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专利原文 04生物技术赵旭刚
United States Patent 5,338,850
Kakamura ,   et al. August 16, 1994
 Pyridinium derivatives AbstractThe present invention provides novel pyridinium derivatives which are effective, for example, in diagnosis of diabetes, diabetic complications aging, diseases accompanied by aging, etc.and also provides an antibody prepared from said derivative as a hapten. The pyridinium derivatives of the present invention are novel compounds represented by the following general formulae ##STR1## wherein R and R' may be the same or different and are optional ly substituted alkyl groups, and their pharmaceutically acceptable salts##STR1## R’ It is possible to conduct the diagnosis of diabetes, diabetic complications, aging, diseases accompanied by aging, etc using a compound of the present invention as an indicator Moreover, it is possible to utilize a compound of the present invention for the evaluation of the pharmaceutical effect of pharmaceuticals effective for diabetes-related diseases, aging and diseases accompanied by aging.  
Inventors: Kakamura; Ko (Hyogo, JP), Hochi; Toshikazu (Hyogo, JP), Ienaga; Kazuhara (Hyogo, JP)
Assignee:Nippon Zoki Pharmaceutical Co., Ltd. (Osaka, JP)
Appl. No.: 08/035,619
Filed: March 23, 1993
  Foreign Application Priority Data 
     
Mar 24, 1992 [JP]  4-100394 
Jun 24, 1992 [JP]  4-191649 
     
 
Current U.S. Class:546/122 ; 436/518; 436/536; 436/547; 436/822; 530/389.8
Current International Class: C07D 471/00 (20060101); C07D 471/04 (20060101); G01N 33/68 (20060101)
Field of Search: 546/122 436/518,536,547,822 530/389.8

Other ReferencesNakamura et al., J. Chem. Soc., Chem. Communications, (14) pp. 992-994 (Jul. 1, 1992)..

Primary Examiner: Dentz; Bernard
Attorney, Agent or Firm: Fisher, Christen & Sabol

Claims



We claim:
1. A pyridinium derivative represented by formula (I) or (II) ##STR4## wherein R and R' may be the same or different and are optionally substituted alkyl groups, and pharmaceutically acceptable salts of the derivatives

2. An antibody prepared from a pyridinium derivative as claimed in claim 1 as a hapter

3. A pyridinium derivative as claimed in claim 1 wherein R and R' are the same.

4. A pyridinium derivative as claimed in claim 1 which is of formula I.
  5. A pyridinium derivative as claimed in claim 1 wherein R and R' may be the same or different and are each an alkyl group or an alkyl group substituted with an amino group, a protected amino group and/or a carboxyl group, as well as pharmaceutically acceptable salts of the derivatives.
6. A pyridinium derivative as claimed in claim 1 wherein R and R' are each an alkyl group substituted with at least one member selected from the group consisting of amino, protected amino, and carboxyl groups.
 
7. A pyridinium derivative as claimed in claim 6 wherein said alkyl group has 1 to 6 carbon atoms
 8. (3R,4S)-3,4-Dihydroxy- 5-{1-[1S,2S,3R-1,2,3,4-tetrahydroxybutyl]}-1,7-di[6-(N-acetyl-L-norleucyl)] -1,2,3,4-tetrahydro-[1,7]naphthyridinium.  9. (3R,4S)-3,4-Dihydroxy-5-{1-[(1R,2S,3R) -1,2,3,4-tetrahydroxybutyl]}-1,7-di[6-(N-acetyl-L-norleucyl)]-1,2,3,4-tetra hydro-[1,7]naphthyridinium.
10. A method for diagnosis of diabetes, diabetic complications, aging and diseases accompanied by aging comprising using a pyridinium derivative of claim 1 as an indicator

11. A method for the diagnosis of diabetes, diabetic complications, aging and diseases accompanied by aging comprising using an antibody which is prepared from a pyridinium derivative of claim 1 as a hapten.

12. A method for the evaluation of the effectiveness of pharmaceuticals as therapeutic agents for diabetes, and for diabetic complications, as preventive agents for aging, and as therapeutic agents for diseases accompanied by aging comprising using a pyridinium derivative of claim 1 as an indicator.

13. A method for the evaluation of the effectiveness of pharmaceuticals as therapeutic agents for diabetes, and for diabetic complications, as preventive agents for aging and as therapeutic agents for diseases accompanied by aging comprising using an antibody which is prepared from the pyridinium derivative of claim 1 as a hapten.

14. A method for the diagnosis of diabetes and diabetic complications comprising detecting antigens or latter-stage, fluorescent Maillard reaction type products associated with or resulting from diabetes or diabetic complications with an antibody as claimed in claim 2

15. A method for the evaluation of the effectiveness of a pharmaceutical for the treatment of diabetes or diabetic complications comprising detecting any changes caused by the treatment in the amount of antigens or latter stage fluorescent Maillard reaction type products which are associated with or result from diabetes or diabetic complications wherein said detection is achieved using an antibody of claim 2.

16. A method for the diagnosis of aging and diseases associated with aging comprising detecting antigens or latter-stage, fluorescent Maillard reaction type products associated with or resulting from aging or diseases associated with aging with an antibody as claimed in claim 2.

17. A method for the evaluation of the effectiveness of a pharmaceutical for the prevention of aging or as a therapeutic agent for diseases associated with aging comprising detecting any changes resulting from administration of said pharmaceutical in the amount of antigens or latter stage fluorescent Maillard reaction type products which are associated with or result from aging or diseases associated with aging wherein said detection is achieved using an antibody of claim 2.
 18. An antibody obtained using a pyridinium derivative as claimed in claim 1 wherein the antibody is an indicator for the diagnosis of diabetes, diabetic complications, aging or diseases accompanied by aging, and is an indicator for the evaluation of the effects of pharmaceuticals or therapeutic agents for diabetes, diabetic complications, the effects of preventative agents for aging, or the effects of therapeutic agents for diseases accompanied by aging.  19. A process for the preparation of pyridinium derivatives as claimed in claim 1 comprising reacting at least one compound of the formula R--NH.sub.2 or R'--NH.sub.2, wherein R and R' may be the same or different and are unsubstituted alkyl groups or substituted alkyl groups, with a hexose or an oligosaccharide composed of one or more hexoses, said reaction being conducted for a sufficient amount of time to obtain at least one derivative as claimed in claim 1.  

Description


FIELD OF THE INVENTION
The present invention relates to novel pyridinium derivatives; an antibody prepared from said derivative as a hapten a method for diagnosis of diabetes, diabetic complications, aging, diseases accompanied by aging, etc. using said derivative or antibody thereof; and a method for evaluation of effects of pharmaceuticals which are effective for such diabetes-related diseases, aging and diseases which accompany old age.

BACKGROUND OF THE INVENTION

In 1968, glycosylhemoglobin (HbAlc) which is one of the minor components of hemoglobin was identified in vivo and was found to increase in patients diagnosed with diabetes. This discovery has aroused interest in the biological meaning of the Maillard reaction and, particularly, in the relationship between aging and diabetes.

The Maillard reaction may be classified into a former stage and latter stage. In the former stage a Schiff base is formed by the condensation reaction between an amino group of a protein and an aldehyde group of a reducing sugar. The Schiff base is stabilized as a result of an Amadori rearrangement. In the latter stage, the former stage product undergoes a long series of reactions, to obtain the latter-stage products of the Maillard reaction. The latter stage products are characterized by fluorescence, a color change to brown, and molecular crosslinking. The fluorescence, which is known as one of the characteristic changes found in the latter-stage products of the Maillard reaction, is significantly higher in diabetic patients than in healthy or non-diabetic persons. The fluorescence is suggested to have a correlation with the onset of diabetic complications such as diabetic nephrosis, arteriosclerosis, nervous disturbance, retinal diseases, cataracts, etc.

However, basic structures for the latter-stage products of the Maillard reaction have not been clarified yet. Accordingly, there have been many ambiguous points with respect to mechanisms for the nonenzymatic saccharization and crosslinking of proteins caused by aging and diabetes.

