JPS61186385A - Deuteroporphyrin derivative and its salt - Google Patents

Abstract

NEW MATERIAL:A compound of the formula (either of R1 and R2 is hydro xyethyl and the other is vinyl) and its salt. EXAMPLE:3-Hydroxyethyl-8-vinyldeuteroporphyrin (HVD). USE:A photosensitizer in diagnosis and therapy for cancers with laser. It has affinity for tumors. PREPARATION:For example, disodium protoporphyrin is treated with a halogen acid, preferably hydrochloric acid usually under stirring for 1hr to give a mixture of HVD and a product obtained by addition of water to the vinyl group in the 8-position (VHD). The mixture is esterified using BF3 as a Lewis acid and methanol and subjected to silica-gel chromatography to effect separation and purification. Then, the product is desterified, converted into sodium salt, further, when needed, neutralized.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a novel deuteroporphyrin derivative, more specifically, a derivative of the following general formula (I) (wherein one of turtle and R7 is -CH-0R). , the other is −
The present invention relates to a deuteroborphyrin derivative represented by C)) CH, CH, and a salt thereof.

[Conventional technology]

In recent years, lasers have been actively applied to medical treatments.
The progress has been particularly remarkable in the treatment and diagnosis of cancer.

Among them, Dougherty et al. have achieved great results in the early detection and treatment of cancer by combining hematoporphyrin derivatives (HPD) and argon dye laser. That is, by utilizing the degree of penetration and orthogonality of the laser into living tissue, a photosensitizer with tumor affinity such as hematoporphyrin derivatives is administered in advance and accumulated in the tumor.
The presence or absence of a tumor is diagnosed by laser irradiation, and the site is selectively destroyed using a low-energy argon dye laser.

Treatment of cancer by administering photosensitizers in this way has been carried out in ultraviolet or radiation therapy for a long time, but in recent years, with the advent of lasers, the development of laser photosensitizers with tumor affinity. has been in the spotlight.

D. Kesael et al. investigated the tumor affinity and laser photosensitization effect of various porphyrin compounds, and provided hematoporphyrin (HP). However, HP is D
The Porphyrins (Tb, ePor) by Olphin
As shown in Phyrins l J vol. 17, published by Kavmic Press, 1978, pp. 297-298, it is difficult to obtain pure products using conventional methods, and in the aforementioned study by Dougherty et al. The HPD used is also a mixture of hematoporphyrin-like substances. This has been a major obstacle in conducting basic research and in applying it to clinical practice.

Recently, many studies have been reported on the isolation of each component of HPD and the accumulation of these components in cancer cells (Da
vid Kesael, Thomas J, Dong
herty: rPorphyrin Photosensitization
initiation) J]. As seen in this document, HPD is produced by reacting HP with sulfuric acid in acetic acid, and most of the product is thought to be acetylated, but HPD is prepared by treating it with a strong alkali before administering it. Its active ingredient is H.
3-hydroxyethyl-8-vinyl deuteroporphyrin (HVD) or 3-hydroxyethyl-8-vinyl deuteroporphyrin (HVD) in which one hydrooxyethyl group of P becomes a lyhinyl group by dehydration reaction with sulfuric acid.
- Vinyl-8-hydroxyethyl deuteroporphyrin (VHD).

[Problem that the invention seeks to solve]

However, to date, there has been no example of isolation of HVD or VHD, and needless to say, N
Physical property data such as MR have also not been reported. Therefore, it has not been determined which of the 3- and 8-position hydroxyethyl groups reacted in the dehydration reaction of HP, that is, whether the product was HVD or VHD.

As described above, the fact that HPD is a mixture of several types of porphyrin compounds with undetermined structures and that its active ingredients have not been identified has been a major obstacle in conducting research and development in this field.

[Means for solving problems]

The present inventor has conducted intensive research to overcome these problems, and has developed a method for treating liver function using protoporphyrin disodium, which is manufactured in large quantities with high purity and is easily available, as a raw material. As a result of a detailed study of the reaction conditions, we succeeded in isolating selective KHVD, VHD, or their salts.
We were able to clarify the physical properties of Furthermore, as a result of evaluating the effects at the cellular level using these specified IVD, VHD, and IJ salts, it was found that they have excellent photosensitizing action, and the present invention was completed.

Therefore, the present invention provides a deuteroporphyrin derivative represented by formula (1) and a salt thereof useful as a photosensitizer.

The deuteroporphyrin derivative or salt thereof of the present invention is
A pure protoporphyrin salt is treated with a halogenated acid and subjected to an addition reaction of water to produce a mixture of HVD and VHD, which is then separated and purified as an ester.
It is produced by deesterification and sodification, and further subjecting it to a neutralization step if necessary.

