KR830000709B1 - Method for preparing 9- (hydroxyalkyl) furin - Google Patents

Abstract

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Description

Method for preparing 9- (hydroxyalkyl) furin

The present invention relates to compounds having the following structural formula.

X is OH, R ² is CH ₃ and R ¹ is alkyl of 1-8 carbon atoms. The compounds are anti-viral and anti-tumor action as immune modulators, also enzyme inhibitors. These compounds can also react with the amine salts of p-azetamidobenzoic acid, and in some cases, can form complex compounds that can enhance the action. This complex has the following structural formula

Wherein Y is an amine salt having p-acetamido benzoic acid of the following structural formula, Z is an integer of 1-10,

Wherein R ³ and R ⁴ are lower alkyl having 1-4 carbon atoms, such as methyl, ether, propyl, butyl, isopropyl or isobutyl, and n is an integer of 2-4.

The immunomodulatory effect is secured by increasing the length of the body of R ¹ (at least methyl to hexyl) as the length increases. R ¹ is n-alkyl, for example methyl, ethyl, n-propyl, n-butyl, n-amyl, n-hexyl, n-heltyl or n-octyl. Representative examples of the amines forming acetamino benzoic acid salts include dimethyl amino ethanol, dimethyl amino isopropanol, diethyl amino ethanol, diethyl amino isobutanol, ditalamino isopropanol, methylethylamino ethanol and diisobutyl Amino-N-butanol, dimethylamino propanol dimethylamino-N-butanol, diisobutyl amino ethanol, dimethyl amino butanol, dibutyl amino-N-butanol, dibutyl amino ethanol, dipropylamino ethanol and diisopropylamino Ethanol is mentioned. Preferred amines are dimethylamino isopropanol.

When Y is present, that is, when Z is a number from 1 to 10, the preferred number of Z is three. However, Z may be 1,2,4,5,6,7,8,9, or 10.

The compounds are hydrochloric acid, sulfuric acid, bromic acid, phosphoric acid, acetic acid, propionic acid, malconic acid, lactic acid, citric acid, tartaric acid, p-, rather than compounds of which Y is an amine salt having p-azetamido benzoic acid. As structural Y ¹ salts having pharmaceutically acceptable acids other than p-azetamibenzoic acid and amines defined above, such as toluene sulfonic acid, adipic acid, maleic acid, succinic acid, methanesulfonic acid, salicylic acid, acetylsalicylic acid and the like It may also be manufactured.

DIP in the abbreviation when Y is present in describing the following compounds. PAcBA stands for dimethylamino-2-propanol-p-acetamido benzoate. For example, Y is 3 when the number 10 in parentheses does not refer to a compound of this acronym. If the number in parentheses is DIP. When teaching PAcBA, the number refers to the number of moles of the Y group present in one mole of 9- (hydroxyalkyl) purine.

The compounds in Table 1 are believed to be pure except for compound 15443, which also contains compounds of the present invention and salts.

Immune modulators are compounds that modulate the immune response. This compound dominates both immunostimulation (immunogenic) and immunosuppression. Of course, immunostimulation is useful for conferring immunity. Immunosuppression is also useful in many fields. It is useful for preventing organ rejection in organ transplants, for example kidney or heart transplants.

The following table shows the immune synergy of the present compounds, plus (+) represents the immune synergy and minus (-) represents the immunosuppressive action. The number 0 indicates no immunosuppression action and no immunosuppressive action of the present compound.

Some of the following tables include many compounds, where the change in X and R ² does not fall within the scope of the compounds claimed herein. The use of the claimed compounds as well as the unclaimed compounds as immunomodulators and antiviral agents as antitumor agents is presented in our application filed on the same date under the name "immunomodulators and antivirus agents". .

Mitosis is a substance that induces cell proliferation that occurs during immunity.

Table 1 shows the definitions of X, R ¹ and R ² as well as the compounds of the present invention as well as the Daron related compounds. The synthesis of A-L mentioned in Table 1 is described in more detail in the future.

