JPH10158265A - Pyrrolonaphthyridinium derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new compound useful for diagnosing diabetes (complications), complications related to dialysis, amyloidosis, aging, diseases, etc., associated with the aging or evaluating pharmacodynamic effects of a medicine effective against the diseases, etc. SOLUTION: This compound is represented by the formula R1 to R3 are each a [(protected) amino or carboxy-substituted]alkyl} or its salt, e.g. 8- hydroxy-1,2,6,7,8,8a-hexahydro-3-(1,2,3-trihydroxypropyl)-1,4,6tris(3- carboxypropyl)- pyrrolo[2,3,4-de][1,7]naphthyridinium. The compound represented by the formula is obtained by the coexistence of an amine component represented by the formula R1 -NH2 , R2 -NH2 or R3 -NH2 with saccharides such as a hexose, an aminosugar or an oligosaccharide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel pyrrolonaphthyridinium derivative, an antibody prepared using the derivative as a hapten, and diabetes, diabetic complications, dialysis-related complications and amyloidosis using the derivative or its antibody. The present invention relates to a method for diagnosing aging, diseases associated with aging, and the like, or a method for evaluating the efficacy of drugs effective for those diseases.

[0002]

BACKGROUND OF THE INVENTION In 1968, glycosylhemoglobin (HbA1c), a small component of hemoglobin, was identified in vivo and found to increase in diabetic patients, which triggered the biological significance of the Maillard reaction, especially aging. The relationship between diabetes and diabetes has been attracting attention. The Maillard reaction is the initial stage from the formation of the Schiff base between the amino group of the protein and the aldehyde group of the reducing sugar until it undergoes Amadori transfer and stabilization, and after a longer period of reaction, it undergoes fluorescence, browning, and molecular crosslinking. And the late stage of transition to the Maillard reaction late product (AGE), which is characterized by The fluorescence, which is known as a characteristic change of AGE, is significantly higher in diabetic patients than in healthy subjects, and is a complication of diabetes, such as diabetic nephropathy, arteriosclerosis, neuropathy, retinopathy, and cataract. It has been suggested that there is a correlation with the onset of the disease. More recently, monocyte, macrophage, mesangial cells and endothelial cells have been found to have scavenger receptors that recognize AGE proteins.
A that GE recognition causes cytokine release, etc.
There are also reports suggesting a relationship between GE and pathologies such as inflammation, capillary occlusion, and arteriosclerosis. In addition, the fluorescence of proteins accumulated in serum increases during dialysis, and it has been pointed out that AGE is associated with dialysis-related amyloidosis.

[0003]

SUMMARY OF THE INVENTION There have been several types of AGEs
Candidate substances are listed, and their structures are being analyzed, but there are still many unclear points such as presence or absence in the living body, differences in immunochemical activity, and relevance to the actual disease state. Is the current situation. The present inventors have proposed important AGs having different structures besides the candidate substances reported so far.
We thought that E might exist and continued our research on AGE. As a result, we found a novel pyrrolonaphthyridinium derivative that is a triamine compound that has not been reported at all, unlike conventional monoamine and diamine compounds. Was.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel pyrrolonaphthyridinium derivative, an antibody prepared using the derivative as a hapten, and diabetes, diabetic complications and dialysis using the derivative or the antibody. An object of the present invention is to provide a method for diagnosing complications, amyloidosis, aging, diseases associated with aging, and the like, and a method for evaluating the efficacy of a drug effective for those diseases.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The novel pyrrolonaphthyridinium derivative of the present invention is a compound represented by the following general formula (I).

Embedded image [Wherein, R ₁ , R ₂ and R ₃ are the same or different and each represents an amino group, an amino group having a protecting group, and / or an alkyl group optionally having a carboxyl group. As the alkyl group for R ₁ , R ₂ and R ₃ in the general formula (I), preferably methyl, ethyl, propyl,
Isopropyl, butyl, isobutyl, sec-butyl,
t-butyl, pentyl, isopentyl, neopentyl,
Examples thereof include linear or branched alkyl groups having 1 to 6 carbon atoms such as t-pentyl, hexyl, and dimethylbutyl.

