JPH0211596A - Nucleotide compound and remedy for viral disease and antiplastic agent containing the same compound as effective component - Google Patents

Abstract

NEW MATERIAL:Compounds of the formula (Y is always H, adenosine, 1-20C primary or secondary alcohol except at terminal phosphate where Y is able to be H; m is 0-4; n is 1-14; K is basic amino acid peptide; x is 1-10) having 2',5'-internucleotide bonds, at least riboseadenine units. USE:A remedy for viral diseases and an antineoplastic agent with low toxicity free from side effects. PREPARATION:Hydroxyl groups at 2' and 3' positions of 2' terminal ribose of 2', 5' linked riboadenylic salt are initially oxidized with sodium periodate under ice cooling and poly-L-lysine is then added to the above-mentioned oxidizing 2', 5' liked riboadenylic salt to be mutually bonded. The resultant 2', 5' linked riboadenylic salt bonded with poly-L-lysine is then subjected to Sephadex column treatment, dialysis, desalting and freeze-drying treatment in order.

Description

[Detailed Description of the Invention] [Industrial Field of Application] The present invention. The present invention relates to a nucleotide compound bound to a basic amino acid peptide, particularly poly-L-lysine or polyornithine, and a therapeutic agent for viral diseases and a tumor agent containing the compound as an active ingredient.

[Technical background and prior art]

Viral infections can be easily overcome under conditions where the body's defense function is fully functional, but when the body's defense function is impaired, it develops and causes severe damage. bring people to death.

Genital herpes disease, which is caused by infection with simplex virus, is one of the viral diseases that has been on the rise in recent years, and is a source of infection for neonatal herpes disease, as well as being closely related to the etiology of uterine problems. It is known that

Also R5 (Re5piratory 5yncyt
ial ) virus is considered to be the most important pathogenic virus for respiratory tract infections in children, especially pneumonia and bronchial inflammation. In adults, upper respiratory tract infections can also cause symptoms such as rhinitis, pharyngitis, or bronchitis. Furthermore, viral encephalitis has a high mortality rate, and even if the patient survives, it leaves behind sequelae, making it a disease with a poor prognosis even among viral diseases.

Currently, therapeutic agents for viral diseases have side effects when administered systemically, and crystal nephropathy (crystal nephropathy) when administered in large doses.
m) Some even cause syndromes. Furthermore, the use of antiviral agents that are activated by the thymidine kinase enzyme of the virus has the problem of promoting the emergence in the future of mutation-resistant strains of the virus that do not have the thymidine kinase enzyme. Therefore, there has been a long-awaited demand for an effective antiviral agent that exhibits as few side effects and toxicity as possible and is less likely to produce resistant mutant strains.

2', 5'-linked riboadenylate has the general formula =ppp
5'A2'(p5'A) (n≧2, p is phosphoric acid, A is adenine, 21 and 2', 5' represents the bonding position) or the general formula: [wherein -Y is It is always a H1 adenosine call, except on the terminal phosphate which can be H.

Ichiho is 0.112.3 or 4.

-n is an integer from 1 to 14. ] is a strong protein synthesis inhibitor discovered in the extract of interferon-treated cells sensitized by a virus (Kerr 1.M, at a
l, Proe, Natl, Ac1d, Set
.

U, S, A,, Volume 25, pp. 256-260 1
978).

2'5'-linked riboadenylate is an interferon-inducing substance21, and is biosynthesized from ATP in the presence of double-stranded RNA by 5'-linked riboadenylate synthetase, and is a 2'5'-adenylate present in cells. By activating acid-dependent endoribonuclease (RNaseL), m
It has the effect of cleaving RNA, suppressing viral protein synthesis and intracellular proliferation of viruses.

Therefore, 215+-linked riboadenylate is considered to be an important mediator of the viral action of interferon. Furthermore, interferon has been recognized to have antitumor effects by suppressing cell proliferation (Klmue et al.
hi A, et at, Eur, J, Bio
-ehem, Vol. 114, p. 5-1O, 1981), the possibility of application of 2'5'-linked lipoadenylate has been suggested not only for its antiviral action but also for its relationship with anti-Ill111g action. Ru.

