University | PYRIDO AD2,3-d BDPYRIMIDIN DERIVATIVES |
| EP0438261A2 (en) * | 1990-01-16 | 1991-07-24 | Takeda Chemical Industries, Ltd. | Condensed heterocyclic glutamic acid derivatives, their production and use |
| WO1994013295A1 (en) * | 1992-12-16 | 1994-06-23 | Agouron Pharmaceuticals, Inc. | Antiproliferative substituted 5-thiapyrimidinone and 5-selenopyrimidinone compounds |
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|---|---|
| CN1159193A (en) | 1997-09-10 |
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