BG100242A - Arylization composition having antiviral effect - Google Patents

Abstract

The arylization composition is used for the treatment of cancer and viral infections. It includes different active compounds each of which consists of aromatic ring with substituent of at least one labile group and substituent of at least one electrophilic group. The preferred active compounds include chlorbenzenesulphonic acids and optionally - halogenized nitrobenzene derivatives. As antiviral compositions the compounds are effective against HIV infections.

Description

The present invention relates to arylated compounds and in particular to phenylated compounds that are suitable for therapeutic agents especially in the treatment of cancer and diseases caused by viral infection.

In its broadest meaning, the invention relates to aryl compounds useful in the treatment of neoplasms or a viral infection such as that caused by HIV.

The aryl compound is in particular a compound with an aryl group whose aromatic ring is preferably carbocyclic and in each case has at least one labile substituent and at least one electrophilic substituent. The carbocyclic or other aromatic ring is preferably monocyclic and in each case the aromatic ring is one which is bonded to parts of one or more carboxylic acids or sulfonic acid together with one or more nitro and / or amino groups and / or one or more halogen substituents. The substituents preferably do not include more than two nitro substituents. The combination of halogen (e.g. chlorine) and nitro substituents, 20 when it comes to a monocyclic aryl compound consisting of a ring bearing a carboxylic acid substituent, is a particularly efficient structure. One example of such a structure is determined by the combination of mono-nitro and mono-chlorine substitution, for example 2-chloro-5-nitrobenzoic acid and 2-chloro-4-nitrobenzoic acid.

According to the invention, the compounds used for the treatment of cancer and disease caused by a viral infection, in particular AIDS (AIDS) have the structure of an aromatic ring with at least one substitute that is easily mobile ( labile) group and at least one substituent which is an electrophilic group. Provided that the compound has two ortho-nitro groups and one para-sulfone group or three symmetric nitro groups and the labile group at position one is one of the groups defined in international application No. WO 91/15200, then its use as an agent is in a concentration greater than 1 * 10 ' ³ mol / liter.

In general, the arylating agent of the invention 35 comprises compounds of the general formula

2.

in which η is an integer but at least 2 and each X is the same or different and is one labile group or one electrophilic group provided that when there are at least two groups X other than nitro, at least one is a labile group and at least one is an electrophilic group.

In addition, it has been found that, according to the putative arylation mechanism, the administration of the compounds allows for high concentrations and, accordingly, dosages. Typically, these concentrations, when administering the compounds of the invention, are at least about 1 * 1 Cr ² mol / liter, 15 which is usually determined at a dose of at least about 5 mg / kg.

In selecting the substituent groups for the compounds according to the invention, one key feature is to provide the middle of the aromatic ring with at least one substituent which is a labile group and at least one substituent which is an electrophilic group. In addition, a group 20 that can be classified as labile under the conditions of a particular ring can be classified as electrophilic under the conditions of an alternative ring. Moreover, when there are at least two nitro substituents, the labile substituent group may be a hydrogen ring. This can be understood as preferred groups - substituents can be 25 defined as those in which at least one X is selected from one of the following groups, namely:

electrophilic groups - SO ₃ H, SO ₃ M (where M is a metal, for example potassium), halogen or NO ₂ ;

labile groups - halogen, SO ₃ H, SO ₃ M (where M is a metal), NH ₂ , substituted NH ₂ for example NHR 1 and NR 2 (where and R ₂ are the same or different and are alkyl, alkyloxy or hydroxyalkyl) , COOH, CONH ₂ , substituted CONH ₂ for example CONHR-1 and CONR-R ₂ (where R _t and R ₂ are defined above) or COOR ₃ (where R ₃ is metal or alkyl).

Thus, the following may be mentioned as major examples of compounds according to the invention, namely:

3.

hlordinitrobenzensulfonovi acids chlorobenzenesulfonic acid dihlorbenzensulfonovi acids aminodinitrobenzensulfonovi acids nitrometilbenzensulfonovi acids glugationildinitrobenzensulfonovi acids nitrohlorbenzensulfonovi acids dinitrobenzensulfonovi acids dinitrohlorbenzeni, omega dinitrofluorobenzene, dihlordinitrobenzeni, trinitrophenol, for example, picric acid, trinitroanilini, trinitrohlorbenzeni, trinitrobenzenesulfonic acid hlordinitrobenzoeni acids dichlorobenzoic acids din itbenzoic acids, nitrochloroanisols, aminodinitrobenzamides, dinitroanilines, dinitrochloroanilines, chlornitroanilines, dinitrofluoranilines.

The above compounds can be basically summarized as compounds of the general formula:

In 'T. X ' g ^x ” (And) 30 p in where X 'is SO ₃ H, SO ₃ M (where M is metal), halogen for example chlorine,

fluorine and T.H., COQ (where Q is hydroxy, amino or substituted amino, or group

OR ₃ in which R ₃ is metal or alkyl), NH ₂ substituted NH ₂ , NO ₂ or OH;

4.

X 'is hydrogen, halogen, glutathione or nitro;

each B is the same or different and is hydrogen, halogen or nitro;

and C is hydrogen, nitro, amino (including substituted amino), halogen, alkyl or glutathione.

Among these compounds with preferred characteristics are the following:

X 'is SO ₃ H, SO ₃ M (where M is a metal), halogen for example chlorine, fluorine, etc., amino, nitro or COOH, and

C is hydrogen, alkyl for example a methyl, amino or nitro group.