In addition, 2',5'-linked riboadenylate is difficult to enter cells due to its ionic nature, and is degraded by 2+,51-phosphogesterase, so it is difficult to enter cells in vivo or in cell extracts. It was pointed out that it has a short lifespan and weak biological activity, and to solve this problem, a more stable 21, sl-linked lipoadenylate derivative was proposed (Japanese Patent Application Laid-Open No. 205394/1983).

However, even this proposed derivative cannot pass through cell membranes in its original form, and some means of introduction into cells is required in order to use it as a drug. Regarding 2', 5'-linked riboadenylate, the microinjection method (Higashi Y, et al.
al, J. Biochem, (Tokyo)
Volume 91, pages 2021-2028, 1982],
Calcium phosphate coprecipitation method (Horanesslan A
, G, + et al.

Vivlogy, Volume 101, pages 81-9o
1980) and hypertonic medium method (Bryan R,G,
et al, FEBSLett, Volume 105, No. 4
7-52 (1979) 2', 5
When only +-linked riboadenylate is introduced into cells, its inhibitory effect on cell proliferation and virus proliferation has been confirmed. However, these methods cannot be applied to living organisms because they cause damage to cell membranes, and in the end, 2',
It has been practically difficult to use 5"-linked riboadenylate or its derivatives as a drug.

Although the general antiviral and antitumor properties of 2', 5'-linked riboadenylate or its derivatives have been taught, there are problems with its use as a drug, and specific The selective and specific action against viruses was unknown.

On the other hand, as a technique for imparting cell membrane permeability to substances that have a large molecular weight or do not permeate the cell membrane, a method has become known in which they are combined with a less toxic carrier, such as poly-L-lysine, and introduced into the ms (Shen W+C,e
tml, Proc, Natl, Acad.

Sct, tl, s, A, 75.1872-1876
, 1978). However, nothing is known about the applicability of this method 21 for 5+-linked riboadenylates or derivatives thereof.

The present inventors found that the above-mentioned means for introducing into cells is 2+,
These substances available for sl-linked riboadenylate and its derivatives and bound to the carriers described above are particularly effective against pathogenic viruses, herpes simplex virus type II and type I,
An antiviral effect has been discovered that greatly inhibits the proliferation of viruses within cells against cells sensitized to respiratory syncytial syncytial virus, poliovirus, and vesicular stomatitis virus.Based on this finding, the present invention has been achieved. did.

[Object of the invention and summary of the invention]

An object of the present invention is to provide a novel nucleotide compound having antiviral and tumorigenic properties, and another object of the present invention is to provide a novel nucleotide compound that can be used as a drug that can be easily and safely introduced into living cells. Another object of the present invention is to provide an antiviral agent and an antitumor agent that have extremely low activity and no side effects. Another purpose is the pathogenic virus, herpes simplex virus type and! The object of the present invention is to provide an antiviral agent that is highly effective against RS virus, respiratory syncytial virus, poliovirus, and vesicular stomatitis virus.

(The following margins) The present invention is based on the general formula (I): In formula C, -Y always represents H1 adenosine, or C1-2. F)
tglffi or secondary alcohol.

The intestines are O1! , 2.3 or 4.

n is an integer from 1 to 15.

K is a basic amino acid peptide.

X is an integer from 1 to 10. ]2'
A nucleotide compound characterized by having at least one ribose adenine unit with a +5'-internucleotide bond.

The present invention also provides general formula (I): except on the terminal phosphate, where -Y is ), always H1 adenosine, or
2.3 or 4.

n is an integer from 1 to 15.

-2 is H or C hydrocarbon or substituted carbon l +-4
0 hydrogen oxide, which is a group bonded to the N atom of the morpholine ring through one of its carbon atoms.

is a basic amino acid peptide.

R' is a group selected from the group consisting of hydrogen, alkyl groups, aromatic groups and other groups.

X is an integer from l to lO. 21
, 51-internucleotide bond, at least one ribose adenine unit, and a terminal morpholine unit, and the present invention also provides treatment for viral diseases using any of the above nucleotide compounds as an active ingredient. It is a therapeutic agent for the treatment of viral diseases caused by viruses selected from the group of herpes simplex virus type II and type I, respiratory syncytial virus, poliovirus, and vesicular stomatitis virus. Particularly effective.