The present inventors have conducted continued studies on the mechanisms of nonenzymatic saccharization and crosslinking of proteins and, as a result thereof, found novel pyridinium derivatives which relate to the basic structure of the latter-stage products of the Maillard reaction. The pyridinium derivatives of the present invention are useful in the diagnosis of diabetes, diabetic complications, aging, and diseases associated with aging. The pyridinium derivatives are also useful for the evaluation of the effectiveness of pharmaceuticals for treatment of diabetes, diabetes related diseases, aging and its accompanying diseases.

SUMMARY OF THE INVENTION

The present invention provides: a) novel pyridinium derivatives and salts thereof, b) an antibody prepared from said derivative as a hapten, c) a method for the diagnosis of diabetes, diabetic complications, aging, diseases accompanied by aging, etc. using said derivative, or an antigen or antibody of said derivative, and d) a method for the evaluation of the effectiveness of pharmaceuticals for treatment of diabetes, diabetes-related diseases, aging, diseases accompanied by aging, etc.

Chemical structures of the pyridinium derivatives of the present invention are believed to be related to the chemical structures of the latter-stage products of the Maillard reaction. The derivatives are useful for detecting Maillard reaction type products associated, for example, with diabetes and aging.

For example, the antibodies of the present invention may be prepared by reacting a pyridinium derivative with a carrier protein to stimulate antibody production in vivo. The antibodies thus produced may be used to detect antigens or latter-stage, fluorescent Maillard reaction type products associated with or resulting from diabetes, diabetic complications, aging, and diseases associated with aging. The pyridinium derivatives of the present invention may be used to detect antibodies associated with diabetes or aging, or diseases which accompany diabetes or aging. Evaluation of the effectiveness of pharmaceuticals or therapeutic agents for the treatment or prevention of these ailments may be performed, for example, by using the antibodies of the present invention to detect any decreases or increases in the amount of antigens or latter stage Maillard reaction type products which result from the treatment.

The pyridinium derivatives of the present invention are represented by the structural formula: ##STR2## wherein R and R' may be the same or different and are unsubstituted or substituted alkyl groups, and R" is hydrogen or a 1, 2 dihydroxyethyl group, and pharmaceutically acceptable salts thereof. The preferred substituted alkyl groups are alkyl groups substituted with at least one member selected from the group consisting of amino, protected amino, and carboxyl groups.

Derivatives of the present invention may be produced by a process comprising combining a compound of the formula R--NH.sub.2 (wherein R and R' have the same meaning as described above) with a hexose or an oligosaccharide consisting of at least one hexose to obtain a mixture, and permitting the mixture to stand for a sufficient amount of time to obtain one or more of the derivatives, and recovering the derivatives from the reaction mixture.

DETAILED DESCRIPTION OF THE INVENTION

Novel pyridinium derivatives of the present invention are the compounds represented by the following general formula (I) or (II): ##STR3## wherein R and R' may be the same or different and represent an optionally substituted alkyl group, as well as pharmaceutically acceptable salts of the derivatives.

The optionally substituted alkyl groups R and R' in the above general formula (I) and (II) may be acyclic and may have a linear or branched structure. The alkyl groups may also be cycloalkyl groups. Examples of the preferred alkyl for R and R' in the above general formula (I) or (II) are linear or branched alkyl groups or cycloalkyl groups having 1 to 6 carbon atom(s) such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl, neopentyl, tert-pentyl, cyclopentyl, hexyl and cyclohexyl groups.

The alkyl groups represented by R and R' may be substituted alkyl groups, that is they may be alkyl groups as described above which further contain one or more functional groups. Among the functional groups, amino, protected amino and/or carboxyl substituents are preferred. With regard to the protective groups for the amino group, those which are commonly used in the field of peptide synthesis, etc. may be utilized. They are for example, acetyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-phenylazobenzyloxycarbonyl, p-methoxyphenylazobenzyloxycarbonyl, tert-butoxycarbonyl, tert-amyloxycarbonyl, p-biphenylisopropyloxycarbonyl, diisopropropylmethyloxycarbonyl and formyl.

The pyridinium derivatives of the present invention include salts of the compounds represented by the general formula (I) or (II). The salts include pharmaceutically acceptable salts with at least one metal, acid, or base. Exemplary salts are alkali metal salts such as sodium, potassium and lithium salts; alkaline earth metal salts such as magnesium, calcium and barium salts; and salts of other metals such as aluminum. Further examples are addition salts with acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, citric acid, lactic acid, hydrobromic acid and trifluoroacetic acid; and salts of ammonia or of organic bases such as amines.

The salts of the present invention can be manufactured by conventional methods from the pyridinium derivatives of the present invention in free form and vice versa.

When stereoisomers such as cis-trans isomers, optical isomers and conformational isomers exist in the compounds of the present invention, the present invention includes any of the isomers.

The compounds of the present invention may be produced, for example, by reacting a compound of the formula R--NH.sub.2 or R'--NH.sub.2 (wherein R and R' are the same groups as described above) with a hexose such as glucose, fructose, galactose or mannose, or an oligosaccharide consisting of the said hexose, to obtain the compound of the present invention. There is no particular restriction for the reaction conditions such as reaction temperature, reaction time, pH, etc. For example, the reaction can be conducted at room temperature and atmospheric pressure or accelerated by heating. Temperatures up to about 40.degree. C. for about three to six weeks may, for example, be used to obtain the compounds of the present invention.

The compounds of the present invention prepared as such may be purified by common means such as distillation, chromatography, recrystallization, etc. and identified by means of NMR, mass analysis, fluorescence spectrum, etc.

The present invention is further illustrated by the following examples wherein all parts, percentages, and ratios are by weight and all temperatures are in .degree.C. unless otherwise indicated:

EXAMPLE 1

Pentylamine (25 ml) and 38.7 g of glucose were dissolved in 1 liter of 250 mM phosphate buffer (pH: 7.4) and allowed to stand at 37.degree. C. for 3 weeks.

The reaction solution was washed with 500 ml of ethyl acetate twice and the resulting aqueous layer was added to a column of Amberlite XAD-2 followed by thorough washing with water. The fraction eluted by methanol containing 10% of acetic acid was concentrated, the concentrate was dissolved in water and the solution was washed with ethyl acetate again. This was acidified with concentrated hydrochloric acid, extracted with n-butanol and the extract was added to a column of Amberlite XAD-2. Acetic acid (20%) was poured down the column and the fractions containing a fluorescent substance were combined and concentrated. This was added to a column of the reversed phase type, washed and eluted with a mixture of methanol and trifluoroacetic acid to give 1.8 g of oil. This was purified by separation by means of high performance liquid chromatography to give 450 mg of the compound 1; 350 mg of the compound 2; 50 mg of the compound 3; and 30 mg of the compound 4 separately, all identified below. Each of the compounds was converted to its hydrochloride salt.

EXAMPLE 2

.alpha.-Acetyllysine hydrochloride (44.9 g) was made to react with 36 g of glucose by allowing the mixture to stand at 37.degree. C. for 6 weeks in accordance with the same manner as used in Example 1. The reaction solution was not concentrated but just added to an ion exchange resin of the sulfonate type, thoroughly washed with water and eluted with 2N aqueous ammonia. The fraction was concentrated, separated/purified using the same resin as used in Example 1 (wherein a mixture of methanol and trifluoroacetic acid was used) and converted to hydrochlorides to afford 37 mg of a pale yellow and syrupy compound 5; 26 mg of the compound 6; and several mg of the compounds 7 and 8, all identified below.

The same procedures were conducted as above using .gamma.-aminobutyric acid and .beta.-alanine to give the compounds 9 to 17, all identified below.

EXAMPLE 3

Instead of glucose, fructose was mixed with pentylamine and the same procedures were conducted as in Example 1 above. As a result of this reaction, the compounds 1 to 4 were obtained similarly as in Example 1.