Compounds which exhibit anticancer and antiviral effects ίο according to the invention can be derived from the number of those with preferred groups, for example as follows:

(i) Compound of general formula

SO ₃ H

where A is hydrogen, halogen for example chlorine, fluorine, etc. or glutathione;

Be hydrogen, nitro or halogen for example chlorine, etc .;

C is hydrogen, nitro, amino (including substituted amino),

halogen, alkyl or glutathione;

and D is hydrogen, halogen or nitro.

The above compounds of general Formula III are preferred because it is suggested that the sulfone group may contribute to an emulsifying effect, which is useful because it increases the solubility of the compounds, which gives better bioavailability in cells (at the cellular level).

Among the above compounds of general formula III, those which are more preferred are:

of 4-chloro-3,5-dinitrobenzenesulfonic acid,

4-chlorobenzenesulfonic acid,

2,5-dichlorobenzenesulfonic acid, 4-amino-3,5-dinitrobenzenesulfonic acid,

3-nitro-4-methylbenzenesulfonic acid,

2-chloro-3,5-dinitrobenzenesulfonic acid,

5.

2-glutathionyl-3,5-dinitrobenzenesulfonic acid,

4-Glugathionyl-3,5-dinitrobenzenesulfonic acid,

3-nitro-4-methylbenzenesulfonic acid,

3-nitro-4-chlorobenzenesulfonic acid,

2.4-dinitrobenzenesulfonic acid.

Among them, the following are particularly preferred:

4-chloro-3,5-dinitrobenzenesulfonic acid, 4-chlorobenzenesulfonic acid,

2.5-dichlorobenzenesulfonic acid, 4-amino-3,5-dinitrobenzenesulfonic acid, 3-nitro-4-methylbenzenesulfonic acid,

2-Chloro-3,5-dinitrobenzenesulfonic acid, (ii) A compound of the general formula:

wherein Halo is halogen for example chlorine, fluorine, etc. and each B is the same or different and is defined above.

Among the above compounds of general formula (IV), those which are particularly preferred are:

1-chloro-2,4-dinitrobenzene,

1-chloro-3,4-dinitrobenzene, 1-fluoro-2,4-dinitrobenzene,

1,2-dichloro-4,5-dinitrobenzene,

1.3-dichloro-4,5-dinitrobenzene.

zo In particular, the following are preferred:

1.3-dichloro-4,5-dinitrobenzene,

-chloro-2,4-dinitrobenzene,

-fluoro-2,4-dinitrobenzene.

6.

(iii) Compound of general formula

where E is SO ₃ H, SO ₃ M (where M is a metal for example potassium), NH ₂ or substituted NH ₂ , halogen or hydroxy.

ίο The following are more preferred among the compounds of general formula V:

2.4.6- Trinitrophenol (picric acid),

2.4.6- Trinitroaniline,

2.4.6- Trinitrochlorobenzene,

2.4.6- Trinitrobenzenesulfonic acid.

Among the above compounds, the first and the third are particularly preferred.

(iv) Compound of general formula:

COQ

where all B is the same or different and B is defined above; G is defined above for group C with the exception of alkyl and glutathione; J is hydrogen or halogen and

Q is hydroxy, amino or substituted amino, or the group OR ₃ in which R ₃ is metal or alkyl.

Among the compounds of general formula VI, those which are more preferred are:

2.4-chloro-3,5-dinitrobenzoic acid, 4-chloro-3,5-dinitrobenzoic acid,

2.5-dichlorobenzoic acid,

2.4-dinitrobenzoic acid,

3.5-dinitrobenzoic acid,

3-nitro-4-chloroanisole,

4-amino-3,5-dinitrobenzamide.

Ί.

Of the above preferred compounds, the last three are particularly preferred.

(ν) Compound of general formula:

ΝΗ ₂ (VII)

wherein all B is the same or different and B is defined above to include both an amino group and substituted group derivatives.

Among the compounds of general formula VII, those which are more preferred are:

2,6-dinitroaniline,

2.4-dinitroaniline,

3.5-dinitroaniline,

2,4-di-nitro-6-chloro-oran yl,

2,6-dinitro-4-chloroaniline,

2-chloro-4-nitroaniline,

2,4-di nitro-5-fluoroanil and the like.

Among them, 2,6-dinitroaniline is particularly preferred.

As noted above where there are at least two nitro substituents the hydrogen ring may admit a labile group. In this connection 25 it may be noted:

(vi) Compound of general formula

NO _{2 to} (VIII) no ₂ which may be:

1.2-dinitrobenzene,

1.3- dinitrobenzene,

8.

1,4-dinitrobenzene.

The compounds of the invention can be prepared by known technical methods for the preparation of benzene-substituted compounds. These methods are described in various standards, for example, in Organic Syntheses 1963, 5 Collective Volume 4, pages 364 to 366 by Harry R. Schultz, published by John Wiley and Sons Inc.

The compounds of the invention are therapeutic agents that can be administered in pharmaceutical compositions (e.g., intravenous - iv, intraperitoneal - ip, oral or sc - subcutaneous use), i. These compositions contain at least one active compound and one solvent. or carrier. Accordingly, the invention also relates to a pharmaceutical composition which comprises as active ingredient at least one compound according to the invention and a pharmaceutically acceptable solvent or carrier (e.g. aqueous solution).

This composition may be in bulk or collected in dosage forms, for example in tablets, capsules, powder, solution or suspension. Soft gel capsules are probably the most comfortable. The composition can be a liposomal composition or applied in a gradual and slow delivery system

of the drug in the body.

The compositions according to the invention can be prepared using the active components defined in the present invention and components in accordance with conventional pharmaceutical practice. The solvents, excipients or carriers, etc. that may be used are well known in the pharmacy and the form chosen for each particular treatment regimen will depend on the case and the physician's choice.