The present invention further provides a therapeutic agent for am, which contains any of the above-mentioned nucleotide compounds as a blind active component, and is particularly effective for treating tumors whose tumor is caused by a virus.

[Detailed description of the invention]

The nucleotide compound of the present invention has a basic amino acid peptide bound to a 2', 5'-linked riboadenylate or a derivative thereof.

The 21.51M combined riboadenylate introduced into the cells is rapidly degraded and inactivated by degrading substances such as phosphatase or phosphogesterase, so 2F, 51 in the mouse L cell extract is rapidly degraded and inactivated. The half-life of conjugated riboadenylate is approximately 20 minutes. Therefore, it is desirable to use derivatives of weirs that have a weak affinity for these 2', 5'-linked riboadenylate-degrading enzymes, have long-lasting activity, and have a stronger effect on protein synthesis. The following derivatives are known. (JP-A-59-205394) General formula: [In the formula, -Y is always H1 adenosine, except on the terminal phosphate, which can be YtfiH, or H1 is 0,
1, 2.3 or 4.

n is an integer from 1 to 15.

n+ is a group selected from the group consisting of hydrogen, alkyl groups, aromatic groups and other groups.

-2 is a H or C hydrocarbon or substituted carbon 1-50 hydrogen, and is a group bonded to the N atom of the morpholine ring through one of its carbon atoms. ] There is...2'
, a 5'-internucleotide bond, at least one ribose adenine unit and a terminal morpholine unit.

In addition, the basic amino acid peptide is bound to 2', 5+-linked lipoadenylate or its derivative as a carrier. It is useful because it neutralizes the negative charge of 2',5+-linked lipoadenylate and increases the permeability of cell membranes.Other carriers include those with low toxicity and small molecules, such as polyester. Ornithine can be used.

Next, the binding reaction between 21, 5+-linked riboadenylate or its derivative and a basic amino acid peptide and the nucleotide compound obtained thereby will be described in detail.

The binding reaction between 2+, sl-bound lipoadenylate and poly-L-lysine is as follows.

First, the hydroxyl groups at positions 21 and 31 of the lipose present at the 21-terminus of the 2', 5'-linked riboadenylate are oxidized with sodium periodate under water cooling to form aldehyde groups. Next, poly-L-lysine is added to this oxidized 2', 5'-linked riboadenylate, a ship base is formed by the aldehyde group and the 6-amino group of poly-L-lysine, and the free aldehyde group is reduced with sodium boron cyanide to complete the bonding reaction with poly-L-lyline.

The 2', 5'-bound lipoadenylate bound to poly-L-lysine is separated from unreacted poly-L-lysine using a Sephadex column and eluted with sodium acetate. Next, the nucleotide compound of the present invention is obtained by dialysis, desalting, and freeze-drying. Furthermore, the proportion of 21, sl-bound riboadenylate bound to poly-L-lysine can be determined by measuring the ratio of absorbance using a spectrophotometer in a sodium acetate buffer. The nucleotide compound obtained as above has the general formula (I): Another nucleotide compound of the present invention obtained as above has the general formula (I): [wherein -Y is Y is always a primary or secondary alcohol of H, adenosine, or C, except on the terminal phosphate, which can be H.

1-20 The proverb is Oll, 2.3 or 4.

n is an integer from 1 to 14.

X is an integer from l to lO. ] This is a compound represented by

In addition, 21, 2, which is one of the derivatives of 51-linked riboadenylate described in JP-A-59-205394,
．． The binding reaction between 5-riboadenylate-morpholinoadenylate nucleotide and poly-L-lysine can also be prepared in a similar manner.

[wherein -Y is always H1 adenosine, or a C gt-class or secondary alcohol, except on the terminal phosphate, where Y can be H.

1-20 ■ is 0, 1, 2.3 or 4.

n is an integer from 1 to 15.

-2 is a H or C hydrocarbon or substituted hydrocarbon, and is a group bonded to the N atom of the morpholine ring through one of its carbon atoms.

K is a basic amino acid peptide.

R' is a group selected from the group consisting of hydrogen, alkyl groups, aromatic groups and other groups.