Compositions of the present invention include mammals (and mammalian cells) including humans, pigs, dogs, livestock, horses, sheep, goats, mice, rabbits, rats, guinea pigs, hamsters, monkeys, and the like. This is useful for dealing with

All parts and percentages are by weight unless otherwise indicated.

All temperatures are given in degrees Celsius unless otherwise noted.

The present composition stone can be constructed essentially, including the described materials, and the method also consists of such steps characterized by the methods described with such materials.

The composition can be administered by injection, in conventional methods, oral, nasal, rectal, vaginal and parenteral. These compositions can be used as injectable solutions or tablets, pills, capsules, etc. dissolved in fire.

TABLE 1

Overview of the chemical properties of 7- (hydroxyl) purines

Other compounds and related p-acetamido benzoic acid salts included within the scope of the present invention are described in Table 1a, wherein the basic structural formulas are as in Table 1. The alkyl groups for R ¹ in Table 1 and 1a are all n-alkyl.

[Table 1a compound]

Techniques for the preparation and use of the compounds of the present invention and related compounds and techniques for preferred carriers

Method A

9- (2-hydroxy-1-propyl) hypoxanthine (NPT 15446)

9- (2-hydroxy-1-propyl) adenine (I, 40 g 20.7 mmol) was suspended in 50% acetic acid (20 ml) and sodium nitrite (4 g, 58 mmol) was added slowly. The mixture was stirred at 25 ° for 3 hours.

The resulting solution was evaporated to dryness, isopropanol was added and this operation was repeated once. The solid residue was boiled in isopropanol and then filtered. The filtrate was evaporated and crystallized by addition of acetone.

Recrystallization from iso-propanol / methanol (98: 2) gave a colorless crystalline product. Yield 3.3g (82%) Melting Point 244-250。UV (H ₂ O: pH 5.5) λmax 250nm

Method B

9- (2-hydroxy-1-propyl) -6-chloropurine

Schaeffer, H.J. Vogel, D. and Vince, R., V. med. Chem. 8.502 (1965): and Schaeffer H.J. And Vince. R., V. med. The method of chem. 10.689 (1967) was adopted.

을 물(300ml)에 붓고 결정성 덩어리로 만들었다. 이것을 여과하여 모으고 물로서 세척한 다음 건조하여 19g의 조 9-(2-하이드록시-l-프로필아미노) 5-아미노-6 클로로피리미딘(II)을 얻었다.A solution of 5-amino-4,6-dichloropyrimidine (I, 20 g, 0.12 mol) in a 1% ethanol solution of isopropanolamine (200 ml) was refluxed for 8 hours. The reaction mixture was evaporated to syrup, ethanol was added, and the mixture was evaporated. This operation was repeated once. Formed city

Was poured into water (300 ml) and made into a crystalline mass. It was collected by filtration, washed with water and dried to give 19 g of crude 9- (2-hydroxy-l-propylamino) 5-amino-6 chloropyrimidine (II).

을 얻고 이것을 고진공 증발에 부하여 과잉의 이소프로파놀 아민을 제거하였다. 키실렌으로 결정화하여 5g의 조물질을 얻었다.Crude Compound II was suspended in triethyl ortho formate (120 ml) and ethanesulfonic acid (5 drops) was added thereto. After 15 minutes all solids were dissolved and the solution was then organic at 25 ° C. overnight. Evaporated in a vacuum

Was obtained and subjected to high vacuum evaporation to remove excess isopropanol amine. Crystallization with xylene gave 5 g of crude.

Method B

9- (2-hydroxy-1-propyl) adenine (NPT 15431)

9- (2-hydroxy-1-propyl) -6-chloropurine (I, 9 g, 42.5 mmol) was dissolved in saturated methanolic ammonia and ammonium chloride (50 mg). The mixture was heated to 130 ° in a gas reservoir for 6 hours.