For example, as illustrated below, the agents of the invention can be administered in a solution of sterile deionized water. Also, if necessary, the dissolution can be facilitated using dimethyl sulfoxide (DMSO) or alternatively alcohol or glycol or vegetable oil. Most preferably, the compounds are administered in corn oil or as a solution in DMSO and sterile water.

The invention further includes, in addition to the abovementioned use cases of the compounds as defined herein, also the preparation of

9.

medicines for the prevention or treatment of cancer or viral infection, for example for the reduction or elimination of cancers.

The dosage regimen for the use of the agents according to the invention can be determined by animal testing as described below. In such trials, the doses that have proven effective are usually around 50 mg / kg, but can increase to 200 mg / kg, even reaching 400 mg / kg or more. In this situation, it should be expected that the typical human dose will be from about 5 mg / kg, usually up to about 20 mg / kg, and possibly generally up to about 40 mg / kg or higher. The concentration of ίο and the dosage must be sufficient to cause the aryl mechanism to be activated.

As can be seen from the particularly preferred compounds

Listed below, the compounds that are most effective are arranged in a presumed order of decreasing effectiveness:

4-chloro-3,5-dinitrobenzenesulfonic acid,

4-chlorobenzenesulfonic acid,

1,5-chloro-2,3-dinitrobenzene,

2,4,6-trinitrophenol (picric acid),

2.4-chloro-3,5-dinitrobenzoic acid,

2.5-dichlorobenzenesulfonic acid,

4-amino-3, 5-dinitrobenzenesulfonic acid,

3-nitro-4-methylbenzenesulfonic acid,

4-chloro-3,5-dinitrobenzoic acid,

2.6- dinitroaniline,

2.4-dinitrochlorobenzene,

2.4-dinitrofluorobenzene,

2.4.6- Trinitrochlorobenzene,

2.4-dichlorobenzoic acid,

2-chloro-3,5-dinitrobenzenesulfonic acid,

2.4-dinitrobenzoic acid.

Particularly preferred compounds are those in which at least one X is selected from:

- substituent labile group (s) - 1 or 2 halogen groups and / or NH ₂ or substituted NH ₂ and / or COOH or substituted COOH and / or alkyl and / or 35 SO ₃ H / SO ₃ M;

10.

- a substitute electrophilic group (s) - 1 or 2 nitro groups and / or SO3H / SO3M and / or 1 or 2 halogen groups.

Furthermore, while the compounds of the invention may be administered at the doses given in the examples above, when they have symmetrically located three nitro substituents, similar to the active agent disclosed in International Application No. WO 91/15200, so and here their concentration in the composition should be greater than 1 x 10 ' ³ mol / l, preferably at least 1 x W ² mol / l.

As can be seen from the results described in Table 8 below, 2-chloro-510 nitrobenzoic acid showed significant anti-tumor activity in vivo.

This could not be supported in vitro, indicating that some compounds of the invention need activation in the patient's body. This compound and some other compounds may also be immunomodulators.

The following animal studies illustrate the remarkable 15 activity of the compounds of the invention.

Animal studies.

The purpose of the studies was to evaluate the antitumor properties of

a group of compounds of structural similarity that can react as arylating agents. Their antitumor activity in vivo was evaluated against two types of ascitic tumors, MAC15A mouse rectal adenocarcinoma, P388 mouse leukemia, and different models of solid tumors. MAC15A acetite tumor cells were transplanted into NMR1 rats by ip - intraperitoneal infection with a cell density of 1 x 10 ⁵ cells in

200 μΙ buffer (Table 1). P388 were ip - intraperitoneally transplanted into BDF1 rats at a cell density of 1 x 10 ⁶ cells in 200 μΙ buffer (Table 2).

Solid tumor models include MAC13 and MAC16 adenocarcinomas in the rectum of mice, B16F1 includes mouse melanoma, and M5076 are reticulum sarcoma cells.

3 Treatment begins 3 days after ip transplantation (ip read all intraperitoneally) or, in the case of solid tumors such as MAC13 and MAC16, when the median tumor volume reaches 40 mm ³ .

The test animals in both cases were located in groups of 5 to 8 animals.

11.

Animals were sacrificed after 12 days or when the tumors were inflamed, or the tumor volume exceeded 1000 mm ³ , or the weight loss exceeded 50%.

Unless otherwise agreed, the 5 compounds used were dissolved in DMSO and diluted with sterile distilled water at a suitable concentration before being applied in a single volume of 200 μΙ solvent.

Antitumor responses were determined by comparing mean survival times or suppression of tumor growth against solvent control. The results obtained are shown in Tables 1 to 8 below.

The preparation of dosage solutions is illustrated as follows. Subjects: 10 experimental animals weighing 22 g each.

Dosage: 50 mg / kg body weight per animal per day ie. 1.1 mg per mouse per day.

The total dosage amount is 55 mg of active ingredient (corresponding to a 5-day treatment regimen).

The total composition is 10 ml of solvent plus 55 mg of active substance, distributed in 50 doses of 1.1 mg each and dissolved in 200 μΙ solution.

T / C% is determined as follows:

The animal survival test T days survives Control - In days T / C% = (T / C) x100 An example of a survival test T = 443 days T / C% - (443/100) x100 = 443 Control C = 100 days

An illustration supported by 158 or more definitions is sufficient to warrant a clinical experiment.

Conclusion

The effect of one group of major halogenated aryl compounds on the growth rate of a number of experimental tumors was evaluated in vivo, and the following was detected:

zo 1. Structurally activated ligaments show maximal activity against MAC15A adenocarcinoma of the rectum of mice in female NMRI mice

12.

one single dose regimen when halogen is maximally activated for nucleophilic attack.

