JPS5894396A - Preparation of 3-deazaguanocine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION During the past decade, a number of nucleoside analogs have been found to have good antitumor and antiviral effects. Of the currently known synthetic nucleotide antiviral and antitumor agents, the most important are generally 5-iodo-
2'-deoxyuridine (IDU), 9-β-D-arabinofuranosyl adenine (ga-A) and 1-β-D
-Arabinofuranosylcytosine (ara-C) 7M
It is believed that there is. However, these compounds as antiviral agents are effective against 6 respiratory diseases in humans! It is only effective against a limited spectrum of viruses, including those that cause (influenza, colds). The only nucleoside congener expected to be effective against these respiratory viruses is U.S. 1%
798209, and the latter compound having this triazole base. certain derivatives,
That is, it has also been found that 1,2.4-triazole-3-calzexamide and 1,2.4-triazole-3-thiocarpthamide have a remarkable antiviral effect. The advent of such compounds and the discovery of their antiviral action created a need for compounds capable of effectively inhibiting viral infections, particularly respiratory disease viruses.

Known nucleoside anti-abscess agents suffer from host toxicity or do not have a broad spectrum of anti-tumor activity.

Furthermore, nucleoside analogues are used to inhibit either viral or tumor growth, and the nucleoside is converted by in vivo metabolism into its corresponding mono- or polyphosphate salts, and the phosphates are used to inhibit either viral or tumor growth. There are 7 inhibitors.

However, a major obstacle to using nucleoside analogs in chemotherapy is that nociceptive cells exhibit low levels of kinase or pyrophosphorylase activity and therefore do not produce effective inhibitors. There is an outbreak of cellular resistance to such compounds. Although this problem is overcome by the use of phosphate nucleosides, such derivatives often do not cross cell membranes well or degrade rapidly in the intercellular fluid, thus rendering them ineffective as inhibitors.

In view of the above-mentioned considerations, the present invention has congeners that are capable of effectively inhibiting viral infection and the development of abnormal tissue, and that have better solubility than existing known antiviral and antitumor agents. It is clear that this is desirable. However, it is extremely difficult to obtain compounds that not only have acceptable activity, but also penetrate cell membranes and reach effective concentrations of viral infection.

In view of the above requirements, the present invention synthesizes 3-deazaguanosine and examines whether such a compound has effective antiviral and antitumor abilities. - leaf lanose, X is H or an alkali metal 1] Compounds according to the invention are prepared according to the following examples according to the table below (temperatures and melting points are given in °Cf) It can be manufactured by K as described in . One compound according to the invention as described is 3-deazaguanine (compound 5) t! It can be made by reacting with a nucleoside phosphorylase at temperatures of about 5 DEG to about 90 DEG C. and about 0 DEG to 50 DEG C. in the presence of J/-1-phosphate.

The starting material 3-deazaguanine (compound 5) is methyl 4 (
5)-acetamido-2-imidazole-5(4)-cal 2-xylate (compound 2), which is reacted with a dehydrating agent such as phosphorous oxychloride, oxalyl chloride, thionyl chloride, etc. under reflux conditions. to prepare methyl (4) 5-acetonitrile-2-imidazole 4)-cal 2-xylate (compound 3), which was then mixed with
Heating with liquid ammonia at a temperature of 150℃
5)-acetonitrile-2-imidazole-5(4)-
Calixamyl-' (compound 4) is obtained. This compound is then reacted with an alkali gold maple carbonate such as sodium carbonate under reflux conditions to form 5-deazaguanine (compound 5).
get. Also, 3-deazaguanine.

Directly from acetonitrile cal I xylate.

Temperatures of about 100 to about 150 C in the presence of ammonia 7, ammonium chloride in aliphatic alcohols such as methanol and more! It can also be produced by processing f0 7 - Example 1 6-amino-1-β-D-ribofuranosylimidazo-[
5-c]Pyridin-4(5H)-one (3-deazaguanosine) Culture samples were made to a final volume of 1. IM Tris Hα during OJll
Buffer (pH 7,5,0,5m >, ribose-1-phos7ate cyclohexylammonium salt, 10 m
M aqueous solution (0,125 itl), 3-deazaguanine (
Compound 5) 1 mM aqueous solution (0.125 d), enzyme suspension [calf lung nucleo-light phosphorylase (calf), manufactured by Sigma Chemical Co., St. Louis, MO;
5pleen nucleoside phosphor
orylase) 0.03a/ and distilled water (0.22
mJ).