The resulting solution was evaporated to dryness and recrystallized from ethanol / acetone. Yield = colorless crystalline product (81%) 6.68 g Melting point 193-194。 UV CH ₂ O: PH 5.5) λ max 260 nm CHCI ₃ : MeOH (5: 1) TLCRf.50.44

Anal. Calcd for C ₈ H ₁₁ N ₅ O: C, 49.73, H, 5.74, N 36.25

Found: C, 49.56, H, 5.62, N 36.22

Method C

9- (1-hydroxyethyl) -6-merphatopurin (NPT 15435)

The method of Schaeffer and Bhargava Biochemistoy 4.71 (1965) was adopted.

9- (1-hydroxyethyl) -6-chloropurine (I, 2 g, 0.1 mol) and thiourea (0.76; 0.1 mol) were dissolved in ethanol (15 ml) and refluxed for 30 minutes. The precipitate formed was collected by filtration and suspended in water to form a slurry.

Neutralization with sodium acetate gave colorless crystals. Yield 1.5 g (76%) Melting point: 278-280. UV (H ₂ O, PH5.5) λmax 320,230 nm

Method D

9-hydroxyethyl hypophosphite (NPT 15425)

The methods of Schaeffer H. J. and Bhargava, P. S Biochemistry 4,71 (1965) were used.

6-Chloro-9-hydroxyethylpurine, III (4 g) was added slowly to warm N NaOH (30 ml) and refluxed for 2 hours. The reaction was cooled in ice and neutralized with glacial acetic acid. After filtration the portion of unreacted compound III is removed. The product was recrystallized from methanol and washed with acetone.

1 g (28%) of colorless crystals;

Melting point 274。; UV (H ₂ O pH5.5), λmax 250 nm

Method E

9- (1-hydroxyl-2-propyl) -6-iodopurine (NPT 15427)

9- (1-hydroxy-2-propyl) -6-chloropurine (I, 1.5 g, 7 mmol) was added to hydroiodic acid (15 mL) at -10 ° with stirring for 45 minutes. The precipitate was filtered off and neutralized with anhydrous sodium acetate at 5 ° and washed with a small amount of fire (three times). Recrystallization from ethanol / water gave colorless crystals.

Yield = 0.9 g (42%) Melting point = 193-194.

UV λmax 276 nm (H ₂ O, pH5.5)

Anal. Calcd for C ₈ H ₉ N ₄ OI Mw = 304.1: C, 31.60 H, 2.98 N, 18.43 I, 41.73

Found: C, 31.53 H, 2.96 N, 18.18 I, 4l.70

Method F

9- (1-hydroxy-2-propane) -6-chloropurine (NPT 15423)

Schaeffer HJ and Schwender CFJ med. The method of Chem 17 , 6 (1974) was used.

A solution of 5-amino-4,6-dichloropyrimidine (I, 6.56 g 40 mmol) and 2-amino-1-propanol (n, 3.3 g, 44 mmol) was added to n-pentanol for 45 hours under N ₂ gas. 288 ml) and tertiary butylamine (96 ml).

상으로 한 다음 에탄올을 4회 가한다음 증발하였다.Evaporate the solution

Phase and ethanol was added four times and then evaporated.

을 트리에틸 오르토포르메이트(150ml) 및 에탄 설폰산(10적) 중이서 현탁하였다. 본 현탁제를 하룻밤 강하게 교반하고 증발 건조하여 에탄올을 가한 다음 본 조작을 3회 반복하였다.Formed city

Was suspended in triethyl orthoformate (150 ml) and ethane sulfonic acid (10 drops). The suspension was stirred vigorously overnight, evaporated to dryness, ethanol was added and this operation was repeated three times.

Crystallization of the colorless product occurs during evaporation. The crystals were filtered, the filtrate was evaporated, ethanol was added, and the procedure was repeated three times to obtain crude (3.6 g).

Recrystallized from 9S% ethanol solution.

UV (H ₂ O, pH5.5) lambda max 265 nm; Melting point 201-203 °; Yield 2.79 (32%)

Found C ₈ H ₉ N ₄ OCI calcd. C, 45.20; H, 4.26; N, 26.36; Cl, 16.68

Found C, 45.11; H, 4.27; N, 26.25; C1,16.71

Method G

9- (1-hydroxy-2-propyl) adenine (NPT 15433)

Schaeffer H. and Schwender C., J. Pharm. Sci., 60, 1204 (1971), also Schaeffer et al. J. med, Chem. 15, and using the method of 456 (1972).