2. The most active compound is 4-chlorobenzenesulfonic acid (T / C% 443) administered at 100 mg / kg body weight per day for 5 days.

3. Against reticular cell sarcoma 2,4-dichloro-3,5-dinitrobenzoic acid shows activity in the daily dosing regimen up to 25 mg / kg body weight in both ip and sc (sc read all over the skin) routes of administration . Accordingly, the amide and methyl ester show a 10-fold increase in toxicity and exhibit no noticeable antitumor activity. However, the acid effectively inhibits the growth of B16 mouse melanoma and MAC16 mouse adenocarcinoma of the rectum.

The conclusion is that this group of compounds exhibits a wide range of activities in the modeling of tumors in mice.

Table 1

Antitumor activity against MAC15A (mouse rectal adenocarcinoma). Structured-activated connection. 5 animals per group.

Dose 100 mg / kg ip per day

Compound Scheme (days) T / C% ^a 4-Chlorobenzenesulfonic acid 1,2,3,4,5 443 4-chloro-3,5-dinitrobenzenesulfonic acid 1,2,3,4,5 414 1,5-dichloro-2,3-dinitrobenzene 1,2,3,4,5 386 2,4,6-trinitrophenol 1,2,3 300 4-amino-3,5-dinitrobenzenesulfonic acid 1,2,3,4,5 286 4-Chloro-3,5-dinitrobenzoic acid 1,2,3,4,5 271 2,4-Dichloro-3,5-dinitrobenzoic acid 1.2 243 2-Glutathionyl-3,5-dinitrobenzenesulfonic acid 1,2,3,4,5 242 3-Nitro-4-methylbenzenesulfonic acid 1,2,3,4,5 229 2,6-dinitroaniline 1,2,3,4,5 214 2,5-Dichlorobenzenesulfonic acid 1,2,3,4,5 212 1,4-dinitrobenzene 1,2 200 1-Chloro-3,4-dinitrobenzene 1,2,3,4,5 200 1-Chloro-2,4-dinitrobenzene 1,2,3,4,5 188

13.

2,4,6-Trinitrobenzenesulfonic acid 1,2,3,4,5 188 2-chloro-4-nitroaniline 1,2,3,4,5 171 2,5-dichlorobenzoic acid 1,2,3,4,5 171 2,4-dinitrobenzenesulfonic acid 1,2,3,4,5 171 1,2-dichloro-4,5-dinitrobenzene 1,2,3,4,5 171 4-Chloro-3-nitrobenzenesulfonic acid 1,2,3,4,5 140 2-Chloro-3,5-dinitrobenzenesulfonic acid 1,2,3,4,5 137 1-Chloro-2,4,6-trinitrobenzene 1.2,3 113 4-glutathionyl-3,5-dinitrobenzene 1,2,3,4 113 2,4-dinitroaniline 1,2 100 2,4-dinitrobenzoic acid 1,2,3,4,5 100 3,5-dinitrobenzoic acid 1,2,3,4,5 100 4-amino-3,5-dinitrobenzamide 1 100 4-chloro-3-nitroanisole 1,2,3,4,5 100 4-chloro-2,6-dinitroaniline 1,2,3,4,5 87 6-chloro-2,4-dinitroaniline 1,2,3,4,5 87 1-fluoro-2,4-dinitroaniline 1 75 1-fluoro-2,4-dinitrobenzene 1 62.5 ^In

a - average value

Τ - experimental group

C - solvent control in - toxic death

Table 2

Antitumor activity against P388 (leukemia in mice) animals in a group. IP treatment days 1 to 5. The dose is for the whole day, T is for the test group, C is the solvent control, a = mean, as described in more detail in the description.

Compound Dose mg / kg t / s% ^a 4-Chloro-3,5-dinitrobenzenesulfonic acid 100 203 4-Chloro-3,5-dinitrobenzenesulfonic acid 50 259

14.

Table 3

Antitumor activity against P388 (leukemia in mice). Treatment of ip with 4-chloro-3,5-dinitrobenzenesulfonic acid (CDNSA).

animals in a group. Daily dose.

Compound Dose mg / kg Scheme days t / s% ^a CDNSA 100 1,2,3,4,5 225 75 1,2,3,4,5 300

a - average

T - experimental group

C - solvent control

Table 4

Antitumor activity against M5076 - reticulum cell sarcoma days after im (im read everywhere intramuscular) transplantation. 7 animals per group. The agent is dissolved in corn oil.

The dose is the daily amount.

Compound Dose mg / kg The way Scheme days % Tumor inhibition, weight 2.4 VA 75 ^a iP 1,4,6,9 ^At 79.88 50 ip 1,4,6,9 57 25 iP 1,2,4,6,9 75 75 Ph.D. 1,4,5,7,9 66 50 Ph.D. 1,2,4,5,6,7,9 76 25 Ph.D. 1,2,4,5,6,7,9 63 2,4 BZ 2.5 ³ iP 1,2,3,4,5,6,7,8,9 51 1,25 ip 1,2,3,4,5,6,7,8,9 34 2.4 VM 1.o ^a ip 1,2,3,4,5,6,7,8,9 41 0.5 ip 1,2,3,4,5,6,7,8,9 39 0.25 iP 1,2,3,4,5,6,7,8,9 42

a - maximum tolerated dose c - two independent experiments; 4 animals did not have a tumor in the second experiment

15.