The physical properties of the resulting compounds 1 to 17 in accordance with the present invention are:

Compound1: (3R,4S)-3,4-Dihydroxy-5-{1-[(1S,2S,3R)-1,2,3,4-tetrahydroxybutyl]}-1,7-dip entyl-1,2,3,4-tetrahydro-[1,7]naphthyridinium

Fluorescence spectrum: Em=380 nm, Ex=463 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=427.

Compound2: (3R,4S)-3,4-Dihydroxy-5-{1-[(1R,2S,3R)-1,2,3,4-tetrahydroxybutyl]}-1, 7-dipentyl-1,2,3,4-tetrahydro-[1,7]naphthyridinium

Fluorescence spectrum: Em=379 nm, Ex=464 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=427.

Compound3: (3R,4S)-3,4-Dihydroxy-5-{1-[(1S)-1,2,-dihydroxyethyl]}-1,7-dipentyl-1,2,3, 4-tetrahydro-[1,7]naphthynidinium

Fluorescence spectrum: Em=380 nm, Ex=464 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=367.

Compound4: (3R,4S)-3,4-Dihydroxy-5-}1-[(1R)-1,2,-dihydroxyethyl]}-1,7-dipentyl-1,2,3, 4-tetrahydro-[1,7]naphthynidinium

Fluorescence spectrum: Em=380 nm, Ex=462 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=367

Compound5:
(3R,4S)-3,4-Dihydroxy-5-}1-[(1S, 2S, 3R-1,2,3,4-tetrahydroxybutyl]}-1,7-di[6-(N-acetyl-L-norleucyl)]-1,2,3,4-te trahydro-[1,7]naphthynidinium

Fluorescence spectrum: Em=379 nm, Ex=461 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=629

Compound6:
 (3R,4S)-3,4-Dihydroxy-5-{1-[(1R, 2S,3R)-1,2,3,4-tetrahydroxybutyl]}-1,7-di[6-(N-acetyl-L-norleucyl)]-1,2,3, 4-tetrahydro-[1,7]naphthynidinium

Fluorescence spectrum: Em=377 nm, Ex=461 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=629

Compound7:
(3R,4S)-3,4-Dihydroxy-5-{1-[(1S)-1,2,-dihydroxyethyl]}-1,7-di(6-(N-acetyl- L-norleucyl)]-1,2,3,4-tetrahydro-[1,7]naphthynidinium

Fluorescence spectrum: Em=379 nm, Ex=461 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=569

Compound8: (3R,4S)-3,4-Dihydroxy-5-{1-[(1R)-1,2,dihydroxyethyl]}-1,7-di(6-(N-acetyl-L -norleucyl)]-1,2,3,4-tetrahydro-[1,7]naphthynidinium

Fluorescence spectrum: Em=378 nm, Ex=462 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=569

Compound9: (3R,4S)-3,4-Dihydroxy-5-{1-[(1S,2S,3R)-1,2,3,4-tetrahydroxybutyl]}-1,7-di( 3-carboxypropyl)-1,2,3,4-tetrahydro-[1,7]naphthyridinium

Fluorescence spectrum: Em=380 nm, Ex=464 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=443

Compound10: (3R,4S)-3,4-Dihydroxy-5-{1-[(1R,2S,3R)-1,2,3,4,-tetrahydroxybutyl]}-1,7-di (3-carboxypropyl)-1,2,3,4-tetrahydro-[1,7]naphthyridinium

Fluorescence spectrum: Em=379 nm, Ex=463 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=443

Compound11: (3R,4S)-3,4-Dihydroxy-5-{1-[(1S)-1,2-dihydroxyethyl]}-1,7-di (3-carboxypropyl)-1,2,3,4-tetrahydro-[1,7]naphthyridinium

Fluorescence spectrum: Em=380 nm, Ex=462 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=383

Compound12: (3R,4S)-3,4-Dihydroxy-5-{1-[(1R)-1,2-dihydroxyethyl]}-1,7-di (3-carboxypropyl)-1,2,3,4-tetrahydro-[1,7]naphthyridinium

Fluorescence spectrum: Em=380 nm, Ex=463 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=383

Compound13: (3R,4S)-3,4-Dihydroxy-5-{1-[(1S,2S,3R)-1,2,3,4-tetrahydroxybutyl]}-1,7-di( 2-carboxyethyl)-1,2,3,4-tetrahydro-[1,7]naphthyridinium

Fluorescence spectrum: Em=379 nm, Ex=463 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=429

Compound14: (3R,4S)-3,4-Dihydroxy-5-{1-[(1R,2S,3R)-1,2,3,4-tetrahydroxybutyl]}-1,7-di( 2-carboxyethyl)-1,2,3,4-tetrahydro-[1,7]naphthynidinium

Fluorescence spectrum: Em=379 nm, Ex=464 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=429

Compound15: (3R,4S)-3,4-Dihydroxy-5-{1-[(1S)-1,2-dihydroxyethyl]}-1,7-di(2-carboxyethy l)-1,2,3,4-tetrahydro[1,7]naphthynidinium

Fluorescence spectrum: Em=380 nm, Ex=463 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=369

Compound16: (3R,4S)-3,4-Dihydroxy-5-{1-[(1R)-1,2-dihydroxyethyl]}-1,7-di(2-carboxyethy l)-1,2,3,4-tetrahydro-[1,7]naphthynidinium

Fluorescence spectrum: Em=378 nm, Ex=462 nm

Mass analysis (SIMS, Glycerol): M.sup.+ (m/z)=369

Compound 17:
Epimer of the compound 9 with respect to carbon atom 3.

Tables 1 to 3 show some examples of the analytical results obtained with .sup.1 H-NMR (400 MHz, CD.sub.3 OD) and .sup.13 C-NMR (100 MHz, CD.sub.3 OD). TMS was used as an internal standard and assignments in .sup.13 C-NMR were based upon the results of measurements of "C-H COSY" and "COLOC". Values in parentheses are coupling coefficients. The assignments marked with !, !!, * and ** have a possibility of replacing each other.
 

             TABLE 1 Compound 1 Compound 2
______________________________________C # .sup.13 C .sup.1 H .sup.13 C .sup.1 H ______________________________________2 49.9 t 3.37 ddd (1, 1, 13) 50.0 t 3.37 ddd (1, 1, 13) 3.68 dd (2, 13) 3.69 dd (2, 13) 3 65.6 d 4.03 ddd (1, 2, 3) 66.1 d 4.02 ddd (1, 2, 3) 4 62.8 d 4.77 dd (1, 3) 63.4 d 4.73 dd (1, 3) 4a 132.9 s 133.2 s 5 145.0 s 145.5 s 6 130.9 d 8.09 br. s 131.4 d 8.14 br. s 8 125.2 d 8.09 br. s 124.8 d 8.09 br. s 8a 144.5 s 144.6 s 9 67.3 d 5.64 br. s 67.8 d 5.53 br. s 10 75.2 d 3.50 br. d (9) 74.9 d 3.84 dd (1, 9) 11 72.9 d 3.84 m 73.6 d 3.85 m 12 65.0 t 3.68 dd (6, 12) 65.1 t 3.66 dd (5, 11) 3.84 dd (3, 12) 3.83 dd (3, 11) 1' 52.3 t 3.41 m 52.5 t 3.42 m 3.54 m 3.58 m 2' 26.6 t 1.66 m 2H 26.7 t 1.66 m 2H 3' 29.3 t 1.3.about.1.45 m 2H 29.4 t 1.3.about.1.45 m 2H 4' 23.3 t 1.3.about.1.45 m 2H 23.3 t 1.3.about.1.45 m 2H 5' 14.4 q 0.94 t (7) 3H 14.4 q 0.94 t (7) 3H 1" 63.2 t 4.49 t (7) 2H 63.2 t 4.49 t (7) 2H 2" 32.3 t 1.99 m 2H 32.4 t 2.00 m 2H 3" 30.1 t 1.3.about.1.45 m 2H 30.1 t 1.3.about.1.45 m 2H 4" 23.8 t 1.3.about.1.45 m 2H 23.8 t 1.3.about.1.45 m 2H 5" 14.6 q 0.94 t (7) 3H 14.6 q 0.95 t (7) 3H ______________________________________