The alkyl group may have an amino group, an amino group having a protecting group and / or a carboxyl group, and the protecting group for the amino group is commonly used in the field of peptide synthesis chemistry and the like. Protecting groups are available and include, for example, acetyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
p-phenylazobenzyloxycarbonyl, p-methoxyphenylazobenzyloxycarbonyl, t-butoxycarbonyl (Boc), p-toluenesulfonyl (Tos), tertiary amyloxycarbonyl, p-biphenylisopropyloxycarbonyl, diisopropylmethyloxycarbo And groups such as nyl and formyl.

In the substance of the present invention represented by the general formula (I), a pyrrolonaphthyridinium derivative in which R ₁ , R ₂ and R ₃ are an alkyl group having an amino group and / or a carboxyl group is a hapten. It can be easily bound to a carrier protein and the like, and is particularly useful when preparing an antibody. As a carrier to be bound to a hapten to prepare an antibody, commonly used carriers such as proteins such as serum albumin and limpet blood chromoprotein and polymers such as polylysine can be used.

The pyrrolonaphthyridinium derivative of the present invention is
Includes salts represented by the general formula (I), for example, hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid, boric acid, formic acid, acetic acid, haloacetate, and propion Acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid,
Benzoic acid, cinnamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, an addition salt with an acid such as sulfanilic acid,
Examples thereof include addition salts with organic bases such as ammonia and organic amines, and salts with alkali metals such as sodium and potassium, alkaline earth metals such as calcium, magnesium and barium, and metals with aluminum and zinc. These salts can be prepared from the free pyrrolonaphthyridinium derivatives of the present invention by known methods or can be converted into each other.

The present invention also provides a compound of the present invention which has stereoisomers such as cis-trans isomers, optical isomers and conformers, or hydrates or complex compounds. Any of the stereoisomers, hydrates,
It also includes complex compounds.

Next, an example of a method for producing the compound of the present invention will be described. Compounds represented by R ₁ —NH ₂ , R ₂ —NH ₂ or R ₃ —NH ₂ (amine components: R ₁ , R ₂ and R ₃ each represent the same group as described above) include, for example, glucose, fructose, The compound of the present invention can be obtained by coexisting with saccharides such as hexoses such as galactose, mannose and deoxyglucose, amino sugars such as glucosamine and galactosamine, and oligosaccharides such as saccharose. or,
The compound of the present invention can also be obtained by carrying out a mixed reaction using a protein, a peptide or the like as an amine component, followed by acid hydrolysis treatment.

The reaction conditions such as reaction temperature, reaction time, pH and the like are not particularly set, and can be set as appropriate. The simplest thing to do is leave it at room temperature,
The reaction can be promoted by heating or the like. The obtained compound of the present invention can be purified by ordinary means such as distillation, chromatography, recrystallization and the like.

[0012]

EXAMPLES Examples of the pyrrolonaphthyridinium derivative of the present invention will be shown below, but the present invention is not limited thereto. Embodiment 1 FIG. 79.2 g of glucose and γ-aminobutyric acid (G
(ABA) 45.3 g in 250 mM phosphate buffer (pH
7.3) It was dissolved in 1100 ml and left standing at 37 ° C for 45 days. Use sulfonic acid type cation exchange resin / DIAI
The solution was added to ON PK-216 (Mitsubishi Chemical), washed with water, and the solution eluted with 2N aqueous ammonia was concentrated to dryness in a 40 ° C water bath under reduced pressure. After the dried matter was dissolved in a small amount of ion-exchanged water, it was added to an AMBERLITE XAD-2 column (organo) equilibrated with the ion-exchanged water, and a passing fraction was collected. After concentrating this fraction, it was added to a DEVELOSIL ODS LOP-45S column (Nomura Chemical) and eluted with a mixed solution of methanol and trifluoroacetic acid. Further, this was subjected to reversed-phase high-performance liquid chromatography / ST.
Separated and purified by R ODS-II column (Shimadzu Techno Research), stereoisomeric compound 1 (65 mg), compound 2
(182.9 mg) and compound 3 (35.5 mg) were each isolated.

