JPH01272568A - Ethylenediamine compound-iron complex - Google Patents

Abstract

NEW MATERIAL:The tris[2-(2-pyridylmethyl)aminoethyl]amine-iron complex of formula I or N,N,N',N'-tetrakis(2-pyridylmethyl)-1,2-ethanediamine-iron complex of formula II. USE:Useful as an active oxygen eliminating agent. It is prepared in the form of drug preferably by including in a fat globule to effect the transfer of the drug to the desired cell part and eliminate the inflammation-causing active oxygen at the site. Concrete symptoms against which the subject compound is effective are chronic rheumatism, collagen diseases, arteriosclerosis, radiation injury, etc. PREPARATION:The compound of formula I can be produced e.g., by heating 2-pyridinocarbaldehyde and tris(2-aminoethyl)amine in argon gas stream in methanol in the absence of molecular sieve, adding sodium borohydride and forming a complex with the resultant compound and ferric chloride.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to an ethylenediamine compound-iron complex, and more particularly to an ethylenediamine compound-iron complex having an active oxygen scavenging effect and an active oxygen scavenger containing the complex as an active ingredient. Regarding.

(Prior art and problems) Active oxygen includes superoxide radical (ot21, hydroxyl radical (・011), -i-term oxygen
(IQ□) and hydrogen peroxide (11□0□), and these active oxygen species are constantly produced as intermediate metabolites of many oxidation reactions in living organisms. In particular, since these active oxygen species themselves are highly reactive active species that have both oxidation and reduction functions, they promote normal cell functions by promoting lipid peroxidation reactions and denaturing nucleic acids and enzymes. It has extremely harmful effects on maintenance and also radiation damage.

It is also known that it plays an important role in in vivo as one of the causative agents of inflammation, cancer, etc.

Among the four types of active oxygen mentioned above, hydroxyl radical (.0(l)) is particularly unstable and short-lived, but due to its violent reactivity, it is a causative agent of various toxicities. The generation of this hydroxyl radical in the living body is caused by the Fe-catalyst
zed Haber -W eiss React
ion (iron-catalyzed Haberbeis reaction).

By the way, as a method for removing active oxygen species, which are considered to be harmful substances such as those mentioned above, from within living organisms, conventional methods include the use of SOD (superoxide dismutase) enzyme or SOD-like active substance, which catalyzes the disproportionation of superoxide radical (toe, ). Various studies have been conducted on (reaction formula 〇 above)
. However, certain compounds can be used to treat various symptoms caused by reactive oxygen species, such as inflammatory diseases (chronic rheumatism, collagen disease, etc.), chronic skin diseases, arteriosclerosis, and drugs.
Although it has been applied to the treatment of cancer, radiation damage, and ultraviolet radiation damage (1-year burns), various problems have been overcome in terms of stability and formulation, and so far no products have been successfully commercialized. There isn't.

Therefore, the present inventors first studied active oxygen in the living body, especially hydroxyl radical (・011), which has the strongest reactivity.
), we believe that if we can find a substance that helps eliminate hydroxyl radicals, it could serve as an effective therapeutic agent for each of the above-mentioned diseases, and we have investigated the development of a system for eliminating hydroxyl radicals.

That is, in general, the non-catalyst −fl ar represented by the following formula (■)
ber-W eiss reaction: lI20□ + 0;
2→・Kano + old 1−+02−1111■ is a very slow reaction and is thought to have almost no meaning in living organisms.

Therefore, from the viewpoint of biological defense, the 1Gl damage caused by hydroxyl radical (leaf) generation in living organisms is due to the aforementioned Fe-
catalyzed 1] arber-W eiss
It is based on the idea that this can be achieved by removing iron in order to make the reaction a non-catalytic Harbor-Weiss reaction.

As a result, certain ethylenediamine-based compounds form metal complexes, that is, Fe"-chelate compounds in vivo, and the Fe-caLalyz described in 1j)
ed II arbcr-W ciss reaction 1(I
It was confirmed that the production of hydroxyl radicals was inhibited as a result, and it was newly discovered that these compounds could serve as promising active oxygen scavengers. No. 222005).