2,4 BA = 2,4-dichloro-3,5-dinitrobenzoic acid,

2,4 BZ = 2,4-dichloro-3,5-dinitrobenzamide,

2.4 BM = 2,4-Dichloro-3,5-dinitrobenzoic acid methyl ester.

5 % Tumor inhibition - weight Treated A gm Control In gm Tumor weight

% inhibition VA-100 *

Table 5

Antitumor activity against B16F1 - murine melanoma 12 days after sc transplantation. 6 animals per group. The drug is dissolved in corn oil. The dose is the amount per day.

Compound Dose mg / kg The way Scheme days % Tumor inhibition, weight 2.4 VA 75 ^a ip 1.5 71,8? 50 ip 1.5 45.56 ^V 25 ip 1.5 13 75 ip 1,3,530 th most common 50 iP 1,3,59 25 ip 1,3,522 th most common 2,4 BZ 2.5 ^a ip 1.2 39 1,25 ip 1,2 17 4VA 100 ip 1.5 39 75 ip 1.5 41 50 ip 1.5 10 4BZ 5 ^a ip 1,3,518 2.5 ip 1,3,518 1,25 ip 1,3,527 th most common 4VM 2.5 ^a ip 1.3 67 1,25 ip 1,2,343

16.

a - maximum tolerated dose c - two independent experiments

2.4 BA = 2,4-dichloro-3,5-dinitrobenzoic acid

2,4 BZ = 2,4-dichloro-3,5-dinitrobenzamide

4 BA = 4-chloro-3,5-dinitrobenzoic acid

BZ = 4-chloro-3,5-dinitrobenzamide

BM = 4-Chloro-3,5-dinitrobenzoic acid methyl ester.

Table 6

Yu antitumor activity against MAC13 - mouse adenocarcinoma of the rectum 12 days after im transplantation. The drug is dissolved in corn oil. The dose is for the day.

Compound Dose mg / kg The way Scheme, days % Tumor inhibition, weight 2.4 VA 75 ^a ip 1.4.5 45 2.4 VA 50 ip 1,2,3,4,5,6,7,8,9 39 2.4 VA according to FIG. 3 ip according to FIG. 3 according to FIG. 3 2,4 BZ 2.5 ^a ip 1,2,3,4,5,6,7,8,9 51 2,4 BZ 1,25 ip 1,2,3,4,5,6,7,8,9 17 2VA according to FIG. 4 ip according to FIG. 4 according to FIG. 4

a - maximum tolerated dose

2.4 BA = 2,4-dichloro-3,5-dinitrobenzoic acid

2,4 BZ = 2,4-dichloro-3,5-dinitrobenzamide

BA = 2-chloro-5-nitrobenzoic acid.

The test results are shown graphically in Figs. 3 and 4

17.

Table 7

Antitumor activity against MAC16, mouse rectal adenocarcinoma sc transplanted 11 days after treatment with 2,4-dichloro-3,5-dinitrobenzoic acid (2.4 BA) was started. The drug is dissolved in 5 corn oil. The tumor volume is at least 40 mm ³ at the start of treatment. 6 animals per group. The daily dose is indicated.

Compound Dose mg / kg The way Scheme, days % Tumor inhibition, weight 2.4 VA 75 ^a ip 1,2,5,8 88 2.4 VA 50 ip 1,2,4,5,8 91

a - maximum tolerated dose.

Table 8

Antitumor activity against B16 mouse melanoma 12 days after sc transplantation into female black C57 mice. 6 animals per group. Daily dose via ip.

Compound Dose mg / kg Scheme, days % Tumor inhibition, weight 2-Chloro-5-nitrobenzoic acid 700 1,2,3,4,5,6 62

In addition, a basic analysis was described below to investigate the antiviral activity of the compounds of the invention, and in particular 4-chloro-3,5-dinitrobenzenesulfonic acid.

Antitumor activity and toxicity studies were further performed for the following compounds, with generally satisfactory results:

C22 - 2,5-dichloro-4-nitrobenzoic acid,

C23 - 2,4-dichloro-5-nitrobenzoic acid,

C24 - 2,6-dichloro-4-nitrobenzoic acid,

C25 - 2-amino-5-nitrobenzoic acid,

C26 - 2-hydroxy-5-nitrobenzoic acid,

C27 - 3,5-dichloro-4-nitrobenzoic acid.

18.

MAIN ANALYSIS (i) Acute infection analysis. High-titer HIV-1 _RF viral colony (human immunodeficiency virus) was propagated in H9 cells with RPMI 1640 (laboratory) supplemented with 10% fetal calf serum, penicillin 5 (100 IU / ml). The cellular debris was removed by low speed centrifugation, the supernatant stored at -70 ° C until needed. In a typical study, C8166 T-lymphoblastoid CD4 + cells were incubated with 10 x TCID50 HIV-1 _RF at 37 ° C for 90 min and then washed three times with phosphate-buffered saline (PBS). An aliquot of cells (2x10 ⁵ ) were resuspended in 1.5 ml of culture medium in 6 ml tubes, with the test compounds pre-diluted (200 μΜ to 0.2 μΜ) immediately added. 20 mM amounts of a solution of each compound were prepared in 70% alcohol. The compounds were stored in powder form and the solutions were prepared immediately before the distilled water experiment, or stored as a fixed amount in a solution of 70%.

alcohol. The final alcohol concentration in the tissue culture medium is 1%. The cells were then incubated at 37 ° C in 5% CO ₂ . 72 hours after infection, 200 μΙ were taken from the supernatant and tested for HIV (Kingchington et al, 1989; Robert et al, 1990) using an antigenic 20 capture ELISA that recognizes all structures of the respective proteins (Coulter Electronics, Luton, UK). The following controls were also used: supernatants were taken from uninfected and infected cells. Infected cells are treated with AZT (manufactured by Roche Products UK,

Ltd), with ddC (also on Roche) and with R031-8959 (on Roche), an HIV 25 proteinase inhibitor. The IC _{50 activities} of 8959, AZT and ddC in infected cells are

1, 10, 20 nM and 200 nM respectively according to FIG. 2. The ELISA readings are counted with a spectrophotometer. Compounds were tested by duplication of each concentration, with the result shown being averaged over at least two tests. This assay evaluates the activity of the compounds by measuring their effect on the inhibition of antigenic levels of the HIV nucleus.