Embodiment 2 FIG. 25.2 g of glucose and 34 g of α-acetyl lysine were added to a 250 mM phosphate buffer 700 m
l, and the same operation as in Example 1 was performed to isolate the stereoisomers of compound 4 (2 mg) and compound 5 (3 mg), respectively.

The physical properties of the obtained compound of the present invention are shown below. In the same manner as in the above example, the first or all carbon atoms were ¹³
A labeled substance was produced using glucose labeled with C and used for structural analysis. Fluorescence spectrum is 650-40 Fl
Ultraviolet absorption (UV) spectra were measured in methanol with a uorescence spectrophotometer (Hitachi) and DU-650 (Beckman). Sputtered ion mass spectrometry (SIMS) was measured using M-80B (Hitachi) using glycerin as a matrix. Nuclear magnetic resonance (NM
R) The spectrum was measured with ARX-500 (Bruker) in heavy water.
Proton has a resonance frequency of 500.13 MHz and ¹³ C has a resonance frequency of 125.77 MHz at 0.00 ppm.
¹ H-NMR spectrum and ¹³ C-NMR spectrum of assignments ¹ H- ¹ H COSY, 2-dimensional NM such HMQC
Determined by R.

Compound 1 8-hydroxy-1,2,6,7,8,8a-hexahydro-3- (1,2,3-trihydroxypropyl)-
1,4,6-tris (3-carboxypropyl) -pyrrolo [2,3,4-de] [1,7] naphthyridinium Fluorescence spectrum: EX _max = 370 nm, EM _max = 450 nm UV spectrum: λ _max = 237, 276, 360 nm SIMS: m / z 527 ¹ H-NMR (D ₂ O) -δppm: 1.79 (2H, m, H-2 "'), 2.04 (2H, m, H-
2 '), 2.13 (2H, m, H-2 "), 2.33 (2H, t, J = 7Hz, H-3"'), 2.44
(4H, t, J = 7Hz, H-3 ', 3 "), 3.35 (2H, m, H-1"'), 3.47 (1H, m,
H-1 '), 3.50 (1H, dd, J = 14,2Hz, H-7), 3.59 (1H, m, H-1'),
3.63-3.69 (4H, m, H-7,10,11,11), 4.33 (1H, m, H-1 "), 4.6
8 (1H, brs, H-8), 4.68 (1H, m, H-1 "), 4.83 (1H, d, J = 14Hz, H
-2), 4.98 (1H, d, J = 1Hz, H-8a), 5.20 (1H, d, J = 9Hz, H-9),
5.36 (1H, dd, J = 14,1Hz, H-2), 7.92 (1H, s, H-5) 13 C-NMR (D 2 O) -δppm: 22.2 (t, C-2 ', 2 "'), 27.1 (t, C-
2 "), 31.5 (t, C-3"'), 32.2 (t, C-3'), 33.3 (t, C-3 "), 4
9.4 (t, C-1 "'), 53.5 (t, C-7), 54.5 (t, C-1'), 58.6 (d, C-
8), 59.8 (t, C-2,1 "), 63.2 (t, C-11), 67.3 (d, C-8a), 6
8.6 (d, C-9), 74.6 (d, C-10), 126.8 (d, C-5), 134.6 (s, C-
2a), 135.3 (s, C-8b), 139.7 (s, C-3), 140.8 (s, C-5a), 1
78.8 (s, C-4 "'), 180.2 (s, C-4', 4")