TABLE 2 Compound 3 Compound 4
______________________________________C # .sup.13 C .sup.1 H .sup.13 C .sup.1 H ______________________________________2 50.0 t 3.37 ddd (1, 1, 13) 50.0 t 3.37 (1, 1, 13) 3.68 dd (2, 13) 3.66 dd (2, 13) 3 65.7 d 4.03 ddd (1, 2, 3) 65.7 d 4.05 ddd (1, 2, 3) 4 63.3 d 4.76 dd (1, 3) 63.7 d 4.69 dd (1, 3) 4a 133.8 s 134.1 s 5 144.6 s 144.6 s 6 130.0 d 8.10 br. s 130.2 d 8.12 br. s 8 125.4 d 8.09 br. s 125.1 d 8.10 br. s 8a 144.1 s 144.6 s 9 69.9 d 5.28 dd (4, 6) 69.9 d 5.18 t (5) 10 67.7 t 3.65 dd (6, 12) 67.6 t 3.79 d (5) 2H 3.78 dd (4, 12) 1' 52.3 t 3.42 m 52.4 t 3.42 m 3.55 m 3.55 m 2' 26.6 t 1.66 m 2H 26.6 t 1.66 m 2H 3' 29.3 t 1.3.about.1.45 m 2H 29.3 t 1.3.about.1.45 m 2H 4' 23.2 t 1.3.about.1.45 m 2H 23.2 t 1.3.about.1.45 m 2H 5' 14.2 q 0.93 t (7) 3H 14.2 q 0.93 t (7) 3H 1" 63.1 t 4.48 t (7) 2H 63.2 t 4.49 t (7) 2H 2" 32.3 t 1.98 m 2H 32.3 t 1.98 m 2H 3" 30.0 t 1.3.about.1.45 m 2H 30.0 t 1.3.about.1.45 m 2H 4" 23.7 t 1.3.about.1.45 m 2H 23.7 t 1.3.about.1.45 m 2H 5" 14.4 q 0.94 t (7) 3H 14.4 q 0.94 t (7) 3H ______________________________________

TABLE 3 Compound 5 Compound 6___________________________________________________________ C # .sup.13 C .sup.1 H .sup.13 C .sup.1 H ___________________________________________________________ 49.9 t 3.35 ddd (1, 1, 13) 50.1 t 3.37 (1, 1, 13) 3.69 dd (2, 13) 3.69 dd (2, 13) 3 65.7 d 4.03 ddd (1, 2, 3) 66.1 d 4.02 ddd (1, 2, 3) 4 63.7 d 4.77 dd (1, 3) 63.4 d 4.74 dd (1, 3) 4a 133.3 s 133.5 s 5 145.3 s 145.7 s 6 131.1 d 8.09 br. s 131.5 d 8.15 br. s 8 124.9 d 8.08 br. s 124.5 d 8.07 br. s 8a 144.5 s 144.6 s 9 67.3 d 5.64 br. s 67.8 d 5.53 br. s 10 75.2 d 3.51 br. d (9) 74.9 d 3.82 dd (1, 9) 11 72.7 d 3.84 m 73.5 d 3.86 m 12 64.9 t 3.70 dd (6, 12) 65.0 t 3.67 dd (5, 11) 3.84 dd (3, 12) 3.83 dd (3, 11) 1' 52.1 t 3.40 m 52.2 t 3.40 m 3.56 m 3.56 m 2' 25.9 t 1.70 m 2H 25.9 t 1.70 m 2H 3' *23.6 t 1.50 m 2H *23.6 t 1.50 m 2H 4' **32.2 t 1.7.about.2.0 m 2H **32.2 t 1.7.about.2.0 m 2H 5' 53.1 d *4.40 dd (5, 9) 53.2 d 4.40 dd (5, 9) 6' !175.1 s !175.1 s Ac--Me 22.5 q **1.96 s 3H 22.5 q 1.96 s 3H --CO !!173.3 s !!173.3 s 1" 62.9 t 4.51 t (7) 2H 63.0 t 4.51 t (7) 2H 2" 31.9 t 2.05 m 2H 31.9 t 2.05 m 2H 3" *23.9 t 1.50 m 2H *23.9 t 1.50 m 2H 4" **32.6 t 1.7.about.2.0 m 2H **32.6 t 1.7.about.2.0 m 2H 5" 53.1 d *4.42 dd (5, 9) 53.2 d 4.42 dd (5, 9) 6" !175.4 s !175.3 s Ac--Me 22.5 q **1.99 s 3H 22.5 q 1.99 s 3H --CO !!173.4 s !!173.4 s ___________________________________________________________
TABLE 4 Compound 9 Compound 10______________________________________C # .sup.13 C .sup.1 H .sup.13 C .sup.1 H_____________________________________2 50.1 t 3.40 ddd (1, 1, 13) 50.2 t 3.40 ddd (1, 1, 13) 3.70 dd (2, 13) 3.71 dd (2, 13) 3 65.9 d 4.04 ddd (1, 2, 3) 66.3 d 4.04 ddd (1, 2, 3) 4 63.1 d 4.78 dd (1, 3) 63.7 d 4.75 dd (1, 3) 4a 133.7 s 133.8 s 5 145.6 s 146.0 s 6 131.5 d 8.33 br. s 131.9 d 8.32 br. s 8 125.6 d 8.13 br. s 125.3 d 8.17 br. s 8a 144.7 s 144.8 s 9 67.6 d 5.65 d (1) 68.0 d 5.53 d (1) 10 75.5 d 3.50 dd (1, 9) 75.2 d 3.85 dd (1, 9) 11 73.0 d 3.85 m 73.7 d 3.86 m 12 65.1 t 3.71 dd (6, 12) 65.2 t 3.66 dd (5, 11) 3.84 dd (3, 12) 3.81 dd (3, 11) 1' 51.7 t 3.46 m 51.9 t 3.45 m 3.55 m 3.55 m 2' 21.5 t 1.91 m 2H 21.5 t 1.89 m 2H 3' 31.1 t 2.44 t (6) 2H 31.2 t 2.42 t (6) 2H 4' *175.7 s *175.9 s 1" 62.7 t 4.55 t (7) 2H 62.7 t 4.53 t (7) 2H 2" 27.9 t 2.30 t (7) 2H 27.9 t 2.29 t (7) 2H 3" 31.4 t 2.46 t (7) 2H 31.4 t 2.44 t (7) 2H 4" *177.2 s *177.2 s ______________________________________
TABLE 5 Compound 17 C
______________________________________
# .sup.13 C .sup.1 H
 2 50.0 t 3.43 ddd (1, 1, 13) 3.70 dd (2, 13) 3 65.9 d 4.02 ddd (1, 2, 3) 4 63.1 d 4.75 dd (1, 3) 4a 134.1 s 5 145.6 s 6 131.9 d 8.32 br. s 8 126.6 d 8.20 br. s 8a 144.3 s 9 67.5 d 5.64 d (1) 10 75.4 d 3.49 dd (1, 9) 11 73.0 d 3.84 m 12 65.2 t 3.69 dd (6, 13) 3.83 dd (3, 13) 1' 48.3 t 3.75 m 3.82 m 2' 31.8 t 2.71 m 2H 3' *173.4 s 4' 1" 58.9 t 4.75 t (7) 2H 2" 36.1 t 3.09 t (7) 2H 3" *175.4 s 4" ______________________________________

Compounds 5 and 6 of the present invention were combined with a carrier protein, for example, bovine serum albumin, keyhole limpet hemocyanin (KLH) and the like, using water-soluble carbodiimide in accordance with the usual coupling reaction to prepare an antibody using the compounds 5 and 6 as haptens. Preparation of each antibody was conducted in rabbits by a method which is commonly used. The antibody prepared as such is capable of recognizing the compound of the present invention in vitro. The antibody is also capable of immunochemically discriminating the saccharized protein prepared in vitro and the crosslinked protein in the basement membrane of the glomerulus of model rats suffering from diabetic nephrosis induced by streptozotocin.