(j) Examination of chronic cellular infection. Chronically infected cells (H9rf) were washed three times to secrete the extracted cell virus and then incubated with the active compounds of the invention (200 to 0.2 μΜ) for four days. The HIV-1 antigen in the supernatant was then measured, 35 using ELISA.

19.

(iii) Toxicity testing. To determine the toxicity of the compounds, ineffective H9 cells were incubated with the respective compounds for four days. The supernatant was removed and the cells were suspended in 200 µg pg medium containing protein hydrolyzate ¹⁴ C. After 6 hours, 5 cells were harvested and ¹⁴ C was measured.

The toxicity test of the compounds in particular consists of the following: aliquots of 2 x 10 ⁵ uninfected cells are cultured together with the compounds in the same medium for 72 hours. The cells were then washed with PBSA and dispersed in 200 μΙ medium containing ¹⁴ C protein hydrolyzate. After 12 hours, cells were harvested and ¹⁴ C was measured. Uninfected and untreated cells are used for control. Toxicity is expressed as inhibition of the increase of ¹⁴ C protein hydrolyzate.

The test results of 4-chloro-3,5-dinitrobenzenesulfonic acid are shown in the accompanying figure 1, where RC is a designation of compound 15 of Radopath Company, which is 4-chloro-3,5-dinitrobenzenesulfonic acid, an agent of the present invention. The results are also summarized in Table 9.

Table 9

Compound IC50 cd ₅₀ ΤΙ 4-Chloro-3,5-dinitrobenzenesulfonic acid 3μΜ 80 μΜ 28.6

Where IC ₅₀ is the concentration of the agent of the invention that causes a 50% reduction in antigenic levels of the HIV nucleus as determined by the Coulter 24 antigen assay and detected by double 25 dilution of the supernatant taken from tubes containing untreated cells with acute infection.

CD ₅₀ is the concentration of the agent that causes 50% inhibition of the cells, measured by increasing the ¹⁴ C protein hydrolyzate. The therapeutic index (TI) is determined by dividing the CD ₅₀ value by the IC ₅₀ value.

The test results of the other compounds of the invention are summarized in the following Table 10.

20.

Table 10

Compound IC50 cd ₅₀ ΤΙ 2-Chloro-3,5-dinitrobenzenesulfonic acid 25 μΜ > 200 μΜ > 8 4-amino-3,5-dinitrobenzenesulfonic acid 20 μΜ 100 μΜ 5 2,4,6-trinitrophenol <0.2 μΜ 95 μΜ > 475 4-Chloro-3,5-dinitrobenzoic acid 30 μΜ 70 μΜ 2.33

Initial tests, performed at about the same time, identified 5-chloro-5-nitrobenzoic acid as at least as effective, if not more, than the compounds tested and described herein.

Following the methodological guidelines given above for determining HIV activity, more intensive studies were performed, the results of which are listed in the following Table 11.

Table 11.1

Structured-activated links that determine activity against HIV virus

Code Compound _______________________________ ^Ag IC50 ^Toh CC50

Group A

P1 Picryl chloride

P2 Picric acid

PZ ______ Picrylsulfonic acid (Na-salt) _____________________________

Group B

Cl 2,4-Dichloro-3,5-dinitrobenzoic acid

C2 2,4-dichloro-3,5-dinitrobenzamide

2,4-Dichloro-3,5-dinitrobenzoic acid C3 methyl ester

C4 4-Chloro-3,5-dinitrobenzoic acid

C5 4-chloro-3,5-dinitrobenzamide

4-Chloro-3,5-dinitrobenzoic acid C6 methyl ester

C7 2-Chloro-3,5-dinitrobenzoic acid

2-Chloro-3,5-dinitrobenzoic acid C8 methyl ester

C9 4-chloro-3-nitrobenzoic acid

C10 2-Chloro-4-nitrobenzoic acid

C11 3,4-Dichlorobenzoic acid

21.

C12 C13 2,5-dichlorobenzoic acid 4-Chlorobenzoic acid G hole C. S1 4-Chloro-3,5-dinitrobenzenesulfonic acid S2 2-Chloro-3,5-dinitrobenzenesulfonic acid S3 4-amino-3,5-dinitrobenzenesulfonic acid S4 4-Chloro-3-nitrobenzenesulfonic acid S5 4-Chlorobenzenesulfonic acid S6 4-Nitrotenylsulfonic acid S7 2,5-Dichlorobenzenesulfonic acid S8 2,4-dinitrobenzenesulfonic acid Group D E1 1-Chloro-3,4-dinitrobenzene E2 1-Chloro-2,4-dinitrobenzene EU 1,2-dichloro-4,5-dinitrobenzene E4 2,4-chloronitrobenzene E5 2,4-dichloronitrobenzene E6 2,5-dichloronitrobenzene E7 3,4-dichloronitrobenzene E8 3,5-dichloronitrobenzene E9 1,5-dichloro-2,3-dinitrobenzene E10 1,2,3-Trichloro-4-nitrobenzene E11 1,2,4-Trichloro-5-nitrobenzene E12 2,4,6-trichlorobenzene E13 2,3,4,6-tetrachloro nitrobenzene E14 pentachloronitrobenzene Table 11.2 IC ₅ o CC5Q P-compounds Antiviral Toxicity Ability SI Selectivity Index Against HIV - 1RF P1 0.6 7 10 5 - 0.4 -

22.