9- (1-hydroxy-2-propyl) -6-chloropurine (I, 2.0 g, 9.4 mmol) was suspended in ethanol / ammonia (30 ml) and ammonium chloride (50 mg) was added as a catalyst and the compound was added to 4.5. Heated at time 130 °;

The solution was evaporated to dryness. The crude product obtained was reconstituted from ethanol to give colorless needles.

Yield = 1.15 g (63%)

Melting Point = 215-216 ° UV (H ₂ O, pH5.5) λmax 260 nm

Method H

9- (1-hydroxy-2-propyl) hypoxanthine (NPT l5443)

9- (1-hydroxy-2-propyl) adenine (I, 4g, 21mmol) is dissolved in 50% acetic acid (20ml) and sodium nitrite (4g, 58mmol) is added and the mixture is 25 for 3-1 / 2 hours. Stir at °. The solution was evaporated to dryness with isopropanol twice. The residue was taken up in isopropanol, filtered to discard the precipitate, and the filtrate was evaporated to form a gel, which was then solidified by adding acetone.

Yield = 3.65 (90%) as colorless crystals, isopropanol / methane (98: condensation = 202-207 °).

TLC / spot Rf-0.3 UV (H ₂ O, pH5.5) in CHCI ₃ : MeOH (5: l) = lambda max 250mn,

Method I

Compound NPT 15417

Schaeffer et al. Journal of Pharrnaceutical Sciences l6: 1204-1210, Method of Method.

This product is Compound XL in Schaeffer et al Table III.

Method J

Erythro-9- (2-hydroxy-3-nonyl) hypophosphite (NPT l5392)

The outline of the synthesis process for the preparation of erythro-9- (2-hydroxy-3-nonyl) hypokixantane (nonylhypoxanthine, XIII) is shown in the first and second process diagrams.

In this reaction step leading to erythro-9 (2-hydroxy-3-nonyl) -6-chloropurine, H. J. Schaeffer and C.F. Schwender, J. Med. An improvement over the method in Chem 17,6 (1974) is pointed out.

Hydrolysis of the 6-chloropurine derivative, which is the final process for obtaining nonylhypokistantan, is carried out by A. Giner Sorolla, C. Gryte A. Bendich and G.B. Brown, J. Brg. The method reported by Chem. 34,2157 (1969) was adopted.

Alternative methods to that described above, namely nitration of erythro-9- (2-hydroxy-3-nonyl) adenylEHNA) (IX) to obtain nonylhypokixanthin (as shown in the second process diagram) This chloro derivative is ammonolyzed and pre-converted to aminopurine (IX, EHNA), followed by nitration to obtain nonylhypokixanthine.

1st process chart

Synthesis of erythro-9- (2-hydroxy-3-enyl) hypoxanthine

Process 1 Acetamidononan-2-one (II)

Process 2 Acet Amidononan-2-one Hydrochloride (III)

Formation of Acet Amidononan-2-one Hydrochloride

Process 3 erythro-3-amino-2-nonanol (IV)

Reduction of Acetamidononan-2-one Hydrochloride

(The numbers under the arrows below indicate the yield)

Process 4 erythro-5-amino-4-chloro-6- (2-hydroxy-3-nonylamino) pyrimidine (VI)

Condensation of erythro-3-amino-2-nonanol with 5-amino-4, 6-dichloro pyrimidine

Step 5 erythro-9- (2-hydroxy-3-nonyl) -6-chloropurine

Ring blockade of erythro-5-amino-4-chloro-6- (2-hydroxy-3-nonylamino) pyrimidine (V)

Step 6 erythro-9- (2-hydroxy-3-nonyl) hypokixanthine

(By gas decomposition of 6-chloropurine derivatives)

2nd process chart

Balance method for preparation of erythro-9- (2-hydroxy-3-nonyl hypokixanthine)