Among the compounds of the present invention represented by the general formula (I) or (II), pyridinium derivatives wherein R and R' are alkyl having amino and/or carboxyl can be easily combined with a carrier protein, etc. as haptens and are particularly useful, for the preparation of an antibody. As to a carrier for combining with a hapten for preparation of the antibody, the commonly-used carriers, such as protein (e.g. serum albumin, KLH) and polymers (e.g. polylsine) may be used. Especially, compounds 5 and 6 of the present invention are believed to exhibit a high relationship with the latter-stage products of the Maillard reaction in terms of chemical structures and, practically, they are of very high utility.

Accordingly, it is possible to conduct diagnosis of diabetes, diabetic complications (e.g. diabetic nephrosis, diabetic arteriosclerosis, diabetic nervous disturbance, diabetic cataracts, diabetic retinal diseases, diabetic minute blood vessel disturbance, etc.), aging and diseases accompanied by aging using the compounds of the present invention as an indicator. Moreover, it is possible to conduct an evaluation of the pharmaceutical effect in in vitro and in vivo systems using a compound of the present invention as an indicator. In addition, the antibody prepared from the compound of the present invention as a hapten can be utilized immunochemically and immunohistochemically in the abovementioned diagnosis and evaluation and are very useful.

* * * * *

美国专利

5,338,850
Kakamura ,   et al. 8 16, 1994
吡啶衍生物专利翻译04生物技术赵旭刚  吡啶衍生物摘要该发明提供一种全新原创的吡啶衍生物,它在糖尿病,糖尿病并发症,衰老,和衰老伴随疾病的诊断方面非常有效,也可提供一种从所谓的吡啶衍生物作为半抗原从而制备的抗体.当前的这个全新的吡啶衍生物由下述的公式所描述: ##STR1##其中RR’可能相同也可能不同,R是个可被选择的可为烷基,也可是药物上可接受的盐.通过使用当前这个原创药物作为指示剂,该发明将使糖尿病,糖尿病并发症,衰老,和衰老伴随疾病的诊断成为可能.更为重要的是 ,可以利用该药物来评价对于糖尿病并发症,衰老,和衰老伴随疾病药理作用的药物效果。 

发明者:

Kakamura; Ko (Hyogo, JP), Hochi; Toshikazu (Hyogo, JP), Ienaga; Kazuhara (Hyogo, JP)
代理人:Nippon Zoki Pharmaceutical Co., Ltd. (Osaka, JP)
Appl. No.: 08/035,619
Filed: 3 23, 1993
 国外优先申请数据 

Mar 24, 1992 [JP]

4-100394
Jun 24, 1992 [JP]4-191649
 

Current U.S. Class:

546/122 ; 436/518; 436/536; 436/547; 436/822; 530/389.8
Current International Class: C07D 471/00 (20060101); C07D 471/04 (20060101); G01N 33/68 (20060101)
Field of Search: 546/122 436/518,536,547,822 530/389.8
 

其他参考文献

Nakamura et al., J. Chem. Soc., Chem. Communications, (14) pp. 992-994 (Jul. 1, 1992)..

第一作者: Dentz; Bernard
律师,代理人或公司: Fisher, Christen & Sabol   

Claims权力要求声明



权力要求

1.
这种嘧啶衍生物药物有公式(I) (II)给出: ##STR4##其中RR’可能相同也可能不同,R是个可被替代选择的烷基,也是药物上可接受的该衍生物盐.

2.
从该嘧啶衍生物正如我再要求1中所说的作为一种半抗原来制备一种抗体

 3.
该嘧啶衍生物 正如我再要求1中所说的其中RR’可能相同。

4..
该嘧啶衍生物 正如我再要求1中所说的满足公式1

5.
该嘧啶衍生物 正如我再要求1中所说的其中RR’可能相同也可以不同,其中每一个烷基被一个氨基,或保护性氨基或羧基或各种生理盐离子所替换。

6.
该嘧啶衍生物 正如我再要求1中所说的其中RR’是两个可替代基团,其中至少有一个被一个氨基,或保护性氨基或羧基或各种生理盐离子所替换。

7.
该嘧啶衍生物 正如我再要求6中所说的其中烷基有1-6个碳原子.

8. ((3R,4S)-3,4-
二羟-5-{1-[1S,2S,3R-1,2,3,4-四羟丁烷]} -1,7-[6-(N-乙酰-L-正(去)亮氨酸)] -1,2,3,4-四氢-[1,7]奈丙胺

9. (3R,4S)-3,4-
二羟-5-{1-[(1R,2S,3R) -1,2,3,4-四羟丁烷]}-1,7-[6-(N-乙酰-L-正(去)亮氨酸)]-1,2,3,4-四氢-[1,7]奈吡啶.

10.
通过使用声明1中的嘧啶衍生物作为一种指示剂.
该方法可用于对糖尿病,糖尿病并发症,衰老,和衰老伴随疾病诊断。

11. 通过使用声明1中的从嘧啶衍生物作为一种半抗原制备的抗体 该方法可用于对糖尿病,糖尿病并发症,衰老,和衰老伴随疾病诊断。
12.
通过使用声明1中的嘧啶衍生物作为一种指示剂。该方法可对治疗糖尿病,糖尿病并发症,衰老,和衰老伴随疾病药物的药效做出评价。

13.
通过使用声明1中的从嘧啶衍生物作为一种半抗原制备的抗体。该方法可对治疗糖尿病,糖尿病并发症,衰老,和衰老伴随疾病药物的药效做出评价。

14.
该方法用于糖尿病诊断,糖尿病并发症组成成分检测抗原,或糖尿病并发症晚期检测,荧光检测糖尿病,糖尿病并发症(依赖和声明2中的从嘧啶衍生物制备的抗体能产生特异反应)。

15.
该方法用于评价治疗糖尿病诊药物效果和由一定的剂量的抗原治疗或对反映后期典型产物引起的糖尿病并发症组成成分变化检测,该典型产物来源于糖尿病,糖尿病并发症应它可以和声明2中的从嘧啶衍生物制备的抗体能产生特异反应从而被探测。

16.
该方法用于诊断衰老,和衰老伴随疾病(通过伴随衰老组成中可检测抗原或后期反应的产物与的抗体能产生特异反应)

17.
该方法或用于对抗衰老药物的药效得评估,或作为药,剂治疗与衰老相关疾病,来检测所谓的在任何剂量上药物变化所引起疗效的改变,或通过使用声明2所提到的抗体与衰老,和衰老伴随疾病的典型物质(抗原)的反应来实现对他们的检测。

18.
该抗体由声明1中的嘧啶衍生物作为抗原来制备,作为诊断糖尿病,糖尿病并发症,衰老,和衰老伴随疾病的指示剂,也可用它来评价治疗上述疾病的药物的药效,或直接作为治疗上述疾病的药物。

19.
制备嘧啶衍生物的过程由声明1所给出:化合一种含有R—NH基团的化合物,公式中. R'--NH.亚基和R--NH.亚基. 其中 R R'可能向同也可能不同,R是个可被选择替换的烷基,例如用己糖 (或寡唐聚合成一种或几种己糖),置换反应足够多的次数即可得到声明一中所说的吡啶衍生物。  

说明


发明的领域:


  
该发明是关于吡啶衍生物和从吡啶衍生物作为半抗原制备的抗体; 和一种方法(诊断糖尿病糖尿病并发症,衰老,和衰老伴随疾病等)和一种评价治疗糖尿病相关的疾病,衰老,老年性或的疾病的方法
发明背景


  
1968年,糖基血红蛋白,它作为血红素蛋白的一小亚基,在体外中被鉴定出来并且发现它在被诊断为糖尿病的病人中含量上升,这一发现,在有重要生物学意义的Maillard反应中,唤起了人们极大的兴趣,尤其是得知它与衰老和糖尿病的关系。