On the other hand, as a method for removing superoxide radical (o”a), which is a type of active oxygen species, the method described above is as follows.
An SOD enzyme is known that catalyzes the disproportionation of M (0”z) (reaction formula (1)) 1. In this case, the SOD enzyme catalyzes the disproportionation of copper,
Zinc-5OD (Cu, Zn-3OD), Manganese-
5OD (Mn-3OD) or iron-3OD (Fe
-300) and is captured in the form of a so-called metal complex. Therefore, when the ethylenediamine compound proposed by the present inventors is used as a metal complex,
Unlike the inhibition of hydroxyl radical production that the ethylenediamine compound itself has, it is thought that SOD activity exists that directly disproportions superoxide radicals.

Therefore, the present inventors generated metal complexes, particularly iron complexes, from these ethylenediamine compounds, and various S.
When the OD activity was measured, it was confirmed that it exhibited excellent effects, and as a result, the present invention was completed. Therefore, the present invention provides an ethylenediamine compound-iron complex having an active oxygen scavenging effect.

Furthermore, the present inventors have applied DDS (drug delivery system) to the actual application of these ethylenediamine compound-iron complexes, and have applied these compounds to, for example, fat microspheres (livid microspheres).
We believe that if we encapsulate it, it will be selectively transported only to desired cell sites, such as inflammation sites, and eliminate active oxygen that causes inflammation on the spot, allowing us to establish effective causative therapy. In addition, as a result, the present invention was completed.

(Structure of the Invention) That is, the present invention relates to an ethylenediamine compound-iron complex, and specifically, a tris[2-(2-pyridylmethyl)aminoethyl]amine-iron complex represented by the following formula: or N, N, N
',N'-tetrakis(2-pyridylmethyl)-1,2
-ethanediamine-iron complex.

The present invention also provides an active oxygen scavenger containing an ethylenediamine compound-iron complex represented by the formula -Ljg as an active ingredient.

The ethylenediamine compound-iron complex provided by the present invention is a novel complex that has not been described in any literature, and it was completely unknown that these compounds have an active oxygen scavenging function.

(Function) The ethylenediamine compound-iron complex provided by the present invention has the chemical formula as shown in F. .

However, the ethylenediamine compound constituting the complex, namely tris[2-(2-pyridylmethyl)aminoethyl]amine (TPAAI and N,N,
N', N'-tetrakis(2-pyridylmethyl)-1,
2-ethanediamine ('rPEN) has the following structural formulas.

(Published 'AA) (Published'
EN) And the iron complex provided by the present invention, that is, Fe
-'rl''AA and Fe-TPEN are equivalent 1:1 complexes in which 1 mol of nitrogen element and 1 mol of iron-r ion form a chelate bond in 1 mol of ethylenediamine compound, respectively. It was confirmed that (see examples below)
.

The present invention also provides an active oxygen scavenger containing at least one of these complexes as an active ingredient, and the scavenger may contain any other ingredients other than these complexes as long as they do not impede the purpose of the present invention. I can do it.

However, in consideration of active oxygen scavenging activity, it is preferable to use DDS in the formulation of the present invention.
Among these, fat emulsions are preferred. For example, these complexes are dissolved in vegetable oils such as large Q oil, sesame oil, and olive oil to create an oil-in-water microemulsion with soybean phospholipids and egg yolk phospholipids, and the emulsion is encapsulated in microspheres. It can be made into a fat emulsion.

(Example) Next, a production example of the ethylenediamine compound-iron complex of the present invention and its active oxygen scavenging action will be described.