X is an integer from l to lO. ], and 2'
A nucleotide compound having a +5'-internucleotide bond, at least one ribose adenine unit and a terminal morpholine unit.

Next, the structure of the nucleotide compound of the present invention was determined as follows.

(1) Structure of 2,5''-linked riboadenylate bound to poly-lourisine A sodium acetate solution (pH: 4・75), the absorption spectrum is 258
It has characteristic absorption peaks at n■ and 222n■, and the results are shown in FIG.

In addition, free poly-lourisine recorded a peak at a wavelength of 212 nm in sodium acetate buffer, but 2',
The peak is 22 due to binding with 5'-linked riboadenylate.
Since there was no change in the absorption peak at 258n due to the adenyl ring when the wavelength was shifted to 2 nm, it was found that there was a bond between the ribose ring and poly-rollidine. Further, the constituent molar ratio of poly-lauridine and 21, sl-bonded ribadenylate is calculated from the ratio of the absorption peaks at 258 nm and 222 nm in the absorption spectrum in a sodium acetate buffer. From this result, the value of -I in the general formula (I) can be determined.

That is, the molecular absorption coefficient (ε258) of 2' + 5' bonded riboadenylate of dimers, trimers, and tetramers is 2
5800.34,500 and al, 600, and the molecular absorption coefficient of poly-lourisine is 20.218, so the following formula: Absorbance (The constituent molar ratio is calculated from the value of 258 nJ and the molecular absorption coefficient of 720,218. Ru.

In addition, 2', 5 bonded to poly-lourisine in the aqueous solution
The absorption peak of the bound ribadenylate was characteristic, rising from around 258 nm and 240 nm, and lacking the absorption peak at 222 nm.

From the above, poly-lourisine is bonded to the ribose ring of 2', 5'-linked riboadenylate, and the absorption spectrum in sodium acetate buffer is 258am and 222n.
The constituent molar ratio of 2', 5'-bonded riboadenylate and poly-lauridine, determined from the ratio of the absorption peaks in (2), is 1:1 to 1.
: The range is 10. That is, 1 to 10 moles of poly-lourisine are bound to 1 mole of 2', 5'-linked riboadenylate. Therefore, it has a structure represented by the general formula (1).

(2) Structure of a derivative of 2T, 5T-bound riboadenylate bound to poly-lauridine 2, one of the above derivatives obtained in Example 2 described below
The tetrameric triphosphate modified at the 1-terminus was prepared from the above (1
), the results showed that the derivative had the same binding site for poly-lysine as in (1).

Similarly, for other derivatives, the binding site of poly-L-lysine and the constituent molar ratio of poly-lolysine and z+, 5T-bound riboadenylate derivatives were determined,
It was confirmed that it has a structure represented by the general formula (1).

The antiviral activity of one of the nucleotide compounds of the present invention, ie, 2T, s+-linked riboadenylate combined with poly-lourisine, is as follows. That is, when human-derived embryonic line germinoblastoma was infected with DNA viruses such as herpes simplex type I virus, herpes simplex type I virus, poliovirus, and vesicular stomatitis virus, the growth-inhibitory concentration was measured, and the concentration was 19M. showed a significant growth-inhibiting effect on all DNA types of viruses, inhibiting the growth of vesicular stomatitis virus by about 25%, poliovirus by about 75%, and herpes simplex virus by 100%. Furthermore, against herpes simplex virus types II and I, a 50% effective concentration was obtained at approximately 28 n14 (28pool/d), and there was no cytotoxicity observed in morphological changes, making it specific and very low. It was confirmed that the virus exerts its effect at certain concentrations.

In addition, when we infected uterine cervical cancer cells with RS virus, which is an RNA type virus, and conducted a test to suppress the growth of this virus using a plaque assay, we found that the 50% target effect was 11.
obtained at a concentration of nM (If pool/mA),
No cytotoxicity was observed. That is, the growth-inhibiting effect was even more effective than that against herpes simplex virus.

Another nucleotide compound of the present invention includes 2'
When a tetrameric triphosphate modified with the goo-end of boadenylate combined with I s' M is used as a derivative,
Similar test results show that herpes simplex virus type II and! 50% targeting effect at a concentration of 0.2 nM against the virus and approximately 40% at a concentration of 0+2 nH against respiratory syncytial virus.
inhibitory activity was observed. Therefore, it was shown that the derivative also has a win-win virus effect.