  
反应可分为第一步和第二步两部分,在第一步蛋白质的氨基和还原糖的醛基发生聚合反应形成西弗碱,西弗碱重排的结果西弗碱被稳定.第二步,第一步反应产物经历一系列再反应后得到了Maillard反应的第二步产物.第二步产物被荧光说标记(与分子铰链一种颜色改变成棕色).该荧光被认为是Maillard反应第二步中的特征的变化,且在糖尿病患者中该荧光被大大增强。该荧光被证实与糖尿病相关的疾病诸如糖尿病肾炎,动脉硬化,神经异常,视网膜疾病,白内障等有关。

  
然而Maillard 反应的第二步产物的结构到目前为止尚未搞清楚.因此由糖尿病,衰老导致的非酶催化的糖基化和蛋白的铰链的反应机制在许多点上仍然存在模倰两可的地方。
  
该发明在蛋白质的无酶催化的糖基化和蛋白的铰链的反应机制方面已经进行了长期持续的研究 ,也由此导致了与Maillard 的反应的第二步产物的基本结构相关的原创的吡啶衍生物被发现. 该吡啶衍生物在糖尿病相关疾病衰老相伴疾病诊断方面非常有用。对于评价糖尿病相关疾病以及衰老相伴疾病的治疗疗效方面该吡啶衍生物同样非常有用。  发明概要 该发明所提供: a) 创新吡啶衍生物和它的盐 b)和由该衍生物作为半抗原所制备的抗体  c) 对糖尿病并发症衰老相伴疾病的一种诊断方法 d)对糖尿病并发症衰老相伴疾病的药物的药效评价方法.

   
吡啶衍生物的化学结构及Maillard 反应的第二步的产物的结构相关该衍生物对Maillard反应产物(例如在糖尿病和衰老疾病中)的检测非常有效
   
例如该发明所提供的抗体可由在免疫细胞中通过吡啶衍生物和载体蛋白一起刺激细胞产生的抗体而制备。因此可以用该抗体来检测抗原和Maillard 反应的第二步产物,而该产物是与糖尿病相关疾病和衰老相伴疾病的产物或与上述疾病相关该吡啶衍生物可以检测上述具有上述特征的抗体,该必定衍生物也可以评价药物的药理效果或者作为一个药物制剂来治疗上述疾病或者抑制上述疾病的恶化例如,通过使用上述发明的抗体来检测上述疾病治疗效果的典型Maillard反应产物或抗原在数量上的增加或减少的变化
  
该吡啶衍生物由下面结构式表示##STR2##其中 R R'可能相同也可能不同,也可能被替代物,或是替代的烷基,R’是羟基或12 二羟甲基,或生理盐离子。##STR2##优先被替代的烷基是下面这类烷基,他们至少被氨基酸,被保护的氨基酸,羧基基团这一组成员中的其中一种替换过。
   
当前该发明物所涉及的所有衍生物都可由下面步骤生产出来,即构建公式R--NH.sub.2所述的化合物(其中R R'如上所述可以相同),用一个组成中至少含一个己糖的己糖(或者由寡糖获得的己糖)经过一系列反应从而获得一个混合物。并且允许经过足够多次的反应来获得更多的衍生物,并且从反应混合物中重获衍生物。    
该发明详细描述

创新的吡啶衍生物就是由下面两个公式(1)或(2)所表示的混合物:##STR3##其中RR'可以相同也可以不同,它们也可以代表一个可被选择性替代的烷基或该衍生物的药理上可被接受的盐离子.
   
在上述公式(1)和(2)中可被随意替代的烷基R R'的结构可能是环状结构也可能是线性结构也可能是分支结构,这个烷基也可能是环状烷基,对于上述公式(1)和(2)中的R R'首选的烷基是拥有1-6C原子诸如甲基、乙基、丙基、异丙基、丁基、异丁基、环丁基,戊基、异戊基、新戊基、己基、环己基的线状的或分支的或环状的烷基。

     R
R'所代表的烷基可能被替代,也就是说他们可能包含一个或两个功能团的如上所述的烷基,在这些功能基团中,氨基酸,被保护过的氨基酸,羧基是优先替代的基团,关于这些通常在多肽合成领域所使用的氨基酸保护性基团也可能会被利用到,举例来说,如醋酸基、苄氧羧基、P-甲基苄氧羧基、P-氯苄氧羧基、P- 硝基苄氧羧基、P-苯基苄氧羧基 P-甲苯基苄氧羧基、        p-甲氧基苯基苄氧羧基, 三丁基氧羧基, 三戊基苄氧羧基,P-二联苯异丙基苄氧羧基, p-2 联苯异丙肾上腺素, 2异丙基甲基羰酰基 甲酸基.

   
该吡啶衍生物包括的盐有下面两个公式给出:(I) (II)。这些盐包括药理上可被接受的含有一个金属离子,一个酸,或者碱的盐。类似的盐有碱金属盐诸如钠盐,钾盐,锂盐;还有碱土金属盐诸如镁盐,钙盐,钡盐;进一步类似的盐有酸性盐,如盐酸盐,硫酸盐,硝酸盐,磷酸盐,蚁酸盐,乙酸盐,乳酸盐,三氟乙酸盐;和氨基酸盐,有机酸盐。
这些盐可以通过传统的方法从吡啶衍生物中制备。
   
当考虑到顺反异构、光学异构、构象异构存在于该复合物中时,该发明也把它们包含在内。
   
该发明复合物可以这样制备例如通过与公式中(R--NH.sub.2 R'--NH.sub.2)的复合物的氨基基团反应得到,用诸如葡萄糖,果糖,麦芽糖或者由寡糖合成的己糖来获得该发明复合物。对反应条件方面本反应没有严格的限制。例如反应可在室温常压下进行,通过加热可以加速反应,在40C需要36周。
   
该发明复合物可以通过常规的方法来纯化,例如蒸馏,色谱,重结晶等,通过核磁共振,质谱,荧光谱来鉴定。
  
由下面的例子可以进一步说明该发明(各部分配比百分数,质量百分数,温度,其他):
例一


   
戊胺(25 ml)、38.7g葡萄糖溶解在1250 Mm 磷酸缓冲液(PH7.4)标准37C  3
  
此反应溶液用500 ml 乙酰乙酸洗两次,将水相转移到离子交换树脂柱中,用水洗脱 ,洗脱液由含有10%的乙酸的甲醇浓缩,浓缩物溶于水再用乙酰乙酸洗涤,用浓盐酸酸化浓缩物,用N-丁醇抽提,将抽提液加入到离子交换树脂,用20%的乙酸洗脱柱子,则包含荧光的浓缩物将被浓缩。 将浓缩物转移到反向柱,用甲醇和三氟乙酸混合物洗脱得到1.8g油状物,通过高效液相色谱分离纯化得到450mg的化合物1350mg的化合物250mg的化合物330mg的化合物4,它们在下文都已被鉴定出来了。每个化合物都已被转化成他的盐酸盐。 例二
   â乙酰赖氨酸氢氯化合物(44.9 g)36g葡萄糖混合与37C反应6周,与例一的方法一样浓缩反应液将他加入到离子交换树脂,用水和2N的氨水彻底洗涤,通过用与例一(其中使用甲醇和三氟乙酸)同样的离子交换树脂来浓缩分离纯化目标混合物且使该物质转化成氢氯化合物即得到37mg的淡黄色的似糖浆的化合物526mg的化合物6,以及少许的化合物78,它们再下文已被鉴定.
  
像上面一样使用同样的过程(使用微克酪氨酸和B-丙氨酸)来产生化合物917
例三


   
用戊胺取代葡萄糖和果糖作为混合物向例一一样进行同样的操作,就像例一一样获得化合物14  1到17化合物的物理性质是  化合物1  (3R,4S)-3,4-2-5-{1-[(1S,2S,3R)-1,2,3,4-4羟基丁烷]}-1,7-2苯基-1,2,3,4-4-[1,7]奈丙氨。

荧光谱: Em=380 nm, Ex=463 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=427.

化合物 2:
(3R,4S)-3,4-2-5-{1-[(1R,2S,3R)-1,2,3,4-4羟丁烷]}-1, 7-2戊基-1,2,3,4-4-[1,7]奈丙氨

荧光谱: Em=379 nm, Ex=464 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=427.