Example 1: [) c 3” -1' P A to 13
Production of C1-! a) Concave Δ or L 2-pyridine carboxyl ahydride: 3 parts 11 and tris(2-aminoethyl)aminotris(2-aminoethyl)ami> 1 part:;l were heated in methanol for 35 hours using a molecular sieve under an argon gas stream. , a Ilj, do. Insoluble materials are removed and the solvent is distilled off. Then, the residue was dissolved in Starral, and sodium borohydride was added thereto in small portions and stirred. After the reaction was completed, the solvent was distilled off, extracted with ether, washed with water, and dried with sodium sulfate.
Mass spectrum (m/e): 420 (M'+
l) NMR spectrum (C[1C11)δ:2.8(
3H,r,), 2.95(1211,s), 4
．． N611. sl.

7.0-7.8 (911, ml, 8.5 (311,
d, d) (b) 倀? A complex was formed between ferric chloride and ferric chloride in a methanol or ethanol solvent using AA (produced in (a)). After the reaction was completed, the solvent was distilled off to obtain a black powdery complex. The formation of the complex is performed by reducing r 11 A from 0 mmol (mM) to 0.
Using 2mM and the corresponding ferric chloride from 0.2mM to OmM, a complex was formed so that the total of each was 0.2mM, and the molar ratio of the two that gave the maximum U■ absorption at 580nm was measured. . As a result, TI'AA
The complex formed from O, 1 mM and ferric chloride 0, ImM [Fe''-published) AAl 3Cl- exhibited the highest absorbance, and a stable complex was produced at a molar ratio of 1:1.

Example 2: Preparation of [Fc"-'rPEN]sony (a) Calculation of TPENq]2-chloromethylpyridine and nitrogen diamine by stirring 1) in methanol at room temperature for 5F-1 , obtained as crystals with a melting point of 110°C.

Elemental analysis value: CaallaaN.

JIW value: C near 3.55, ll: 6.65,
N:19. BO actual value: C near 3. old, ll:6
．． 66, N: 19.77Mass spectrum (m/
e): 424 (M”), 332.212 NMR spectrum (CDC1, lδ: 2.9 (4H, s), 3.
9 (8tl, sl, 7.0~7.8 (12111m
18,5+411. d, d) (b) During the production of [Fe”-TPENISO], a corresponding amount of ferrous sulfate was added at room temperature, stirred for 5 minutes, and after the reaction was completed, the solvent was distilled off, and a yellow powdery complex was obtained. In other words, TI'EN obtained in Fa) is converted to 1.O from OmM.
1.mM and corresponding ferrous sulfate (FeS04). OmM is used from omM, and the total of each is 1. om
A complex was formed so that M was obtained, and the molar ratio of the two that gave the maximum Uv absorption at 416 nm was measured. As a result, l
The complex formed from 'PEN O, 5mM and Fe5t'40.5mM showed the highest absorbance, and a stable complex was produced at a molar ratio of l:l.

Example 3: Fc-'PAA and Fe obtained in Examples 1 and 2 above
- The catalytic rate constant of TPEN in the SOD disproportionation reaction system was determined.

Measurements of rate constants were performed using a rotating disk electrode device. in 0.1M Tris buffer ipH 7,5).

s xlO-'M triphenylphosphine oxide (j), 0 to - with respect to the saturated calomel electrode (reference electrode)
1.2 ■, and the current value (il) generated at that time was measured. Mercury amalgam was used as the electrode, electric → rotational speed ω = 94.25 (rad-sec-'), sweep speed was 50 mV/sec. If id is the ammeter in the absence of Fc-TPAA or Fe-TPEN, and 1 is the current M in the presence of a certain concentration (test compound 1), a quintic equation is established. ☆: One.

Δ: constant, B: constant, ω: reaction rate constant, ω: electrode rotation speed, kinematic viscosity: I-, 30.1 under the above conditions.The obtained reaction rate constant is shown in the table.

Table 1 Rate constant Compound pfl k(M-'s-')S
Ol) 7.0 2. OX 109 Example 4
: Fe-TPAA and Fc-'rPEN
Prepare the following solutions with SOD activation.