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

Example I: 2+, =, attached to poly-L-lysine
Preparation of nucleotide compounds consisting of l-linked riboadenylate ■ Coupling reaction of poly-lysine with 2', 5'-linked riboadenylate 2', 5'-linked riboadenylate tetramer triphosphate (Seikagaku Corporation) 40 pMol (90 pg)
It was dissolved in μl of distilled water for injection and placed under water cooling. To this, 0.1M sodium acetate buffer (pH: a
, 75) 4 μl was added under water cooling and stirred with a touch mixer. The reaction was continued for 30 minutes while occasionally stirring under water cooling.

Next, poly L-lysine (MW) was added to this reaction solution.

Viscosity: 22+000, manufactured by Sigma)
14 nMo1 is dissolved in 40 pJ! 0.2 of
Add 20 μl of 0.2 M sodium phosphate buffer (pH 78.0) in which 2 pMol of sodium boronate cyanide (manufactured by Hanui Chemical Co., Ltd.) and M sodium phosphate buffer (pH: 8.0) are dissolved, and the mixture is heated to room temperature. The mixture was left to stand for 2 hours to complete the binding reaction between poly-L-lysine and 2', 5'-linked riboadenylate.

■Sephadex G50 [Fine 220
-80 p, manufactured by Pharmacia Fine Chemicals (Sweden)], add 150 p of distilled water for injection, and add 100 p.
Gels were prepared by swelling for 3 hours at °C. This gel was packed into a glass column with a diameter of lα and a height of 40α.
．． Equilibration was performed with 1M sodium acetate buffer (pH: 4.75). The buffer was run at a flow rate of approximately 1-74 minutes and fractionated every 2+d. The void volume of the column is
0 of blue dextran (MW: 2 x 106)
．． When measured using 100 μE of a 1% aqueous solution, approximately 1
It was 4-.

Next, 100 μl of the reaction solution of 21,51-linked riboadenylate bound to poly-L-lysine was layered on the column.
Fractionation was carried out under the same conditions as the buffer. The absorbance of the obtained fraction was measured using a spectrophotometer (manufactured by JASCO Corporation) at a wavelength of 258 nm to obtain a fraction of 2', 5'-bonded riboadenylate bound to poly-L-lysine. .

■Purified cellulose tube (seamless, 20/32, diameter:
160n (manufactured by Sanko Junyaku Co., Ltd.) was charged with the fraction obtained in step (1) above, dialyzed overnight in distilled water at 4°C, desalted, and freeze-dried to obtain one of the nucleotide compounds of the present invention. Six freeze-dried products containing 75 μg (50 nMo1) of nucleotide compounds consisting of 2+, sl-linked riboadenylate bound to poly-L-lysine were obtained. Recovery rate is 90
% or more.

In addition, this freeze-dried product 75 ug (50mMo1)
Poly L- 2+ , s bound to lysine
When the concentration of +l-bound riboadenylate was determined, it was found to be 100
It was 9M.

and poly-
According to the above calculation formula, the constituent molar ratio of lysine is 1+6720,218 0.7 / 41,600, and this compound has X in the general formula (I) above.
The compound had a value of 5.

Example 2: 2+, 5+ bound to poly L-lysine
Preparation of a nucleotide compound tube consisting of a 21-terminally modified tetrameric triphosphate of a derivative of bound lipoadenylate In Example 1, instead of 60 nMo1 of 2',5' bound lipoadenylate tetrameric triphosphate, 21 of its derivatives
- terminally modified tetrameric triphosphate 60neo
A lyophilized product 67p of this derivative bound to poly L-lysine was prepared in the same manner as in Example 1 except that 1 was used.
g (45nMo+) was obtained.

The 2'-terminally modified tetrameric triphosphate has the following structural formula.

(Left below) Example 3: Nucleotide compound consisting of poly-L-lysine-bonded zl, s+-bonded riboadenylate! Inhibitory effect on pure herpesvirus type II and summer type Infectious titer was measured using a plaque assay to confirm the inhibitory effect on virus proliferation.