As the halogenated acid, common hydrochloric acid is preferably used, and the reaction is carried out by mixing and stirring for about 1 hour. When doing this, the reaction product is obtained as a mixture of 7'jI (VD) by adding water to the vinyl group at the 3-position and 9VHD by adding water to the vinyl group at the 8-position. depends largely on the acid concentration, and is shown in Figure 2 below.
As shown in Figure 4, the reaction with 35-thihydrochloric acid is very slow;
On the other hand, with 40% hydrochloric acid, the raw materials disappear quickly, but HP, which is a product of further reaction, is observed to be produced. The water utilization reaction is completed by adding the reaction solution to an aqueous solution of caustic soda and neutralizing it. After neutralization, the precipitate is collected in a furnace, washed with water, and then dried under reduced pressure.

In order to separate the hydration reaction product into each component, there is a method of converting it into an ester form and then subjecting it to chromatographic separation.

used. However, under general esterification conditions using methanol and hydrochloric acid, etc., metathurisis occurs simultaneously with the esterification of carboxylic acid, and the generated hydroxyethyl group is converted into a methoxyethyl group. Therefore, the present inventor selected BF as the Lewis acid and attempted esterification, and found that no conversion to a methoxyethyl group occurred and only the desired ester derivative of the carboxylic acid was obtained. After esterification is carried out in this manner, chromatographic separation is carried out. The dimethyl ester of protoporphyrin, which is the raw material, can be easily separated by fractionation using a solvent. HP dimethyl ester has similar physical properties to HVD dimethyl ester and vHD dimethyl ester, and separation using silica gel chromatography is required to separate these mixtures.

After making the water-use reaction product into an ester with BF3 and methanol using the above method, this was subjected to thin-layer chromatography or high-performance liquid chromatography to test the product ratio, and the hydrochloric acid concentration in the water-use reaction was 36%. When the reaction temperature is 20°C, the raw protoporphyrin salt will attenuate to 50 ts or less in 1 hour, and the generation of HP due to excessive reaction can be suppressed to 5 ts or less, so these reaction conditions are suitable. It has been found.

Nine HVD dimethyl esters, V
HD dimethyl ester can be converted into HVD disodium or VHD disodium by further deesterification and sodium conversion. By further subjecting this sodium salt to a neutralization step, the desired deuteroborphyrin derivative CI) can be obtained.

[Effect]

The deuteroborphyrin derivative (1) or a salt thereof of the present invention has tumor affinity and photosensitizing action.

〔Effect of the invention〕

As stated in the paper, according to the present invention, HVD%V)ID or a salt thereof can be obtained as a simple substance. This can be achieved by selecting specific reaction conditions, i.e. by using a specific concentration of hydrochloric acid in the water utilization reaction of protoporphyrin, and by also performing esterification in the separation step of the product by a combination of BF and methanol. became. Furthermore, H.V.D.
By isolating , VHD, etc., it has become clear that HVD disodium has a higher photosensitizing effect than HPD, as shown in Table 1 below.

〔Example〕

Next, the present invention will be explained with reference to Examples.

Example 1 (1) High purity protoporphyrin disodium 1.0?
was suspended in 50°C of water, added to 450R1 of 40% hydrochloric acid prepared separately, and stirred at 20°C for 1 hour. After the reaction,
Immediate reaction solution 5% NaOH aqueous solution 3.9! and further add an aqueous solution of sodium 5-thiacetate to adjust the pH to 3. At this time, a precipitate is deposited, which is collected in F, washed with water, and then dried under reduced pressure. In this way, 4 reaction products were obtained.
It is dissolved in a mixed solution of ether solution 3- containing 7% BF and etherate and methanol 100-, and after reacting at 40°C for 1 hour, triethylamine is added to neutralize. Add dichloromethane to the resulting neutralized solution, transfer to a separating funnel, add water and shake to mix. After washing with water three times, separate the dichloromethane layer, dehydrate it with sodium sulfate, add the same amount of methanol, and gradually distill off the dichloromethane using an evaporator. When crystals begin to precipitate, distill off. Stop and let it cool. The precipitated crystals were removed and protoporphyrin dimethyl ester 0.4? was recovered. On the other hand, the p solution was subjected to the next reaction.

(The solvent of the filtrate obtained in IO (I) is distilled off using an evaporator, and the residue is dissolved in dichloromethane and subjected to silica gel chromatography (WAKOGEL C-200, 501). The chromatography is divided into four fractions. The fraction is protoporphyrin dimethyl ester. The second fraction (confirmed to be single by silica gel thin layer chromatography test) was collected, the solvent was distilled off, and the residue was recrystallized from toluene. 0.11? (yield 10.7) of HVD dimethyl ester was obtained.