50mM phosphate buffer (p
H7, 8) (This will be referred to as Solution A hereinafter.) 0.1mM cytochrome C-containing Δ solution 0, 5mM xanthine-containing A solution xanthine oxidase was diluted 70 times with A solution. C-containing liquid 30μ
! and xanthine content? &3OOμI, then add XO solution so that the absorbance at 550 nm changes by 0.02 per minute, and then add test solutions having the following concentrations. The inhibition rate of cytochrome C8 element by the system was determined.

To 7: (1) Fc-TPAA: 5. 10. 20 and 30uMf2) Fe-TPEN: 0.25.0.
5. 1, 0.2 and 34M In each case, a control was conducted in which no test compound was added.

The damage rate was calculated from the formula below.

The inhibition rate at each concentration was plotted and shown in Figure 1 (Fe-T
PAAI and FIG. 2 (Fc-TPEN).

As is clear from the results, at low concentrations of the test compound, the inhibition rate was recorded as a linear linear function in a dose-dependent manner. The concentration was determined as follows.

(1) Fe-TPAA: IC-, = 7.5 μM
(2) Fe-TPEN: ICF,, = 0.8
Until now, several types of complexes, mainly Cu complexes, have been known to have SOD-like activity in vitro, but based on this IC value, Fe-1'PAA, Fe-TPEN
The '1 organ was found to be one of the most active.

Example 5: Fe-TPAA and Fe-'rPEN
Influence of serum albumin on the superoxide radical dismutation activity of metal complexes In general, the complex structure of metal complexes is easily changed by proteins or various ions in the living body.
The activity observed in tro was observed in viv.

Fe-TPAA, Fe-T, which is often lost in
In order to examine whether PF and N can effectively retain their active oxygen scavenging activity in vivo, SOD activity was examined in the presence of serum albumin, a representative protein. SOD activity was determined by the xanthine-xanthine oxidase-cytochrome C method described by Moe, and ICao was used as one unit.

h me yu Prepare each of the following solutions.

50mM phosphate buffer (p
i7. l'l) (This will be referred to as Solution A hereinafter.) Solution A containing 0, ImM cytochrome C0, Solution A containing 5mM xanthine. /ml) Add A solution 27 to the cell.
Add 30μl of cytochrome C-containing solution and 300μl of xanthine-containing solution, and add 550n of XO solution.
Add so that the absorbance at m changes by 0.02 per minute. Furthermore, the test compound Fe-TPAA or F
e-TPEN is added so that the absorbance at 550 nm is 50% compared to the case where there is no change, that is, only ICaoR is added. Whether this 3011 activity is affected by live serum albumin or not, 0. Old, 0.1.0.25.0.5
and 1. The test was conducted in the presence of Omg/ml of M.

As a result, even in the presence of 1 mg/ml, Fe-TPAA
, the SOO activity of Fe-TPEN was not affected at all, while the complexes Cu(DIPS), Cu(salicylaL), which have conventionally known representative SOD activities
e) It was revealed that z lost its activity in the presence of O and 1 mg/ml of bovine serum albumin.

The details are shown in Figure 3.

Example 6: Escherichia coli (E, col) by Balacote 19
i) Fc-TPAA and Fc against proliferation I'll book
Effects of e-TPEN Balaquat, which is widely used as a herbicide, promotes the production of superoxide radicals through the oxidation-reduction reaction of baraquat radicals. It is known that the superoxide radical to:2) produced by baraquat further produces a hydroxyl radical (.DH), which exhibits high Izz properties.

Therefore, inhibiting baraquat toxicity ultimately inhibits the (J:, formation) of active oxygen.

Therefore, E inhibited by baracote toxicity.

The effect of the compound of the present invention on the growth of E. coli was observed.

・Post-cultured E. coli (10' cells/ml
) 600 u i in Vogel Bonner medium □ JgSO4・
7HzUO, 2g.

citric acid -11*02. Og,
XJIIPO410, Og.

NaN1141IPO4・411z03.5g, vi
Tamin B+21.0mg.