化合物 3
: (3R,4S)-3,4-2-5-{1-[(1S)-1,2,-羟乙基]}-1,7-2戊烷-1,2,3, 4-4-[1,7]奈丙氨

荧光谱: Em=380 nm, Ex=464 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=367.

化合物 4:
 (3R,4S)-3,4-2-5-}1-[(1R)-1,2,-2羟乙烷]}-1,7-2戊烷-1,2,3, 4-4-[1,7]奈丙氨

荧光谱: Em=380 nm, Ex=462 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=367

化合物5:
 (3R,4S)-3,4-2-5-}1-[(1S, 2S, 3R-1,2,3,4-4羟丁烷]}-1,7-2[6-(N-乙酰-L-亮氨酸)]-1,2,3,4-te 3-[1,7]奈丙氨

荧光谱: Em=379 nm, Ex=461 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=629

化合物6:
(3R,4S)-3,4-2-5-{1-[(1R, 2S,3R)-1,2,3,4-4羟丁烷]}-1,7-2[6-(N-乙酰-L-亮氨酸)]-1,2,3, 4-4-[1,7]奈丙氨

荧光谱: Em=377 nm, Ex=461 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=629

化合物 7
: (3R,4S)-3,4-2 -5-{1-[(1S)-1,2,-2羟乙烷]}-1,7-2(6-(N-乙酰- L-亮氨酸)]-1,2,3,4-4 -[1,7]奈丙氨

荧光谱: Em=379 nm, Ex=461 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=569

化合物8:
(3R,4S)-3,4-2-5-{1-[(1R)-1,2,2羟乙烷]}-1,7-2 (6-(N-乙酰-L –正亮氨酸)]-1,2,3,4-4-[1,7]奈苯胺

荧光谱: Em=378 nm, Ex=462 nm

质谱(SIMS, Glycerol): M.sup.+ (m/z)=569


化合物 9:
(3R,4S)-3,4-2-5-{1-[(1S,2S,3R)-1,2,3,4-4羟丁基]}-1,7-2( 3-3羰丙缩醛)-1,2,3,4-4-[1,7]奈丙氨

荧光谱: Em=380 nm, Ex=464 nm

质谱(SIMS, Glycerol): M.sup.+ (m/z)=443

化合物 10:
 (3R,4S)-3,4-2-5-{1-[(1R,2S,3R)-1,2,3,4,-4羟丙烷]}-1,7-2 (3-羰基丙缩醛)-1,2,3,4-4 -[1,7]奈丙氨

荧光谱: Em=379 nm, Ex=463 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=443

化合物 11:
(3R,4S)-3,4-2 -5-{1-[(1S)-1,2-2羟乙烷]}-1,7-2(3-羰基丙缩醛)-1,2,3,4-4 -[1,7]奈丙氨

荧光谱: Em=380 nm, Ex=462 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=383


化合物 12
: (3R,4S)-3,4-2-5-{1-[(1R)-1,2-2强乙烷]}-1,7-2 (3-羰基丙缩醛)-1,2,3,4-4 -[1,7]奈丙氨

荧光谱: Em=380 nm, Ex=463 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=383

化合物 13:
(3R,4S)-3,4-2-5-{1-[(1S,2S,3R)-1,2,3,4-4羟丁基l]}-1,7-2( 2-羰基乙烷)-1,2,3,4-4 -[1,7]奈丙氨

荧光谱: Em=379 nm, Ex=463 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=429

化合物14:
(3R,4S)-3,4-2羟基 -5-{1-[(1R,2S,3R)-1,2,3,4-4羟丙烷]}-1,7-2( 2-羰乙烷)-1,2,3,4-4 -[1,7]奈丙氨

荧光谱: Em=379 nm, Ex=464 nm

质谱(SIMS, Glycerol): M.sup.+ (m/z)=429

化合物15
: (3R,4S)-3,4-2 -5-{1-[(1S)-1,2-4羟乙烷]}-1,7-2 (2-羰乙烷 l)-1,2,3,4-4[1,7]奈丙氨

荧光谱: Em=380 nm, Ex=463 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=369

化合物16:
 (3R,4S)-3,4-2-5-{1-[(1R)-1,2-2羟乙基]}-1,7-2(2-羰基乙烷 l)-1,2,3,4-4 -[1,7]奈丙氨

荧光谱: Em=378 nm, Ex=462 nm

质谱 (SIMS, Glycerol): M.sup.+ (m/z)=369

化合物 17
:  化合物9的差相异构体      1-3显示了由sup.1 H-NMR (400 MHz, CD.sub.3 OD) and .sup.13 C-NMR (100 MHz, CD.sub.3 OD).核磁共振谱分析一些例子的结果,TMS 被用作内部标准基于sup.13 C-NM"C-H COSY" and "COLOC的重排的分析结果。括号内的值为偶联系数。
..用!, !!表示重排, * 和 ** 又互相取代的可能性
.  表 1化合物 1化合物2_ ________________________________________________________ C # .sup.13 C .sup.1 H .sup.13 C .sup.1 H ________________________________________________________  2 49.9 t 3.37 ddd (1, 1, 13) 50.0 t 3.37 ddd (1, 1, 13) 3.68 dd (2, 13) 3.69 dd (2, 13) 3 65.6 d 4.03 ddd (1, 2, 3) 66.1 d 4.02 ddd (1, 2, 3) 4 62.8 d 4.77 dd (1, 3) 63.4 d 4.73 dd (1, 3) 4a 132.9 s 133.2 s 5 145.0 s 145.5 s 6 130.9 d 8.09 br. s 131.4 d 8.14 br. s 8 125.2 d 8.09 br. s 124.8 d 8.09 br. s 8a 144.5 s 144.6 s 9 67.3 d 5.64 br. s 67.8 d 5.53 br. s 10 75.2 d 3.50 br. d (9) 74.9 d 3.84 dd (1, 9) 11 72.9 d 3.84 m 73.6 d 3.85 m 12 65.0 t 3.68 dd (6, 12) 65.1 t 3.66 dd (5, 11) 3.84 dd (3, 12) 3.83 dd (3, 11) 1' 52.3 t 3.41 m 52.5 t 3.42 m 3.54 m 3.58 m 2' 26.6 t 1.66 m 2H 26.7 t 1.66 m 2H 3' 29.3 t 1.3.about.1.45 m 2H 29.4 t 1.3.about.1.45 m 2H 4' 23.3 t 1.3.about.1.45 m 2H 23.3 t 1.3.about.1.45 m 2H 5' 14.4 q 0.94 t (7) 3H 14.4 q 0.94 t (7) 3H 1" 63.2 t 4.49 t (7) 2H 63.2 t 4.49 t (7) 2H 2" 32.3 t 1.99 m 2H 32.4 t 2.00 m 2H 3" 30.1 t 1.3.about.1.45 m 2H 30.1 t 1.3.about.1.45 m 2H 4" 23.8 t 1.3.about.1.45 m 2H 23.8 t 1.3.about.1.45 m 2H 5" 14.6 q 0.94 t (7) 3H 14.6 q 0.95 t (7) 3H 表2 化合物3 化合物 4________________________________________________________ C # .sup.13 C .sup.1 H .sup.13 C .sup.1 H________________________________________________________ 2 50.0 t 3.37 ddd (1, 1, 13) 50.0 t 3.37 (1, 1, 13) 3.68 dd (2, 13) 3.66 dd (2, 13) 3 65.7 d 4.03 ddd (1, 2, 3) 65.7 d 4.05 ddd (1, 2, 3) 4 63.3 d 4.76 dd (1, 3) 63.7 d 4.69 dd (1, 3) 4a 133.8 s 134.1 s 5 144.6 s 144.6 s 6 130.0 d 8.10 br. s 130.2 d 8.12 br. s 8 125.4 d 8.09 br. s 125.1 d 8.10 br. s 8a 144.1 s 144.6 s 9 69.9 d 5.28 dd (4, 6) 69.9 d 5.18 t (5) 10 67.7 t 3.65 dd (6, 12) 67.6 t 3.79 d (5) 2H 3.78 dd (4, 12) 1' 52.3 t 3.42 m 52.4 t 3.42 m 3.55 m 3.55 m 2' 26.6 t 1.66 m 2H 26.6 t 1.66 m 2H 3' 29.3 t 1.3.about.1.45 m 2H 29.3 t 1.3.about.1.45 m 2H 4' 23.2 t 1.3.about.1.45 m 2H 23.2 t 1.3.about.1.45 m 2H 5' 14.2 q 0.93 t (7) 3H 14.2 q 0.93 t (7) 3H 1" 63.1 t 4.48 t (7) 2H 63.2 t 4.49 t (7) 2H 2" 32.3 t 1.98 m 2H 32.3 t 1.98 m 2H 3" 30.0 t 1.3.about.1.45 m 2H 30.0 t 1.3.about.1.45 m 2H 4" 23.7 t 1.3.about.1.45 m 2H 23.7 t 1.3.about.1.45 m 2H 5" 14.4 q 0.94 t (7) 3H 14.4 q 0.94 t (7) 3H ______________________________________