Human-derived embryonic fibroblastoma (12th to 13th passage) was cultured in a monolayer in a plastic petri dish (24yell, manufactured by NUNK) in Eagle's HEM medium 1111+++jl supplemented with 8% bovine serum 1 (manufactured by Gibeo). .

After the intercellular spaces have completely disappeared, discard the medium and clean the cell surface.
Washed twice. After washing, an aqueous solution of 2'+5'-bonded riboadenylate bound to the same poly-L-lysine as in Example 1, adjusted to each covering concentration in the Hi range of θ to 100 OpM, was poured into each hole (tell) of the Petri dish for 1tR1. Add 3
7@() for 3 hours.

To this culture, a diluent l- of herpes simplex virus summer type (strain K2S) and summer type (strain 0), which had been thoroughly washed once with MEM i and adjusted to 30 PFU (plaque forming units), was added. I did MN. The Petri dishes were shaken every 10 minutes at 37°C for 1 to 1.5 hours to adsorb these viruses to the cells. After washing twice with growth medium, lysed 0.8% agar culture (MEM i containing 2% calf serum) was added.
(including soil) was layered on the petri dish after adsorption. After the agar after overlay solidifies, it is cultured at 37°C for 2 days to allow infection to grow, form plaques, and neutral red (
The cells were stained by adding an appropriate amount of (neutral, red) and further incubated at 37°C for 1 hour. This was fixed by adding an appropriate amount of 5% formalin solution, the fixative was washed, the number of formed plaques was measured, and the growth inhibition rate (%) was calculated according to the following formula.

The number of plaques in the control was 31.5 (average value) and 41 for herpes simplex virus summer type and summer type, respectively.
．． 5 (average value).

In addition, cell attributes were evaluated by observing morphological changes in cultured cells due to virus infection, dividing the cells into three levels: +, +, and -.

The average of the results of these two measurements was as shown in Table 81.

(Margin below) (Number of control plaques) Table 1 (Note 1) According to Table 1, the drug concentration for herpes simplex virus type 1 and type 1 is the concentration of 21,51-bound riboadenylate bound to poly-L-lysine. , 2*, 5* conjugated lipoadenylate with poly L-lysine at a concentration of 250
μ support (250pool/+ag) completely inhibits the proliferation of herpes simplex virus type 1 and type I, and the 50% effective concentration is approximately 28mM (approximately 28p) for both type I and type I.
ool/mj). Cytotoxicity is IpMll1
It was observed only when it was above 30 degrees. Also, 50% shown in Table 1
The selective toxicity coefficient calculated from the effective degree (ED) is
For herpes simplex virus type 1 and type I, it was 0.5-170.028 == 18-36.

Example 4: Effect of a nucleotide compound consisting of 21,51-linked riboadenylate bound to poly-thi-lysine on the proliferation of RS virus. In the same manner as in Example 3, the infectious titer was measured by the plaque assay method, and the virus proliferation was measured. The inhibitory effect was confirmed.

In Example 3, human cervical cancer He1a was used instead of human fetal lI ostoblasts (!2 to 13 passages).
JI cells were used, and herpes simplex virus type 1 (K2S strain) adjusted to 30 PFtl (plaque forming unit)
and RS virus (T, ong strain) adjusted to 100 PFU was inoculated instead of type ■ (strain 0), the culture temperature was changed from 37°C to 35”C, and the growth of infected cells was inhibited after agar solidification. Example 3 except that it was carried out for 3 days
Plaques were formed in the same manner as above, and the growth inhibition rate (%) was calculated.

The results were as shown in Table 2.

Table N2 Antiproliferation effect on RS virus (blank below) (Note 1) The drug concentration is 1 degree of poly-L-lysine-bound 21,51-linked riboadenylate.According to Table 2, poly-L-lysine
2',5'-linked riboadenylate with lysine attached is
A concentration of 500 nM (500 pMol/d) completely targets the proliferation of RS virus, and the 50% effective concentration is 11
nM (11pool/-). Cytotoxicity is 1
Observed at 9M or higher. Also, the 50 shown in Table 2
The selective toxicity coefficient, calculated from the % effective concentration (ED), was 1-270.01:91-182, indicating a higher potency of 21,51-conjugated riboadenylate against respiratory syncytial virus than against herpes simplex virus. It is characterized by high

What is the growth-inhibiting effect on human-derived cervical cancer cells? μM
observed at concentrations above.