5.0 g of glucose (in 10,100 O each) added to 20 ml.

Each test solution having the concentration of the vestibule was added to this solution, cultured at 37°C, and growth of E. coli was observed over time.

In addition, the growth of E. coli was measured at 600 nm [J V
It was done as an absorption.

Table 2 Sample No. PQ” fmM) Fe-TP
AA (mMll 0 0 2 0.7 0 3 0.7 3 4 0.7 5 5 0.7 7 6 0.7
The change in absorbance was determined every 101 hours, and the measurement was carried out for up to 15 hours. The absorbance at 500 nm was expressed as a logarithm on the vertical axis, and time was plotted on the horizontal axis, as shown in FIG.

As is clear from the results, it is clear that Fe-TPAA significantly impairs baraquat toxicity.

(b) Fe-TPEN! '-6: Except for using each test solution with the concentration shown in Table 3 below.
d(a).

Table 3 Sample No. PQ"tμM) Fe-'rPE
N (μM)2 0.7 0 30.7100 4 0, 7 300 6 0, 7 500 Obtain the change in absorbance every hour, and continue for up to 7 hours.
As in (a), the absorbance is expressed logarithmically on the vertical axis and is shown in Figure 5.

As is clear from the results, it is clear that Fe-[PEN causes significant baraquat toxicity at low concentrations.

Example 7: Escherichia coli IE, coli due to baracote toxicity
) Effect of Fe-TPAA and Fe-TPEN on lethality As described in Example 6, superoxide radical (0τ2) produced by baraquat further generates hydroxyl radical (·01).

This shows high toxicity. Therefore, inhibiting balacote toxicity means inhibiting the production of primarily produced superoxide radicals (oral).

Therefore, the inhibitory effect of the compound of the present invention on baraquat toxicity was observed.

E. coli (IQ” cells/m cultured overnight using L method)
1) 10μm, 1.0mM containing 0.5% glucose
Add phosphate buffer (pt17.4) l Oml. To this solution, add 0.5mM of PQ'' + Balaquat and test compounds such as O, ImM and 1.OmM for Fe-'PAA, and 5.0mM for Fe-TPF and N, and incubate at 37°C for up to 60 minutes. The culture solution is sampled a total of three times every 20 minutes to measure the number of viable E. coli bacteria.

The medium for the plate used to measure the number of E. coli colonies is
Agar 2%, bacLoLrypton 1%, Na
A material containing 14% C was used.

The survival percentage of E. coli with the elapse of the slime culture time was graphed, and FIG. 6 shows the results for Fe-TPAA, and FIG. 7 shows the results for Fe-TPEN.

As is clear from the results in the figure, the complex of the present invention, Fe
-TPAA and Fe-TPEN are found to inhibit baraquat toxicity. Among them, F e -T
It is found that PAA and Fe-TPEN inhibit baraquat toxicity. Among them Fe-rl)
AΔ is lower than that of Fe-TPEN, and Baraquat 1 has a lower concentration than Fe-TPEN.
It is understood that it has a significant active oxygen scavenging effect.

Example 8: Effect of Fe-TPAA on the induction of SOO enzyme in E. coli by baraquat (7) When E. coli is grown in a nutrient-rich medium in the presence of baraquat, the toxicity of superoxide produced by baraquat causes biological damage. To protect against this, E. coli uses superoxide radicals (0
``2) is known to induce SOD that eliminates Fc-TPAA.If Fc-TPAA were to express an SOD-like effect within cells, this SOD induction would not occur.This point is explained below. We considered this.

E. coli cultured overnight in ljL (10' cells/m
l) Add 600 g of I to 20 ml of the above-mentioned Vogel Bonner medium plus yeast extract. Each test solution having the concentration of the vestibule was added to this solution, and cultured at 37°C for 8 hours. After culturing, the cells are collected, washed with 50mM phosphate buffer, and then suspended in 50mM phosphate buffer containing O, ImM ED1'A. Cell 1y- by ultrasonic cell disrupter
saLe is prepared and SOD activity is measured by the xanthine-xanthine oxidase-cytochrome C method.