Step 1a erythro-9- (2-hydroxy-3-nonyl) adenine

Ammonolysis of Erythro-9- (2-hydroxy-3-nonyl) -6-chloropurine

Step 2b erythro-9- (2-hydroxy-3-nonyl) hypoxanthine

Nitrification of erythro-9- (2-hydroxy-3-nonyl) adenine (IX)

3-acetamidononan-2-one (II)

A mixture of 2-amino-1-caprylic acid (I, 200 g, 1.26 mol) and pyridine (640 ml) in acetic anhydride (970 ml) was heated on a boiling water bath for 4 hours. The reaction mixture was evaporated in vacuo and the residue was separated 6-8 times between 5% aqueous solution of NaHCO ₃ (400 ml) and ether (400 ml). The mixed etheric extract was dried over anhydrous MgSO ₄ and then evaporated to dryness to afford 154 g (70%) of crude 3-acet amidononan-2-one.

3-amino-2-nonanone hydrochloride (III)

The crude product (II) (154 g) obtained in the above-mentioned operation was dissolved in concentrated hydrochloric acid solution (1.540 ml), refluxed for 2 hours, and evaporated to dryness in vacuo. The resulting solid was recrystallized from a warm solution in EtOH (200 ml) and then cooled to 25 °. Ether (600 ml) was added to the solution. White crystalline precipitation appears. The suspension is left overnight at 5 °. The precipitate was collected and washed as ether (once as 100 ml) to give 125 g (67%) of white crystalline product. Melting point 112 °

If this crystalline material is not white or has a low melting point, it should be recrystallized from tetrahydrofuran with charcoal. For the first iteration of this procedure 150 ml of hydrofuran was used for 100 g of crude hydrochloride (III).

Erythro-3-amino-2-nonanol (IV)

3-amino-2-nonanol hydrochloride (43.8 g, 0.226 moles) was dissolved in anhydrous methanol (150 ml) and cooled to -10 ° in an ice salt bath.

1) Potassium borohydride (24.4 g, 0.45 mol) was added in small portions over 2-3 hours. The mixture was kept at -10 ° N -15 ° for 3 hours, 3), 4) allowed to slowly reach room temperature (22 °) and stirred overnight at room temperature (20 hours).

)하고 물(150ml)과 클로로포름(150ml)간에서 분리하였다.The mixture is then evaporated to dryness in vacuo.

), And separated between water (150 ml) and chloroform (150 ml).

The aqueous layer was further extracted three times with chloroform (100 ml each). The chloroform layer was dried over MgSO ₄ and evaporated in vacuo to give a pale yellow, oily product. The solution was distilled in high vacuum at 95 ° -100 ° (0.15 mmHg) to give 26.4 g of pure erythro-3-amino-2-nonanol. Yield 75%, Melting Point 81 ° -86 °

By cooling the solution of product III, a precipitate is produced. This precipitation does not affect the result of this reaction.

In this respect the method differs from that of Schaeffer et al. Schaeffer adds acetic acid at the same time as KBH ₄ while maintaining the pH at 5-6. Neutralization resulted in the loss of KBH ₄ and found that pH of 5 or more was resistant. More importantly, the simultaneous addition of acetic acid and KBH ₄ (as suggested by Schaeffer) results in a very difficult reaction to adjust.

The temperature rises significantly and results in loss of yield and / or degradation of the quality of the production rate.

It is recommended that effective agitation be used to ensure a proper reaction that completes when all small lumps and portions of potassium borohydride are lost. Cooling at 0 ° is insufficient as described by Schaefer et al (Method D, Row 4 and 55). Maintaining this reaction well below 0 ° is an improvement. Keeping well below -10 ° is always an improvement. It is always best to keep it below -10 °.

If the temperature is above -10 °, inherent losses in yield will result.

Erythro-5-amino-4-chloro-6- (2-hydroxy-3-nonylamino) pyrimidine (VI)

4.6-dichloro-5-aminopyrimidine (V, 24.6, 0.15 mol) and erythro-3-amino-2-nonanol (IV, 26.2 g, 1-pentanol (1.080 ml) and tributylamine (35 ml) 0.164 mol) was prepared with stirring at 25 °. The formed suspension was heated to reflux under nitrogen gas for 28 hours (solution was left for about 1/2 hour). Sigongpum of this time the reaction product of the exhibited UV λmax 267 and _{297nm (H 2 O, pH5.5)} .