Example 5: Growth inhibitory effect of poly-L-lysine-linked 2', 5'-linked riboadenylate nucleotide compound on herpes simplex virus summer type and summer type, poliovirus, and vesicular stomatitis virus human-derived fetal fibroblasts Make cells plastic 9
M-layer culture was performed in Eagle's MEM medium supplemented with 2% calf serum (manufactured by Gibco) using 6 wells (W) from NUNK. Poly-lysine-conjugated 2″, 5″-linked riboadenylate (2
% of amended serum-containing MEM medium) was added to each well of the petri dish, and incubated at 35°C for 3 hours.
Cultured for hours. After that, it was washed once with MEN medium, and herpes simplex virus summer type (KOS strain) and summer type (0 strain) were isolated.
Poliovirus type I (Strain Mahone Y) and various diluted viruses of vesicular stomatitis virus (multiplicity of infection: Mol = 0.01) were inoculated and adsorbed for 1 hour in a 35" (under gentle shaking). After washing with HEM medium, 1-1 MEN medium containing 2% calf serum (manufactured by Gibco) was added to each tell, cultured at 35°C, and each sample collected orally was - Frozen cells were frozen at 80°C. Frozen cells were frozen and thawed three times and incubated at 3000 rpm and 4°C for 10
After centrifugation for a minute, the virus in the supernatant virus solution was identified according to a conventional method, and the virus titer was measured by a virus infectivity titer assay.

These results were as shown in FIGS. 2 and 3.

A, B, C, and D in Figures 2 and 3 are for herpes simplex virus summer type (K2S strain), its summer type (0 strain), vesicular stomatitis virus, and poliovirus (Mahoney strain), respectively. , in the figure (−・
-) is the control, and the vertical axis is the virus titer (TCD/
-logarithm value), and the horizontal axis shows the elapsed time (hours) after adsorption of each virus.

According to Figure 2, 2', 5 bound to poly L-lysine
'Conjugated riboadenylate completely inhibits the growth of herpes simplex virus summer type and summer type at a concentration of lμ;
Excellent antiviral activity was confirmed. Furthermore, according to Figure AA3, the virus titer for other open type A viruses, vesicular stomatitis virus and poliovirus, decreased by approximately 78% compared to the control after 2 days of culture after virus infection. , 1g! It was found to have a significant effect on suppressing virus proliferation.

This also indicates that a culture period of 2 days after virus infection is preferable for assaying virus growth inhibition.

Example 6: Propagation of a nucleotide compound consisting of a tetrameric triphosphate modified at the 21-terminus of a 2',5'M conjugated riboadenylate coconductor bound to poly-L-lysine against herpes simplex virus summer type and summer type. In suppressive effect Example 3, 21 bound to poly-L-lysine
The virus suppressive effect was confirmed in the same manner as in Example 3, except that the poly-L-lysine-bound derivative obtained in Example 2 was used instead of the 51-bound lipoadenylate.

The results were as follows when compared with the results in Table 1 for Example 3.

The above derivative with poly-L-lysine attached has a concentration of 1
pM (1 pMol/i), completely targets the proliferation of herpes simplex virus summer type and summer type, and the 50% effective concentration (Eel) is approximately 0.2 for both herpes simplex virus types II and l. 2',5'-linked riboadenylate tetramer 3 of Example 3 at a low concentration of mM.
It was found to be more than 100 times more active than phosphoric acid.

This result shows that the correlation between proliferation and ejection of herpes simplex virus lff1 and type II and concentration is similar to that between inhibition of protein synthesis and concentration in vitro using L cell extracts. Also, the improvement in activity is 2
Tetrameric triphosphate modified at the 2', 5'-end
'This suggests that it confers low damage to riboadenylate-degrading enzyme.