Compound 2 8-hydroxy-1,2,6,7,8,8a-hexahydro-3- (1,2,3-trihydroxypropyl)-
1,4,6-tris (3-carboxypropyl) -pyrrolo [2,3,4-de] [1,7] naphthyridinium Fluorescence spectrum: EX _max = 370 nm, EM _max = 450 nm UV spectrum: λ _max = 240, 277, 360 nm SIMS: m / z 527 ¹ H-NMR (D ₂ O) -δppm: 1.82 (2H, m, H-2 "'), 2.06 (2H, m, H-
2 '), 2.12 (2H, m, H-2 "), 2.35 (2H, t, J = 7Hz, H-3"'), 2.46
(4H, t, J = 7Hz, H-3 ', 3 "), 3.33 (1H, ddd, J = 14,7,7Hz, H-
1 "'), 3.42 (1H, ddd, J = 14,7,7Hz, H-1"'), 3.48 (1H, m, H-
1 '), 3.49 (1H, dd, J = 14,2Hz, H-7), 3.61 (1H, m, H-1'), 3.
68 (1H, dd, J = 14,1Hz, H-7), 3.69 (1H, ddd, J = 9,3,3Hz, H-1
0), 3.73 (1H, dd, J = 3,3Hz, H-11), 3.76 (1H, dd, J = 3,3Hz, H
-11), 4.39 (1H, m, H-1 "), 4.68 (1H, d, J = 2Hz, H-8), 4.79
(1H, m, H-1 "), 4.89 (1H, d, J = 14Hz, H-2), 5.01 (1H, d, J = 2H
z, H-8a), 5.06 (1H, d, J = 14Hz, H-2), 5.21 (1H, d, J = 9Hz, H-
9), 7.95 (1H, s , H-5) 13 C-NMR (D 2 O) -δppm: 21.8 (t, C-2 "'), 22.2 (t, C-2'),
26.8 (t, C-2 "), 31.5 (t, C-3"'), 31.8 (t, C-3'), 32.0 (t,
C-3 "), 49.8 (t, C-1"'), 53.9 (t, C-7), 54.3 (t, C-1'), 5
8.8 (t, C-1 "), 60.4 (d, C-8), 61.1 (t, C-2), 63.7 (t, C-1
1), 67.7 (d, C-8a), 69.0 (d, C-9), 74.8 (d, C-10), 127.3
(d, C-5), 134.5 (s, C-2a), 135.1 (s, C-8b), 141.1 (s, C-
3,5a), 178.0 (s, C-4 ', 4 "'), 178.8 (s, C-4")

Compound 3 8-Hydroxy-1,2,6,7,8,8a-hexahydro-3- (1,2,3-trihydroxypropyl)-
1,4,6-tris (3-carboxypropyl) -pyrrolo [2,3,4-de] [1,7] naphthyridinium Fluorescence spectrum: EX _max = 373 nm, EM _max = 452 nm UV spectrum: λ _max = 239, 278, 360 nm SIMS: m / z 527 ¹ H-NMR (D ₂ O) -δppm: 1.84 (2H, m, H-2 ″ ′), 2.03 (2H, m, H-
2 '), 2.12 (2H, m, H-2 "), 2.35 (2H, t, J = 7Hz, H-3"'), 2.46
(4H, m, H-3 ', 3 "), 3.31 (1H, ddd, J = 14,7,7Hz, H-1"'), 3.3
9 (1H, ddd, J = 14,7,7Hz, H-1 "'), 3.46 (1H, dd, J = 14,1Hz, H-
7), 3.50 (1H, m, H-1 '), 3.78 (1H, dd, J = 14,3Hz, H-7), 3.7
0-3.79 (4H, m, H-10,11,11,1 '), 4.32 (2H, m, H-8,1 "), 4.7
8 (1H, m, H-1 "), 4.79 (1H, d, J = 10Hz, H-8a), 4.83 (1H, d, J =
14Hz, H-2), 5.08 (1H, d, J = 14Hz, H-2), 5.22 (1H, d, J = 9Hz,
H-9), 7.93 (1H , s, H-5) 13 C-NMR (D 2 O) -δppm: 21.8 (t, C-2 "'), 22.1 (t, C-2'),
26.6 (t, C-2 "), 31.3 (t, C-3"'), 31.6 (t, C-3'), 31.8 (t,
C-3 "), 49.6 (t, C-1"'), 54.0 (t, C-7), 55.7 (t, C-1'), 6
0.2 (t, C-2), 60.4 (t, C-1 "), 63.5 (t, C-11), 63.9 (d, C-
8), 69.0 (d, C-9), 70.4 (d, C-8a), 74.7 (d, C-10), 127.7
(d, C-5), 134.1 (s, C-2a), 135.2 (s, C-8b), 141.4 (s, C-5
a, 3), 177.7 (s, C-4 "'), 177.7 (s, C-4'), 178.8 (s, C-4")