상으로 농축한 다음 0.1mm 및 100°에서 유욕중에서 증발하여 점액성인 액체를 얻고 여기에 n-헥산(450ml)을 가하였다. 혼합물을 1시간 환류한 다음 뜨거운 황색 헥산 상징액을 환저 플라스크 저부의 액체로부터 분리하였다.The formed solution was heated in a water bath under 10 mm pressure.

Phase was concentrated and then evaporated in an oil bath at 0.1 mm and 100 ° to give a mucus liquid, to which n-hexane (450 ml) was added. The mixture was refluxed for 1 hour and then the hot yellow hexane supernatant was separated from the liquid at the bottom of the round bottom flask.

Residual hexane from the pale brown oil formed was evaporated in vacuo and dissolved in chloroform (150 ml). The chloroform solution was extracted eight times (250 ml each time) with a saturated aqueous solution of NaHCO ₃ . The chloroform layer was then separated, dried (with sodium sulfate or magnesium sulfate) and then evaporated at 40 ° (water bath) under high vacuum (0.1 mm) to give a pale brown oil which solidified by cooling. The material can be used directly in the next process or it can be purified as follows.

The oil formed was dissolved in 75-100 ml of chloroform and n-hexane (about 300 ml) was added to precipitate a white crystalline solid which was filtered from the cooled solution (extraction was carried out 4-8 until no carbon dioxide was released further). Times). This treatment was repeated two more times.

Yield: 23.3 g (54%) UV lambda max 267,297 (H ₂ O, pH5.5) Melting point 113-116 °

Erythro-9- (2-hydroxy-3-nonyl) 6-chloropurine

을 트리에틸오로토 포르메이트(106ml) 및 클로로포름(34ml) 중에 용해하고 용해를 유효하게 하기 위해서 에탄설폰산(10적)을 가하였다. 25°에서 하룻밤 방치후 본 용액을 진공하에 증발하여 시

으로 만들었다. 수득량 11.7g(정량적) 조 에리스로-9-(2-하이드록시-3-노닐)-6-클로로푸린(Ⅶ)으로 구성된 본 시

을 다음 공정에서 사용하였다. λmax 264nmPreparation from the above-mentioned operation consisting of erythro-5-amino-4-chloro-6- (2-hydroxy-3-nonyl amino) pyrimidine (11.48 g, 40 mmol)

Was dissolved in triethylortho formate (106 ml) and chloroform (34 ml) and ethanesulfonic acid (10 drops) was added to effect dissolution. After standing at 25 ° overnight, the solution was evaporated under vacuum

Made with. Yield 11.7 g (quantitative) of crude erythro-9- (2-hydroxy-3-nonyl) -6-chloropurine

Was used in the next process. λmax 264nm

Erythro-9- (2-hydroxy-3-nonyl) hypoxanthine

(By hydrolysis of 6-chloropurine derivatives)

A suspension of erythro-6-chloro-9- (2-hydroxy-3-nonyl) purine (Ⅶ, 4.0 g, 13.4 mmol) in 0.5 N NaOH (40 ml) was refluxed for 2 hours and cooled. Neutralization with glacial acetic acid and cooling afforded a crystalline precipitate of erythro-9- (2-hydroxy-3nonyl) hypokixanthine. It was filtered and dried.

Yield: 3.8 g (quantitative), melting point 196 UV lambda max (pH5.5) 251 nm

The crude product thus obtained was homogeneous by paper chromatography (3 solvents) and showed negative in the test for Cl- (copper and salt minerals; sodium dissolution, acidification and nitric acid temperature).

The crude product was recrystallized three times from the ethanol solution (see Purification) to obtain colorless crystals.