Example 7: Growth inhibitory effect on RS virus of a nucleotide compound consisting of a tetrameric triphosphate modified at the 2''-end of a 21,5'-linked lipoadenylate derivative bound to poly-L-lysine. -zl with one lysine attached
, Instead of 5'-linked lipoadenylate, a nucleotide consisting of a tetrameric triphosphate modified at the 2'-terminus of a 2',5'-linked riboadenylate derivative bound to the same poly-L-lysine as in Example 2. The virus proliferation inhibitory effect was confirmed in the same manner as in Example 4 except that the compound was used.

The results were as follows when compared with the results in Table 2 of Example 4.

The above derivative completely inhibited the growth of RS virus at a concentration of 1 pH. Even at a concentration of 0.2 μM, approximately 4
Comparing 8 nM, which showed a virus proliferation inhibition rate of 0%, with 8 nM, which showed a virus proliferation inhibition rate of 40% in Example 4, it was confirmed that the above-mentioned derivative had an activity about 40 times more active.

This indicates that the above-mentioned derivatives have a significant viral transmission effect not only on DNA viruses but also on RNA viruses.

In addition, the nucleotide compound of the present invention can be used as a co-combinant agent to enhance the concomitant use of interferon,
It can be used as a more useful antiviral and @tumor drug.

The nucleotide compound of the present invention exhibits high solubility in aqueous solutions and can be used in various dosage forms, and can be administered intralesionally, locally in an artery, intracerebrospinal fluid, and for ocular infections. Possible uses include eye drops for upper respiratory tract infections, nasal drops and nasal and throat sprays for upper respiratory tract infections.

In the field of dermatology, it can also be applied locally to the site of infection as an ointment, cream or aerosol to external tissues.

〔Effect of the invention〕

(2) To impart cell membrane permeability to 2', 5'-linked riboadenylate and its derivatives, thereby enabling their use as drugs against viruses and enemy tumors.

■It is possible to provide a virus-repelling agent that exhibits little toxicity and in particular does not cause the emergence of virus mutation-resistant strains.

- Providing a curative agent that is highly effective at very low concentrations against viral diseases caused by herpes simplex virus types 1 and 1, respiratory syncytial virus, poliovirus, and vesicular stomatitis virus.

■It is caused by a virus! 1111! To provide a therapeutic drug that exhibits little toxicity and sometimes does not cause the appearance of virus mutant V4i# strains.

[Brief explanation of the drawing]

Figure 1 shows the sodium acetate solution (pH: 4.75).
FIG. 2 is a chart showing the absorption spectrum of poly-L-lysine-bound riboadenylate in ), FIG. 1 is a chart showing changes over time in virus titers against vesicular stomatitis virus and poliovirus.

Claims (6)

[Claims] (1) General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, -Y is always H, adenosine, or C_1_-_2, except on the terminal phosphate where Y can be H.
It is a primary or secondary alcohol of _0. m is 0, 1, 2, 3 or 4. n is an integer from 1 to 14. K is a basic amino acid peptide. x is an integer from 1 to 10. ] A nucleotide compound characterized in that it has a 2',5'-internucleotide bond and at least one ribose adenine unit. (2) General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, -Y is always H, adenosine, or C_1_-_2, except on the terminal phosphate where Y can be H.
It is a primary or secondary alcohol of _0. m is 0, 1, 2, 3 or 4. n is an integer from 1 to 15. -Z is H or a C_1_ to_4_0 hydrocarbon or substituted hydrocarbon, and is a group bonded to the N atom of the morpholine ring through one of its carbon atoms. K is a basic amino acid peptide. R' is a group selected from the group consisting of hydrogen, alkyl groups, aromatic groups and other groups. x is an integer from 1 to 10. ] A nucleotide compound characterized in that it has a 2',5'-internucleotide bond, at least one ribose adenine unit and a terminal morpholine unit. (3) A therapeutic agent for viral diseases containing the nucleotide compound according to claim 1 or 2 as an active ingredient. (4) Contains the nucleotide compound according to claim 1 or 2 as an active ingredient, and contains herpes simplex virus type I, herpes simplex virus type II, RS (Respirator).
y syncytial) virus, poliovirus, and vesicular stomatitis virus. (5) A tumor therapeutic agent containing the nucleotide compound according to claim 1 or 2 as an active ingredient. (6) A therapeutic agent for tumors caused by viruses, which contains the nucleotide compound according to claim 1 or 2 as an active ingredient.