Compound 4 8-Hydroxy-1,2,6,7,8,8a-hexahydro-3- (1,2,3-trihydroxypropyl)-
1,4,6-tris [6- (N-acetyl-L-norleucyl)]-pyrrolo [2,3,4-de] [1,7] naphthyridinium Fluorescence spectrum: EX _max = 374 nm, EM _max = 452 nm UV spectrum: λ _max = 237, 277, 360 nm SIMS: m / z 782 ¹ H-NMR (D ₂ O) -δ ppm: 1.40 (6H, m, H-3 ′, 3 ″, 3 ″ ′), 1.68 (2
H, m, H-2 "'), 1.72 (4H, m, H-2', 2"), 1.84 (6H, m, H-4 ', 4 ",
4 "'), 1.98 (3H, s, AcO''-Me), 1.99 (3H, s, AcO'-Me), 2.0
0 (3H, s, AcO "-Me), 3.41 (1H, m, H-1 '), 3.53 (2H, m, H-
1 "'), 3.61 (1H, dd, J = 14,1Hz, H-7), 3.62 (1H, m, H-1'),
3.74 (1H, dd, J = 14,1Hz, H-7), 3.79 (3H, m, H-10,11,11),
4.23 (3H, m, H-5 ', 5 ", 5"'), 4.42 (1H, m, H-1 "), 4.70 (1H,
s, H-8), 4.70 (1H, m, H-1 "), 4.89 (1H, d, J = 14Hz, H-2), 5.
07 (1H, d, J = 1Hz, H-8a), 5.22 (1H, d, J = 9Hz, H-9), 5.41 (1
H, dd, J = 14,1Hz, H-2), 7.95 (1H, s, H-5) 13 C-NMR (D 2 O) -δppm: 22.6 (q, AcO'-Me, AcO "-Me , AcO "'-
Me), 22.8 (t, C-2 '), 23.1 (t, C-2 "', 3 ', 3"'), 25.4 (t, C-
3 "), 26.0 (t, C-2"), 30.8 (t, C-4 '), 30.9 (t, C-4 "'), 3
1.3 (t, C-4 ") 49.9 (t, C-1 '), 53.4 (d, C-5', 5"'), 53.6
(d, C-5 "), 54.6 (t, C-7,1"'), 58.5 (d, C-8), 60.5 (t, C-
1 "), 60.8 (t, C-2), 63.1 (t, C-11), 67.3 (d, C-8a), 68.6
(d, C-9), 74.4 (d, C-10), 126.6 (d, C-5), 134.0 (s, C-2
a), 134.3 (s, C-8b), 140.1 (s, C-3), 140.7 (s, C-5a), 17
5.1 (s, C-6 '), 175.1 (s, C-6 "'), 175.2 (s, C-6"), 176.6
(s, AcO'-CO), 176.7 (s, AcO "'-CO), 176.8 (s, AcO" -CO)