Average value 0.5 6 12 P2 38 67 2 RE > 200 > 200 Against HIV -1 III B. P1 0.6 7 11.6 1 7 7 Average value 0.8 7 9

Against chronically infected cells

P1 0.9 7 8 2 12 6 Average value 1.5 9.5 6

Table 11.3

C-compounds IC50 Anti-virus To ability CC50 toxicity SI Selectivity Index C1 Against HIV-III B. 5 70 14 36 70 2 33 70 2 35 60 2 Average value 27 70 3 C1 Against HIV -1 RF 7 60 8.5 - - 56 16 56 3.5 Average value 11.5 57 5

Against chronically infected cells

C1 16 30 2 16 95 6 Average value 16 63 4

23.

Against HIV-1 III B. C2 2 70 35 NW 0.3 7 23 C4 40 100 2.5 30 70 2.3 Average value 35 85 2.4 C5 5 50 10 C6 5 60 12 C7 23 150 6 5 > 200 > 10 Average value 22 > 175 8 C8 10 60 5 C9 > 200 > 200 - C10 > 200 > 200 - C11 > 200 > 200 - C12 > 200 > 200 - Table 11.4 Fr. tr about ss ₅₀ SI S-compounds Antiviral Toxicity Index of ability selectivity Against HIV -1 RF S1 20 100 5 19 60 3 Average value 20 80 4 S2 NR S3 NR S4 > 200 > 200 - S5 > 200 > 200 - S6 > 200 > 200 - S7 > 200 > 200 - S8 40 100 2.5 30 70 2 Average value 35 75 2.4

24.

Table 11.5

E-compounds IC50 Antivirus ability CC ₅₀ Toxicity SI Selectivity Index Against HIV- 1 RF E1 4 10 2.5 E2 4 13 3 EU 4 7 1.5 E4 80 > 200 1.5 E5 180 > 200 1 E6 110 > 200 2 E7 > 200 > 200 - E8 120 > 200 1.5 E9 ND E10 > 200 90 E11 > 200 > 200 E12 > 200 > 200 E13 > 200 80 E14 > 200 > 200

The present invention is disclosed in the specification with various specific details, which make it clear that the number of various modifications in which it can be implemented is numerous in the spirit and scope of the claims that follow. For example, the functional groups may be in different positions other than those given in the examples of the description.

Claims (16)