表 3化合物 5 化合物 6
________________________________________________________C # .sup.13 C .sup.1 H .sup.13 C .sup.1 H ________________________________________________________ 2 49.9 t 3.35 ddd (1, 1, 13) 50.1 t 3.37 (1, 1, 13) 3.69 dd (2, 13) 3.69 dd (2, 13) 3 65.7 d 4.03 ddd (1, 2, 3) 66.1 d 4.02 ddd (1, 2, 3) 4 63.7 d 4.77 dd (1, 3) 63.4 d 4.74 dd (1, 3) 4a 133.3 s 133.5 s 5 145.3 s 145.7 s 6 131.1 d 8.09 br. s 131.5 d 8.15 br. s 8 124.9 d 8.08 br. s 124.5 d 8.07 br. s 8a 144.5 s 144.6 s 9 67.3 d 5.64 br. s 67.8 d 5.53 br. s 10 75.2 d 3.51 br. d (9) 74.9 d 3.82 dd (1, 9) 11 72.7 d 3.84 m 73.5 d 3.86 m 12 64.9 t 3.70 dd (6, 12) 65.0 t 3.67 dd (5, 11) 3.84 dd (3, 12) 3.83 dd (3, 11) 1' 52.1 t 3.40 m 52.2 t 3.40 m 3.56 m 3.56 m 2' 25.9 t 1.70 m 2H 25.9 t 1.70 m 2H 3' *23.6 t 1.50 m 2H *23.6 t 1.50 m 2H 4' **32.2 t 1.7.about.2.0 m 2H **32.2 t 1.7.about.2.0 m 2H 5' 53.1 d *4.40 dd (5, 9) 53.2 d 4.40 dd (5, 9) 6' !175.1 s !175.1 s Ac--Me 22.5 q **1.96 s 3H 22.5 q 1.96 s 3H --CO !!173.3 s !!173.3 s 1" 62.9 t 4.51 t (7) 2H 63.0 t 4.51 t (7) 2H 2" 31.9 t 2.05 m 2H 31.9 t 2.05 m 2H 3" *23.9 t 1.50 m 2H *23.9 t 1.50 m 2H 4" **32.6 t 1.7.about.2.0 m 2H **32.6 t 1.7.about.2.0 m 2H 5" 53.1 d *4.42 dd (5, 9) 53.2 d 4.42 dd (5, 9) 6" !175.4 s !175.3 s Ac--Me 22.5 q **1.99 s 3H 22.5 q 1.99 s 3H --CO !!173.4 s !!173.4 s ________________________________________________________

 表 4化合物9 化合物10     ___________________________________                    C # .sup.13 C .sup.1 H .sup.13 C .sup.1 H ______________________________________ 2 50.1 t 3.40 ddd (1, 1, 13) 50.2 t 3.40 ddd (1, 1, 13) 3.70 dd (2, 13) 3.71 dd (2, 13) 3 65.9 d 4.04 ddd (1, 2, 3) 66.3 d 4.04 ddd (1, 2, 3) 4 63.1 d 4.78 dd (1, 3) 63.7 d 4.75 dd (1, 3) 4a 133.7 s 133.8 s 5 145.6 s 146.0 s 6 131.5 d 8.33 br. s 131.9 d 8.32 br. s 8 125.6 d 8.13 br. s 125.3 d 8.17 br. s 8a 144.7 s 144.8 s 9 67.6 d 5.65 d (1) 68.0 d 5.53 d (1) 10 75.5 d 3.50 dd (1, 9) 75.2 d 3.85 dd (1, 9) 11 73.0 d 3.85 m 73.7 d 3.86 m 12 65.1 t 3.71 dd (6, 12) 65.2 t 3.66 dd (5, 11) 3.84 dd (3, 12) 3.81 dd (3, 11) 1' 51.7 t 3.46 m 51.9 t 3.45 m 3.55 m 3.55 m 2' 21.5 t 1.91 m 2H 21.5 t 1.89 m 2H 3' 31.1 t 2.44 t (6) 2H 31.2 t 2.42 t (6) 2H 4' *175.7 s *175.9 s 1" 62.7 t 4.55 t (7) 2H 62.7 t 4.53 t (7) 2H 2" 27.9 t 2.30 t (7) 2H 27.9 t 2.29 t (7) 2H 3" 31.4 t 2.46 t (7) 2H 31.4 t 2.44 t (7) 2H 4" *177.2 s *177.2 s ______________________________________

 
表 5化合物17  
         ______________________________________                 C # .sup.13 C .sup.1 H ______________________________________                2 50.0 t 3.43 ddd (1, 1, 13) 3.70 dd (2, 13) 3 65.9 d 4.02 ddd (1, 2, 3) 4 63.1 d 4.75 dd (1, 3) 4a 134.1 s 5 145.6 s 6 131.9 d 8.32 br. s 8 126.6 d 8.20 br. s 8a 144.3 s 9 67.5 d 5.64 d (1) 10 75.4 d 3.49 dd (1, 9) 11 73.0 d 3.84 m 12 65.2 t 3.69 dd (6, 13) 3.83 dd (3, 13) 1' 48.3 t 3.75 m 3.82 m 2' 31.8 t 2.71 m 2H 3' *173.4 s 4' 1" 58.9 t 4.75 t (7) 2H 2" 36.1 t 3.09 t (7) 2H 3" *175.4 s 4" ______________________________________

   
化合物56可以和载体蛋白结合,例如小牛血清蛋白,KLHkeyhole limpet hemocyanin)和类似的蛋白。使用化合物56作为半抗原,使用水溶性糖(根据通常的偶联反应来制备抗体),通过一般的方法在野兔体内制备抗体,制备的该抗体在体外能够识别上述化合物,他也有识别糖化蛋白的免疫性,它也能够对链尿菌素诱导的糖尿病性肾炎模式老鼠的肾小球糖蛋白。
  
在由公式12所表达的发明物混合物中吡啶衍生物中R R'是带有一个氨基或者羧基的烃链,它可与载体蛋白很容易的结合作为一个半抗原并且对于抗体的制备特别有效。作为一个半抗原结合从而制备抗体的载体蛋白,通常所使用的载体蛋白即可。例如小牛血清蛋白KLH多聚体。尤其是化合物56被认为在化学结构上与Maillard 反应的第二步反应产物存在高度的相似性。
    
因此,将它作为指示剂来诊断糖尿病及其并发症(糖尿病肾炎,糖尿病神经性疾病,白内障,视网膜疾病,动脉硬化,糖尿病引起的毛细血管疾病等等),衰老并发症是有可能的。更为重要的是,将它作为指示剂在体内外用它评价药效是可能的。加之,该抗体(由该发明所提供的化合物作为半抗原所制备得到)可用于上述提到的诊断和评价中并且非常有效。   * * * * * 04生物技术班赵旭刚
 


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