Compound 5 8-hydroxy-1,2,6,7,8,8a-hexahydro-3- (1,2,3-trihydroxypropyl)-
1,4,6-tris [6- (N-acetyl-L-norleucyl)]-pyrrolo [2,3,4-de] [1,7] naphthyridinium Fluorescence spectrum: EX _max = 376 nm, EM _max = 452 nm UV spectrum: λ _max = 239, 278, 360 nm SIMS: m / z 782 ¹ H-NMR (D ₂ O) -δ ppm: 1.39-1.48 (6H, m, H-3 ', 3 ", 3"' ), 1.
66 (2H, m, H-2 "'), 1.73 (4H, m, H-2', 2"), 1.89 (6H, m, H-
4 ', 4 ", 4"'), 1.98 (3H, s, AcO "'-Me), 1.98 (3H, s, AcO'-M
e), 2.00 (3H, s, AcO "-Me), 3.39 (1H, m, H-1"'), 3.44 (1H,
m, H-1 "'), 3.50 (1H, m, H-1'), 3.57 (1H, dd, J = 14,2Hz, H-
7), 3.65 (1H, m, H-1 '), 3.73 (1H, dd, J = 14,1Hz, H-7), 3.8
1 (3H, m, H-10,11,11), 4.27-4.35 (4H, m, H-1 ", 5 ', 5",
5 "'), 4.75 (1H, brs, H-8), 4.78 (1H, m, H-1"), 4.94 (1H,
d, J = 14Hz, H-2), 5.08 (1H, brs, H-8a), 5.09 (1H, dd, J = 14,
1Hz, H-2), 5.20 (1H, d, J = 9Hz, H-9), 7.95 (1H, s, H-5)

[0020]

As described above, several candidate substances for AGE have been reported so far, and research is ongoing. The present inventors and their co-workers have also found pyridinium derivatives and naphthyridinium derivatives as disclosed in JP-A-6-73057 and JP-A-8-48686. Was the basic mother nucleus. The pyrrolonaphthyridinium derivative of the present invention is different from conventional compounds in that a triamine condensed tricyclic ring is used as a basic mother nucleus and has a novel structure in which there are three sites capable of cross-linking with in vivo proteins. A completely new A
It is very noteworthy as a GE candidate substance.

Therefore, similarly to the AGE compounds which have been reported so far, the compounds of the present invention are used as indicators for diabetes and diabetic nephropathy, diabetic arteriosclerosis, diabetic neuropathy, diabetic cataract, diabetic retinopathy, Diagnosis of diabetic complications such as diabetic microangiopathy and the like, aging and diseases associated therewith can be diagnosed, and in vitro and in vivo test systems can be used to evaluate drug efficacy using the compound of the present invention as an index. In addition, an antibody prepared by using the compound of the present invention as a hapten can be utilized immunochemically and immunohistochemically in the aforementioned diagnosis and evaluation of drug efficacy, and is extremely useful. As described above, the pyrrolonaphthyridinium derivative of the present invention is a novel substance which is a condensed tricyclic skeleton of a triamine which is clearly different from known AGEs. Biological activity is suggested, and different utility can be expected.

Claims (6)

[Reference number] PC-260 [Claims] 1. A pyrrolonaphthyridinium derivative represented by the general formula (I) and a salt thereof. Embedded image [Wherein, R ₁ , R ₂ and R ₃ are the same or different and each represents an amino group, an amino group having a protecting group, and / or an alkyl group optionally having a carboxyl group. ] 2. An antibody prepared using a pyrrolonaphthyridinium derivative represented by the above general formula (I) as a hapten. 3. A method for diagnosing diabetes, diabetic complications, dialysis-related complications, amyloidosis, aging, and diseases associated with aging, using the pyrrolonaphthyridinium derivative represented by the general formula (I) as an index. 4. Use of an antibody prepared using a pyrrolonaphthyridinium derivative represented by the above general formula (I) as a hapten, for the treatment of diabetes, diabetic complications, dialysis-related complications, amyloidosis, aging, and diseases associated with aging. Diagnostic method. 5. A therapeutic agent for diabetes, a therapeutic agent for diabetic complications, a therapeutic agent for dialysis-related complications, a therapeutic agent for amyloidosis, an anti-aging agent, an aging agent, using the pyrrolonaphthyridinium derivative represented by the general formula (I) as an index. For assessing the efficacy of drugs for treating diseases associated with the disease. 6. A therapeutic agent for diabetes, a therapeutic agent for diabetic complications, a therapeutic agent for dialysis-related complications, and a therapeutic agent for amyloidosis using an antibody prepared using the pyrrolonaphthyridinium derivative represented by the general formula (I) as a hapten. , Anti-aging drugs, drug efficacy evaluation method for the treatment of diseases associated with aging.