Melting point 202 °, calcd for C ₁₄ H ₂₂ N ₄ O ₂ (Ⅷ): C, 60.41 H, 7.97 N, 20.13

Found: C, 60.47 H, 7.86 N, 20.08

Purification of Eryristo-9- (2-hydroxy-3-nonyl) hypokisane

Crude nonylhypokisane is purified by recrystallization. The crude material is heated by dissolution in about 6-10 times ethyl alcohol by weight and the same volume of water is added.

The solution was treated as charcoal in Erlenmeyer and filtered through celite on hot. The solution is evaporated with continued stirring on a hot plate.

Twisted water is added to replace the evaporated capacity until abundant precipitation appears. The solvent must be continuously evaporated to remove all ethyl alcohol with repeated additions of water, with a weight of 8-12 times the weight of the material. The loss in material is about 10% for each recrystallization.

The crude material was slightly yellow or pink and dissolved at 192 °, but recrystallized twice, raising the melting point to 202 ° and obtaining a colorless crystalline material.

Eryristo-9- (2-hydroxy-3-nonyl) adenine HCI (IX)

Crude oil-based erythro-9- (2-hydroxy-3-nonyl) -5-chloropurine (6.15 g) from the above-mentioned formulations was carried out in a stainless steel reservoir for 1 hour 80-100 °. Was dissolved in saturated methanolic ammonia (300 ml) and ammonium chloride (1 g). After cooling the solution was evaporated to dryness in vacuo. Methanol was added and evaporated to third time (3 times) to remove excess ammonia.

The syrup-like residue was dissolved in anhydrous alcohol, and dry hydrochloric acid gas was generated to maintain the temperature below 20 ° (as an ice bath).

The mixture was cooled to 5 ° after passing through 1/2 hour hydrochloric acid.

The precipitate was triturated with calcined glass filter, washed with cold methyl alcohol and dried in air. Yield 6.0 g (92%) Melting point 173-175。 Decomposition UV λmax 260 nm (in H2O, pH5.5)

Balance method for preparation of erythro-9- (2-hydroxy-3-nonyl) hypoxanthine

(By deamination of char)

Sodium nitrite (5.6 g 71 mmol) was added to a solution of erythro-9- (2-hydroxy-3-nonyl) -atenin (IX, 4.0 g, 14 mmole) in 50% acetic acid (20 ml) and N HCI (3.2 ml). The mixture was slowly added at 25 ° while stirring.

The mixture was stirred at 25 ° for 2 hours. The UV spectrum was then measured. The solution was neutralized with 2N NaOH when the UV lambda max reached 250 nm. The resulting precipitate was filtered off and washed as H ₂ O.

Yield = 3.03 g (75%) m.p. = 195.

The analytical construction was recrystallized three times from water to obtain the desired product.

Melting point 202。 Analytical calcd for C ₁₄ H ₂₂ N ₄ O ₂ : C, 60.40 H, 7.96 N, 20.l3

Found: C, 60.40 H, 7.90 N, 20.12

Method K

Compound NPT 15426

H.J. Schaeffer and S.F. Schwender, J. Med. The method of Chem. 17: 6 (19764) was used.

Method L

Manufacture of NPT 15410

9- (2-hydroxy-3-nonyl) -6-hydroxypurine 0.1 mmol NPT 15392 (27.9 mg) and 0.3 mmol 2-hydroxypropyl dimethyl ammonium 4- (acetylamide) benzoate (DIPPAc 13A) (77.lmg) was precisely weighed and dissolved in 105 ml of 0.25% sodium carbonate (Na 2 CO 3) to give a 0.1% solution of NPT 15410 (compound formed from (DIP.PAcBA) in NPT 15392 and 1: 3 molar ratio).

Evidence for Complex Formation

A review of the solubility of phases conducted with NPT 15392 and DIP PACBA shows that the solubility of NPT 15392 increased with increasing concentration of DIP PAcBA under constant pH conditions.

This is an indication of the interaction that takes place in solution to produce a complex.

Other complexes are formed by using ratios of 1: 1 and 1: D instead of molar ratios of 1: 3 (NPT 15392 and DIP-PAcBA).