The sample is kept constant at 25°C. Cary-14 (Cary
14) Changes in absorption were followed by checking the UV spectra every 5 minutes by recording with a spectrophotometer needle.

After 20 minutes, no more changes were observed.

λmaxx (pH 7,5) from 262 nm to 270
λ with increasing extinction coefficient. ax
Peeling Fi298nm (pH 7,5) 't% Shoulder. Nucleo! The highest conversion of 13-deazaguanine in the reaction mixture to
M"14XIO-'~lXlO"M and the concentration of Nucleo//de phosphorylase is 60-9
It was obtained when it was 0 μg/rLl.

Furthermore, in order to confirm that the product thus obtained was 3-deazaguanosine, the following operation was also carried out. 3-deazaguanine (compound 5) (4゜5m9.0.05 mmol) in 0.5M'7)) Lis H (1 buffer, pH 7.5,
(300m). Ribose-1-phosphate dicyclohexylammonium salt (42,81119,
0.1 mmol) and nucleo-side phosphorylase (elF, 2! units/In9) were added and the reaction was incubated for 25 min.
The mixture was stirred at ℃ for 2 hours. The − of the bath solution was adjusted to 4.5 using Hα and then the charcoal column (
100 to 200 mesh). Column Hlo (
200 ml) and then rinse the product tube with ethanol/H
1O/concentrated NH4OH (45: 45: 10) (
300+j! ) was eluted. The eluent was evaporated to dryness in vacuo, leaving an amorphous pale green material. Yield: 8.1!
(95%). It was confirmed by UV spectrum and TLCK to be identical to the synthesized 3-deazaguanosine of Example 7 (Compound 11).

Generally, 3-deazaguanine (compound 5) is about 5 to about 9
, advantageously a pH in the range 7-8? Enzyme concentration approximately 0.05
~about 0.5 da/-1 advantageously about 0°05 to about 0.111
9/ILf"l' and a temperature within the range of about 0 to about 50°C (
Advantageous temperatures range from about 25 to about 35 degrees Fahrenheit (about 25 to about 35 degrees Fahrenheit), at which the enzyme nucleolase dephosphorylates. 3-deazaguanine is 5
Concentrations greater than X10-5 M, advantageously about lX10-'
Satisfactory results have been obtained when M is present at a concentration of 1 and ribose-1-phosphate is present at a concentration of about 3.times.10' to about 1.times.1o-' SM. The time required for the reaction is generally from about 0.1 to about 2 hours, advantageously from about 0.5 to about 1 hour. The enzyme may be of animal, tissue or bacterial origin. The main bacterial origin is E.

coli) and yeast, but also include bovine lung, latte liver, calf liver, calf thymus, bovine liver, monkey brain, horse liver, calf pancreas, human red blood cells, fish skin and fish muscle. Various animal origins exist.

The enzyme may be a purified protein or may be present in an active metabolically active bacterial or fungal cell. If present in active metabolic bacterial or fungal cells, a process using said cells or mutants of said cells can lead to the production of 3-deazaguanosine from 3-deazaguanine.

2'-0-acylated (e.g. C-acyl) analogs are Falbriard.
) and other co-deposition “Biohimica et Biofijica Φ
Actor (Biochxm, et Blophys,
Acta) Vol. 148, 99 Negative (1967), the free nucleotide 41 can be synthesized with the corresponding acid anhydride or acyl halide, such as acetic anhydride, acetyl chloride, propionyl chloride, anhydrous By reacting with propionic acid, butyric anhydride, zotyryl chloride, pareryl chloride, nomaleylic anhydride, hebutanoic anhydride, heptanoyl chloride, hexanoyl chloride, hexanoic anhydride, octanoyl chloride, octanoic anhydride, balmitoyl chloride, palmitic anhydride, etc. There is one thing I can confirm. This type of compound is expected to have antiviral activity due to its structural similarity to the above-mentioned nucleocarbons, and is described in "Biochemical and Biophysical Research Communications" co-authored by Hoeksema (1) and others. (Bioche
+n1caland Biophysical Re5
Nucleo≠≠ The oral absorption pattern of Nucleo≠41 in humans was determined by considering the report by Nucleo≠≠ (1961), Vol. 6, p. 213 (1961)
It is also expected that the acetylation of F will help.