Patent Claims An arylating agent for treating neoplasms or a viral infection, characterized in that it is a compound having the structure of an aromatic ring having at least one substitute labile group and at least one substitute electrophilic group. An arylating agent according to claim 1, characterized in that it is a compound of the general formula: X ¹ X ⁴ where one of the substituents X ¹ to X ⁶ is a labile group, at least one of the remaining X is an electrophilic group, the others being the same or different and being hydrogen or another substituent. An arylating agent according to claim 2, characterized in that when X ¹ is a labile group, one of the substituents X ² to X ⁶ is an electrophilic group, the others being each independently hydrogen or a substituent, provided that when X ² and X ⁶ are nitro groups, X ⁴ is neither a nitro group, nor a sulfonic acid group, nor a sulfo group, and X ¹ is not a labile group of the type defined below, namely: hydroxy group, amino group, sulfo group a group, a carboxy group, a methyloxy group, a halogen or a hydrazyl group of the formula wherein A is hydrogen or a single electron a nitrogen atom, Y is hydrogen or an organic group, a Z is an organic group, or Y and Z together with the adjacent nitrogen atom to form a nitrogen-containing heterocycle. The agent according to claim 2, characterized in that one of the substituents X ¹ to X ⁶ is a labile group and one of the remaining substituents is an electrophilic group, wherein the others are the same or different and are 2. hydrogen or substituents, at least two of which are other than nitro, but at least one is a labile group and at least one is an electrophilic group. An agent according to claims 2, 3 and 4, characterized in that at least one of the substituents X ¹ to X ⁶ is an electrophilic group or one 5 labile group selected from the following: electrophilic groups - SO ₃ H, SO ₃ M (where M is metal), halogen or NO ₂ ; labile groups are halogen, SO ₃ H, SO ₃ M (where M is metal), optionally substituted NH ₂ , COOH, optionally substituted CONH ₂ and COOR ₃ , where R ₃ is metal or alkyl. An alkylating agent according to any one of the preceding claims 1 to 5, characterized in that it is a compound of the general formula wherein X ⁷ is SO ₃ H, SO ₃ M (where M is metal), hydrogen, COQ (where Q is a hydroxy, amino or substituted amino group, or an OR ₃ group in which R ₃ is metal or alkyl), 20 NH ₂ , substituted NH ₂ , NO ₂ or OH; X ⁸ is hydrogen, halogen, glutathione or nitro; X ⁹ , X ¹⁰ and X ¹¹ are each hydrogen, halogen or nitro; and X ¹² is hydrogen, nitro, an optionally substituted amino group, alkyl or glutathione. An alkylating agent according to claim 6, characterized in that it is a compound of the group with the substituents as follows: 25 (a) X ⁷ is SO ₃ H; X ⁸ is hydrogen, halogen or glutathione; X ⁹ and X ¹⁰ each are independently hydrogen, halogen or nitro; X ¹¹ is hydrogen and X ¹² is hydrogen, nitro, an optionally substituted amino group, halogen, alkyl or glutathione; (b) X ⁷ is halogen; X ⁸ , X ⁹ , X ¹⁰ and X ¹² are each independently hydrogen, halogen or nitro, and X ¹¹ is hydrogen; z (c) X ⁷ is SO ₃ H, SO ₃ M (where M is a metal), NH ₂ or a substituted NH ₂ , halogen or hydroxy group; X ⁸ is a nitro group; X ⁹ is hydrogen; X ¹⁰ is hydrogen, X ¹¹ is a nitro group and X ¹² is a nitro group; (d) X ⁷ is an optionally substituted amino group, and X ⁸ to X ¹² are each independently hydrogen, halogen or nitro. 3. An alkylating agent according to claim 6, characterized in that the substituents are as follows: X ⁷ is a group of formula COQ, in which Q is a hydroxy, substituted NH ₂ group or of formula -OR ₃ , in which R ₃ is alkyl or metal; X ⁸ is hydrogen or halogen; X ⁹ and X ¹⁰ are each hydrogen, halogen or 5 nitro; X ¹¹ is hydrogen and X ¹² is hydrogen, nitro, a substituted amino group or halogen. An alkylating agent according to claims 1 to 8, characterized in that each of the group of compounds is as follows: 9.1 2-Chloro-5-nitrobenzoic acid or ίο 9.2 2,4-Dichloro-3,5-dinitrobenzoic acid or its alkyl esters, 9.3 4-Chloro-3,5-dinitrobenzoic acid or its alkyl esters, 9.4 2,5-dichlorobenzoic acid, 9.5 2,4-dinitrobenzoic acid, 9.6 3,5-dinitrobenzoic acid, 15 9.7 3-Nitro-4-chloroanisole, 9.8 4-amino-3,5-dinitrobenzamide, 9.9 4-chloro-3,5-dinitrobenzamide, 9. 10 2,4-dichloro-3,5-dinitrobenzamide, 9.11 4-Chloro-3,5-dinitrobenzenesulfonic acid, 20 9.12 4-Chlorobenzenesulfonic acid, 9.13 2,5-Dichlorobenzenesulfonic acid, 9.14 4-Amino-3,5-dinitrobenzenesulfonic acid, 9.15 3-Nitro-4-methylbenzenesulfonic acid, 9.16 2-Chloro-3,5-dinitrobenzenesulfonic acid, 25 9.17 2-Glugathionyl-3,5-dinitrobenzenesulfonic acid, 9.18 4-Glutathionyl-3,5-dinitrobenzenesulfonic acid, 9.19 3-Nitro-4-methylbenzenesulfonic acid, 9.20 3-Nitro-4-chlorobenzenesulfonic acid, 9.21 2,4-dinitrobenzenesulfonic acid, 9.22 4-chloro-3,5-dinitrobenzenesulfonic acid, 9.23 2,4-dinitrochlorobenzene, 9.24 3,4-dinitrochlorobenzene, 9.25 2,4-dinitrofluorobenzene, 9.26 1,2-Dichloro-4,5-dinitrobenzene, 35 9.27 1,3-Dichloro-4,5-dinitrobenzene, 4. 9.28 1,5-Dichloro-2,3-dinitrobenzene, 9,29 2,4,6-Trinitrophenol (picric acid), 9.30 2,4,6-Trinitroaniline, 9.31 2,4,6-trinitroporbenzene, 5 9.32 2,6-dinitroaniline, 9.33 2,4-dinitroaniline, 9.34 3,5-dinitroaniline, 9.35 2,4-dinitro-6-chloroaniline, 9.36 2,6-dinitro-4-chloroaniline, 9.37 2-chloro-4-nitroaniline, 9.38 2,4-dinitro-5-fluoroaniline, 9.39 1,2-dinitrobenzene, 9.40 1,3-dinitrobenzene or 9.41 1,4-Dinitrobenzene. An arylating agent according to any one of claims 1 to 5, characterized in that the hydrogen ring is a labile group when the compound is of general formula (VIII) no ₂ 11. Administration of compounds containing aromatic structure 25 ring containing at least one labile group and at least one electrophilic group as a means of treating and preventing a cancer and precancerous condition or disease caused by a viral infection. 12. Administration of a compound selected from the group of the following compounds: 12.1 2,4,6-Trinitrophenol, zo 12.2 2,4-Dichloro-3,5-dinitrobenzoic acid, 12.3 4-Chloro-3,5-dinitrobenzoic acid, 12.4 1,5-dichloro-2,3-dinitrobenzene, 12.5 2-Chloro-5-nitrobenzoic acid, 12.6 4-chlorobenzenesulfonic acid, 35 1 2.7 4-Chloro-3,5-dinitrobenzenesulfonic acid, 5. 12.8 4-chloro-3,5-dinitrobenzamide, 12.9 2,4-Dichloro-3,5-dinitrobenzamide. for the treatment and prophylaxis of cancer and precancerous conditions or of a disease caused by a viral infection. 5 A pharmaceutical composition comprising, as an active ingredient, a compound according to any one of the preceding claims, as well as pharmaceutically acceptable solvents or carriers. Use of the compounds according to any one of claims 1 to 12 for the preparation of medicaments for the prophylaxis or treatment of cancer or precancerous conditions or viral infection. 15. A method of treating a) a disease caused by a viral infection or b) cancerous or precancerous conditions by reducing or eliminating the tumor formation, wherein the patients are treated with one effective amount of a compound described in any one of 15 claims 1 to 12 or with a composition as described in claim 13. 16. Administration of a chlorine or nitro-benzenesulfonic acid type compound; or of chlorine or nitro-benzoic acid; or chlorine or nitrobenzamide as a pharmaceutical for treating viral infection or neoplasms.