Synergy of Biological Activity by DIP · PAcBA

Of the material stones described in Table 1, NPT 15392 and NPT 15446 are new. Also new are the DIP-PAcBA salts shown in this table, namely, 15428,15437,15447,15432,15434,15444,15418 and 15410. NPT 15392, NPT 15417, and NPT 15426 all alone have significant anti-influenza action. As an example (in the case of NPT 15392), the addition of DIP-PAcBA salt to NPT15392 to form 15410 does not elevate anti-influenza action.

In the case of 15417, the addition of DIP.PAcBA salt to form 15418 enhances anti-influenza action. An overview of the correlational capacity of DIP-PAcBA salts to enhance different biological activities is given below.

Animal testing on mice as NPT 15392 and NPT 154l0:

Effects of In Vivo Stimulation of Splenocyte Proliferation by CONCANAVALLN A

The purpose of this experiment was to determine the effect of rat in vivo testing on the collateral action of splenocytes isolated from rats with compounds NPT 15392 and 15410 and to examine their proliferative response to the mitotic substance concanavalin A (Con A). It was evaluated by an in vitro test.

Way

Animal testing

Nine Balb / C mice weighing 18-20 gms and 8-9 weeks old were divided into three groups. One group was orally administered NFT 15392 twice a day morning and evening at a dose of lOmg / kg body weight. The second group treated NPT 15410 at a dose of 20 mg / kg body weight, similar to the previous method wave. The third group administered physiological saline only to be used as placebo control.

In vitro Spleen Cell Assay: Cell Specimen

The next day, each group was sacrificed for experimentation, the spleen was removed, and then turned together. The spleens were sliced and the cells washed in 1d MI-1640 P medium (Grand Island Bio1ogicaIs) supplemented with 2mm glutamine and antibiotics.

Cell concentration of each sample was measured as CouIter Counter and adjusted to 5 × 10 6 cells as RPMI medium.

Microtiter assays were performed with 0.2 mM cultures containing 5 × 10 5 cells and large A (Con A) or large A and compounds of the indicated concentrations.

All analyzes were carried out as six simulations and remarked by blind analysis with blastoma. The assay plate was incubated at 37 ° in 5% CO 2 for 4 days. 0.5 ml of HTdR (10uci / m16ci / mmoIe) was added to each culture during the last 18-20 hours of culture. Cultures were hydrated by the method of cell proliferation with bound 3HTdR measured as a multi-automated Sembler Harvester (MASH) unit and Becknan LS8000 liquid scintillation counter.

The results are plotted as the ratio of activity in cultures treated with Concanavalin A (ConA) or Concanavalin A (ConA) and compounds to the empty culture.

Compound 15392, or 154l0, increases the incident response of splenocytes in vitro to large A (ConA) stimuli at sub-optimal mitotic concentrations (5 μg / ml) in vivo. The observed stimulation rate was 120: 1 for splenocytes isolated from mice treated as 15392 compared to 40: 1, the ratio of splenocytes isolated from mice treated with placebo. When the present cells were stimulated with a more optimal concentration of A (ConA) (10 μg / ml) there was no significant difference due to treatment with Compound 15392 or 15410.

Con A-stimulated preparations were also tested for effects seen by treatment in vitro as NPT15392 and 15410 / μg / m1. Bi-unbalance shows a marked ability to enhance ConA stimulation, particularly in suboptimal mitotic concentrations (5 μg / mI) and in dose ranges less than 1 μg / m1. At 5 μg μA of ConA, the stimulus by NPT15392 is 2.8 times larger than that of Con A tan Germany and 3.3 times for NPT l5410.

These results indicate the immunomodulatory effects of these compounds on splenocyte proliferation. Pretreatment of animals with these compounds will sensitize the cells to concomitant mitogenic stimuli, while simultaneously exposing the cells in vitro after mitotic stimulation will enhance the proliferative response.

Claims (1)

A process for preparing a compound of formula (I), wherein the compound of formula (II) is hydrolyzed in alkaline or diazotized under acidic conditions.

R ¹ in the above formula is alkyl having ¹ to 8 carbon atoms.