Table 4 Sample No. PQ” (mM) Fe-TP
When only AA(mM)PQ'' was present, SOD was induced approximately twice as much.On the other hand, when Fe-TPAA was present at 0.
I ~1.0mM S by PO"+
No induction of OD occurred, and the enzyme level was almost unchanged from normal. This indicates that Fe-TPAA functions as a substitute for the SOD enzyme within E. coli cells.

Formulation Example: Fat emulsion 0 g of Fe-TPAA Ig was added to 20 g of Japanese Pharmacopoeia Q oil and dissolved while heating. Add to this 2.4g of purified fattening phospholipids
After adding 5 g of glycerin and stirring vigorously while heating to dissolve, appropriate clear distilled water is added and a rough emulsion is prepared using a Polyton homogenizer.

This rough emulsion was further high-pressure emulsified using a Manton-Gawarin type homogenizer, and then distilled water was added to completely crush the liquid.
00ml to obtain a fine fat emulsion.

Fat emulsions were similarly obtained by varying the content of Fe-TPAA.

Note that Fe-TPEN could also be made into a fat emulsion.

(Effects) As is clear from the descriptions below, the ethylenediamine compound-iron complex of the present invention has been shown to be effective in vitro and i.
It was found to have a good active oxygen scavenging effect in both n vivo tests, and is understood to be extremely effective in treating various diseases. An active oxygen scavenger containing the complex as an active ingredient, such as a fat emulsion as a specific example of its formulation, is an application of DDS and can be said to have great medical utility value. . It goes without saying that the preparation of the formulation is not limited to fat emulsions, and other formulations may be used.

[Brief explanation of the drawing]

FIGS. 1 and 2 are Fe-
The results of TPAA and Fe-TPEN are shown, and FIG. 3 shows the results of Fe-TPAA and Fe-TPEN in the test of Example 5.
Comparing the effects of e-TP E N with conventional compounds,
4 and 5 show the results of the complex of the present invention on the growth of E. coli in Example 6, FIGS. 6 and 7 show the survival rate of E. coli in Example 7, and FIG. FIG. 7 is a diagram showing fl[l+constant results of the current amount of the test in Example 3. Patent applicant Nippon Redary Co., Ltd. Agent Patent attorney Yukichi Odajima Patent attorney Yoji Esumi Figure 1 Figure 2 0 1 2 3 4 (μM) Fe-TP
Addition amount of EN Figure 3 0 0.5 1.0
(m9/ml) Addition amount of bovine serum albumin: Fe-TPEN Roux-4: Fe-TPAA Sea: Cu (diisopropyl salicylate) 2-o: Cu (salicylate) 2 Figure 4 Q 5 10 (hours) Incubation time ←-→: Sample No., 1 Seek: Sample No., 4 tow: Sample No., 2 b-Roux Sample No., 5--4-Sample No., 3
M: Sample No. 6--Mallet; Sample No.
7 Fig. 5 0 1 2 3 4 5 6 7 (hours) Incubation time ←-→; Sample No. 1 0-o: Sample No.
4 to 1 = Sample No. 12 Sa-ku: Sample No.
5-Ichishu; Sample No. 3 Re-→: Sample No. 6 Fig. 6 0 20 40 60 (min) Incubation time o-+): Control to: PQ”0.5mM Fig. 7 0204060 (min) Incubation time o-O: Control to (:PQ”0.5mM+Fe-TPEN5.OmM
←−→:PQ”0.5mM

Claims (1)

[Claims] 1) Tris[2-(2-pyridylmethyl)aminoethyl]amine-iron complex represented by the following formula: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or ▲ Numerical formulas, chemical formulas, tables, etc. There is an N,N,N',N'-tetrakis(2-pyridylmethyl)-1,2-ethanediamine-iron complex shown by ▼. 2) An active oxygen scavenger containing the complex according to claim 1 as an active ingredient.