Elemental analysis value CsaH4ON40a (MW=eo&'tx
>Calculated value (chi) C near 1.03. H:6,62. N
:9.21 actual value (chi) C near 0.71. H:6.5
5. N:9.11Mass:Ca1cd 608
Found 609 (M+11NMR (400MHz
in CD (Js l Ppmlo, 48 (
IH,s, 5-CHllo, 17(IH,a, 1O-
CH) 10.04 (IH, s, 2O-CH) 10.02 (IH, s, 15-CH) 8.32.8.29.8.27.8.24 (IH, d
d, 8”′-CI(+, 6.53.6.51 (I
H, d, 3l-C) ()6.39.6.35 (IH
,d, 8l-CH,)6.20.6.17 (IH,
d, s”-CH,)4.39.438.4.37 (
4H, t, 13", 17'-C horse) 3.69 (
3H,s, 7-CH8)3.66 (3H,a, 2
-CH31&66 (6H,s, 13", 17" -varnish fru0CH3) 3.62 (6H,s, 12
CHs, 18 CHs) 3.29. λ27. &
25 (4H, t, 13", 1'7"-CH,)!
25.2.23 (3H, d, 3”-CH,)-3,
94 (2H, s, N-H) (...) The third fraction of silica gel chromatography (confirmed to be single by silica gel thin layer chromatography test) was collected, the solvent was distilled off, and the residue was Recrystallize from toluene to obtain VHD dimethyl ester. IP (yield 9.7) was obtained.

Elemental analysis value Cs5H4ON<Os Calculated value (%) C near 1.03. H:6.62. N:
9.21 Actual value (chi) C near 0.69. H:6.4
3. N:8.97Mass: Calcd 608
Found 609NMR (400MHz in
CDCl2. ) ppm10.11 (IH,s,
5-CH)9.95 (II(,s, 1O-CIH
)9.87 (LH,s, 2O-CH)9.86 (
LH,s, 15-CH)8.21. 8.18. 8
．． 16. 8.14 (IH, dd, 3rd Emperor-CH
J6.32.6.28 (IH, d, 3”-OHri6
．． 15.6.14 (IH, d, 3m-OH,)6.
13.6.12 (IH, d, 8l-OH-14,3
2,4,30(2H,t, 13'-Ca-)4.29
．． 4.28 (2H, t, 17'-C horse) 3.71
(3H,s, 7-CH,)3.68 (3H,s,
2-CH,)3.66 (6H,s, 13", 17"
-z Stell0CHs ) 3.50 (3H,s, l2
-CH,)3.38 (3H,s, 15-aH3)3
．． 24.3.22.3.20 (4H,t, 13jL
, 17"-0 formula)% formula%) (Iv) The fourth fraction of silica gel chromatography (confirmed to be single by silica gel thin layer chromatography test) was collected, the solvent was distilled off, and the residue was Recrystallize from toluene to obtain HP dimethyl ester 0.02? (yield 1.8)
I got it.

Elemental analysis value Cyo formula t N4 os Calculated value (%) C: 68.99. H: 6.76, N:
8.94 actual value (ch) C: 68.41. H:6.8
2. N: 8.77NMR (400MHz, in CD
CA3) ppm10.11 (IH, d, 5-C
H) 10.02 (IH, d, 1O-CH) 9.75
(2H,d, 2O-CH,15CH16,12(I
H, q, 3'-CH) 6.03 (IH, q, 8'-CH) 4.21 (
2H,t, 13凰-CHt14.14(2H,t,
17 凰-CHt) 3.67 (6H, m, 13”
, 17”-ester OCH,)3.42 (6H,
s, 2-0M3.7-CH,)3.31 (3H,d
d, l2-CH,) 3.26 (3H, dd, l8-CH
, ) 3.18 (4H, tt, 13", 17"-CH,
) Example 2 High purity protoporphyrin disodium 1. o? water 5
- and suspended in concentrated hydrochloric acid (35.3% +495-
and react with stirring at 20°C. This reaction solution is sampled every 30 minutes, and 5 d of each solution is added to an acetic acid-sodium acetate buffer solution of pH 5.0 for neutralization, and the resulting precipitate is collected in a furnace, washed with water, and dried under reduced pressure after death.

After drying, an ether solution containing 471BFs etherate was dissolved in methanol 1o- containing 3'16, and 471BFs etherate was dissolved at 45°C.
Esterification takes place after stirring for 0 minutes.

After the reaction, the reaction solution 1d was added to a solution containing dichloroethane (25114K) ethylamine IR1, and then transferred to a separating funnel and washed with water three times. After washing with water, apply a dichloroethane solution dehydrated and dried with sodium sulfate to a silica gel thin layer plate of 50μ! Spot and develop using a solvent containing 3% methanol in dichloroethane as a developing solution. This operation was performed for each sample at each time, and observations were made until 2 hours and 30 minutes later. Each thin layer chromatograph thus obtained was subjected to a thin layer scanner to calculate the proportion of each component. An example of the analysis results obtained using the thin layer scanner is shown in Fig. 1. Further, the results of investigating the degree of attenuation of the raw material in the water utilization reaction and the degree of production of HVD, VHD, and HP by the method of converting it into an ester form are shown in FIGS. 2 to 4.