From the foregoing discovery, acid-added clays can be prepared by suspending 3-deazaguanosine in water and reacting it with a suitable acid reactant, such as hydrochloric acid, sulfuric acid, etc., by standard methods known to those skilled in the art. is obvious to those skilled in the art.

Similarly, basic salts are produced by reaction with alkali metal bases such as sodium hydroxide.

REFERENCE EXAMPLE This example describes the use of 3-deazaguanosine (compound 6) in order to determine its in vitro antiviral potency. 1nduc
ed cytopathogenic effect
-) (CPE) method [“Applied Micro/ζ
Iology ° (Applied Mic'robio
22, pp. 797-δ01 (1971
)]

Simply put, the CPE method extracts vitamins, amino acids,
It involves dissolving the antiviral agent in a cell culture medium consisting of serum, buffer, penicillin, streptomycin, and indicator dye. Virus suspended in cell culture medium
B cells or RK-13 cells were added to the monophase and then 1 equivalent of the antiviral agent was added within 15 minutes. Infected cells were classified by microscopic examination. Control substances for each experiment include cell control substances (cells and cell culture medium only), virus control substances (cells and viruses and cell culture medium), and toxicity control substances (cells and chemical drugs and cell culture medium).

Said °Applied Micro;Iology.

Sitwell et al.'s Virus grade level (VR), detailed in
virus rating system
'Used to evaluate the effectiveness of PE inhibition. A VR greater than 0.5 indicates significant antiviral activity;
．． A VR of less than 5 has only a small antiviral activity, compared with the data on 3-deazaguanosine and 6-amino-3-β-D-ribonylanosyl imidazo C4,5-C] -Viridi 8-5H
) 4-on in vitro tie 7'l Herzos Simpress 1.0 0.4 Type 3 Adeno
0.8 0.0 type 139
0.7 0.1 Type 3 influenzae 0.5 0.0 Vaccinia 0.9 0.2 *Cytotoxicity is 72 hours at 37°C. It was expressed as it (μg/mJ) if no appreciable change occurred.

KB cells: Human nasopharyngeal cancer.

The reference examples show that 3-deazaguanosine exhibits a wide range of antiviral activity and is particularly effective against viruses that cause respiratory diseases.

In some cases, the compound is tested in vitro with the comparison compound 1-β-
D-lyzefuranosyl-1.2.4-triazole-3-
It showed significantly higher activity than lactate xamide. 3-
Structurally similar to deazaguanosine and generally increased solubility of salts of active pyryl compounds -1'l
1 addition salts are also expected to exhibit antiviral activity.

Next, embodiments of the present invention will be listed.

(2) the enzyme is present at a concentration of about 0.05 to about 0.5 Da/d; Level 1 exists,
The method described in item 2 above.

(3) 3-deazaguanine is present at a concentration of at least about δXIO'M, and the ribose-1-phosphate is present at a concentration of about 3X
3. The method of claim 2, wherein the molecule is present at a concentration of 10' to about 1X10''M.

The method according to item 4 above, wherein the f41 PH is about 7 to about 8↑ and the temperature is 25°C to about 35°C1.

51

Claims (1)

[Claims] 1. 6-Amino-1-β-D characterized in that 3-deazaguanine is reacted with #nucleoside phosphorylase in the presence of lysate-1-phosphate at a temperature of about 5 to about 9° C. and about 0 to 50° C. -Production method of ribofuranosylimidazo-[4,5-c]pyridin=4(5H)-one (3-deazaguanosine).