Example 3 High purity protoporphyrin disodium 1.0? water 1
50-thihydrochloric acid 4QQ suspended in 00d and prepared separately
Add to R1 and react with stirring at 20°C.

This reaction solution was sampled every 30 minutes, and the subsequent operations were carried out in the same manner as in Example 2. The results are shown in a graph for the reaction with 40% hydrochloric acid. (Figures 2 to 4). The same graph also shows the results of the reaction with 36-thihydrochloric acid.

The following has become clear from the above examples.

In order to produce the desired HVD%VHD, the optimum concentration of hydrochloric acid is 36%, and at 35%, more than 50% of the raw material remains even if the reaction is carried out for 1 hour and 30 minutes or more. On the other hand, 4
At a hydrochloric acid concentration of 0%, the raw material disappears quickly, but after 30 minutes the production ratio of HVD%VHD decreases and HP is produced. In this case, as time passes, more parts remain at the origin of thin layer chromatography. Therefore, HVD, VH
In order to selectively produce D, it is preferable to react at a hydrochloric acid concentration of 36% for 1 hour, an example of which is shown in Example 1. Under these reaction conditions, more than 50 units of raw material are lost, while nearly 50 units of HVD and VHD are produced in total, and only about 5 units of HP are still produced.

Also, the compound that appears at the origin in thin layer chromatography analysis does not appear.

Therefore, in the water-use reaction of protoporphyrin with hydrochloric acid, it has been found that the type and proportion of reaction products greatly depend on the acidity of the reaction solution, and from this, HV
To selectively obtain D%VHD, the hydrochloric acid concentration is 369
It was concluded that 6 was the best.

Example 4 Each of the 9 HVD dimethyl ester, VHD dimethyl ester and HP dimethyl ester obtained in Example 1 was dissolved in 50 mm of toluene, a methanol solution of 20 η of sodium hydroxide was added to this solution, and the mixture was boiled and refluxed for 2 hours. . After the reaction, the precipitated crystals were collected and dried under reduced pressure.
D disodium 0.9? , VHD+) IJ'y
0.95 y-1HP disodium 1.01 was obtained.

Example 5 DAB-induced rat ascites liver cancer (AH-79741-derived cultured cell line (JTC-16) was cultured at 37°C in DM-170 medium, and in the mid-logarithmic growth phase, HVD disodium, VHD disodium, HP disodium or HPD
After adding 5μ437Ill of each and incubating for 24 hours, the wavelength 630
It was irradiated with a nm argon laser. The results are shown in Table 1.

Table 1 (Note) 廿 Cell destruction + cell process disappearance ± cell vacuolization - no change As shown in Table 1, no change was observed in cells without additives after 3 minutes of laser irradiation, but HVD disodium Reacts in a shorter time, VHD disodium is H
HP disodium caused cell vacuolization after 3 minutes of irradiation to the same extent as PD.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram when the components of the reaction product obtained in Example 2 were analyzed using thin layer chromatography using a thin layer scanner. Figures 2 to 4 are diagrams showing reaction curves when protoporphyrin disodium is subjected to water utilization reactions. Figure 2 is a diagram showing the production curves of HVD and VHD, and Figure 3 is a diagram showing the production curves of protoporphyrin disodium. FIG. 4 is a drawing showing the attenuation curve, and FIG. 4 is a drawing showing the HP generation curve. Applicant: Sato Pharmaceutical Research Institute Co., Ltd. Agent: Patent Attorney Ariga Royal Family ゛°-゛[ (-Patent Attorney Nobu Ono Keishinia. 1. Bow: Gishu.-2 Fig. 1-A: Delot?rufilinzomethyl ester (area ratio 38
．． 9%) F::HVD'l methyl ester (l 17.
4%) E': VHD zomethyl ester ('17.
0%) F: HP (# 10.1%) X: (12,0%) Origin: ('14.6%) Reaction time Figure 3 Figure 4

Claims (1)

[Claims] 1. The following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, one of R_1 and R_2 is ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, and the other is -CH=CH_2 ) Deuteroporphyrin derivatives and salts thereof. The deuteroporphyrin derivative and its salt according to claim 1, which is 2,3-hydroxyethyl-8-vinyl deuteroporphyrin. The deuteroporphyrin derivative and its salt according to claim 1, which is 3,3-vinyl-8-hydroxyethyl deuteroporphyrin.