SK145795A3 - Arylating medicaments - Google Patents

Abstract

Various arylating agents having activity in the treatment of cancer and viral infection are disclosed. The active compounds include an aromatic ring having at least one labile leaving group and at least one electrophilic group. Preferred active compounds include chlorobenzenesulphonic acids and optionally halogenated nitrobenzene compounds. In an anti-viral context, the active compounds have efficacy against HIV infections.

Description

(57) Annotation:

Arylating agents, in particular phenylating agents, which are useful as compounds having a therapeutic effect, in particular for the treatment of cancer and diseases caused by a viral infection, such as HIV. Further disclosed are pharmaceutical compositions containing the compounds and their use in therapy.

the

Arylating agents

Technical field

The invention relates to arylating agents, in particular phenylating agents, which are useful as compounds having a therapeutic effect, in particular in the treatment of cancer and diseases caused by viral infection.

in the broadest sense, the invention relates to arylating agents which can be used in the treatment of neoplasms or viral infections such as HIV. In particular, the arylating agent should be a compound having an aryl group, the aromatic ring of which is preferably carbocyclic and which, in a particular case, has at least one labile substituent and at least one electrophilic substituent. The carbocyclic or other aromatic ring is preferably monocyclic and in some cases the aromatic ring is usually one having one or more carboxylic or sulfonic acid residues simultaneously with one or more nitro and / or amino groups and / or one or more halogen atoms as substituents. Preferably, the substituents comprise no more than two nitro substituents. Combinations of a halogen atom (e.g., a chlorine atom) with a nitrosubstitute, particularly in conjunction with a monocyclic arylating agent having a ring with a carboxylic acid type substituent, is primarily an effective structure. An example of such a structure is the combination of a mono-nitroso substituent and a mono-chloro substituent (for example, 2-chloro-5-nitrobenzoic acid and 2-chloro-4-nitrobenzoic acid).

SUMMARY OF THE INVENTION

The invention thus relates to a compound for use in the treatment of cancer or a disease caused by a viral infection, in particular AIDS, which compound has an aromatic ring structure with at least one substituent of the easily cleavable group type and at least one electrophilic group type substituent. are two ortho-nitro groups and a para-sulfone group or three symmetrical nitro groups and a labile group at position one is that defined in WO 91/15200 (International Specification); it is used in a concentration higher than 1 x 10 ³ mol / liter.

Generally speaking, the compound of the invention has the general formula I

C x] (I) wherein n is an integer and is at least 2 and X is the same or different and is a labile or electrophilic group, provided that there are at least two X groups that are different from the nitro group, at least one of them is a labile group and at least one is an electrophilic group.

In addition, it is believed that what is considered to be an arylation mechanism typically involves the use of relatively high concentrations and hence high doses. In general, such concentrations for use of the compounds of the invention will be 1 x 10 ^-a mol / liter, which in dosage terms is usually at least about 5 mg / kg.

When selecting a substituent grouping for a compound of the invention, the essential feature is to ensure that there is no link between a particular aromatic ring and at least one substituent as a labile group and at least one electrophilic substituent. In addition, a group to be classified as labile in relation to a particular ring may alternatively be classified as electrophilic with another ring. Further, where there are at least two nitro groups as substituents, the labile substituent may be a hydrogen atom in the ring.

For the sake of clarity, it is possible to define preferred substituent groups as those wherein at least one X is selected from any of the following groups, in particular from:

electrophilic groups such as SO ₃ H or SO ₃ M, in which M represents a metal atom, for example potassium? or a halogen atom and a nitro group;

labile groups such as halogen, SO ₃ H or SO _and M, in which H represents a metal atom; amino, substituted amino, for example NHR ¹ , NR ¹ R ³ , wherein R ¹ and R ³ are the same or different and are alkyl, alkoxy or hydroxyalkyl; a carboxyl group, CONH _a , substituted with CONH _a , for example CONHR ¹ , CONR ¹ R ³ , (R ¹ and R ³ have the same meaning as above) and further a COOR ³ group in which R ³ is a metal atom or an alkyl group.

Thus, general examples of the compounds of the invention include, but are not limited to:

chlórdinitrobenzénsulfónové acid, acetic acid salt, dichlorobenzene acid aminodinitrobenzénsulfónové acid nitrometylbenzénsulfónové acid * glutationyldinitrobenzénsulfónové acid nitrochlórbenzénsulfónové acid, dinitrobenzenesulfonic acid, dinitrochlorobenzene, dinitrofluorobenzene, dichlórdinitrobenzény, trinitrophenol, such as picric acid, trinitroanilíny, trinitrochlórbenzény, trinitrobenzenesulfonic acid, chlórdinitrobenzoové acid, dichlorobenzoic acid, dinitrobenzoic acids, nitrochloroanilines, aminodinitrobenzamides, dinitroanilines, dinitrochloroanilines, chloronitroanilines, dinitrofluoroanilines.

The above compounds can be included in the group of compounds of formula II

X '

E (II) wherein

XX 'is SO ₃ H or SO _and M, wherein M is a metal atom or a halogen atom such as chlorine, fluorine and the like; a COQ group wherein Q is a hydroxyl group, an amino group or a substituted amino group, or an OR ³ group wherein R ³ is a metal atom or an alkyl group; amino, substituted amino, nitro or hydroxyl;

represents a hydrogen atom, a halogen atom, a glutathione or a nitro group, the substituents B are the same or different and represent a hydrogen atom, a halogen atom or a nitro group; and

C represents a hydrogen atom, a nitro group, an amino group including a substituted amino group, a halogen atom, an alkyl group and glutathione.

The following characteristics are preferred in these compounds:

X 'is SO _and H or SO _and M, wherein M is a metal atom; a halogen atom such as chlorine, fluorine and the like, amino, nitro or carboxyl groups,

C is a hydrogen atom, an alkyl group such as a methyl group, an amino group or a nitro group.

Compounds of the invention which exhibit anti-cancer and antiviral effects can be further subdivided into a number of preferred moieties, for example as follows:

a) a compound of formula III

(III) wherein it represents a hydrogen atom, a halogen atom, for example a chlorine atom, a fluorine atom and the like or glutathione;

represents a hydrogen atom, a nitro group or a halogen atom, for example a chlorine atom and the like,

C represents a hydrogen atom, a nitro group, an amino group including a substituted amino group, a halogen atom, an alkyl group or a glutathione; and

D represents a hydrogen atom, a halogen atom or a nitro group.

Said compounds of formula III are preferred because the SO _and H groups are believed to be involved in an emulsifying effect which is useful in increasing the solubility of the compounds, which in addition contributes to a better bioavailability in the cell walls.

Among the compounds of formula II, the following are most preferred:

4-chlorobenzenesulfonic acid,

2,5-dichlorobenzenesulfonic acid,

4-amino-3,5-dinitrobenzenesulfonic acid,

3-nitro-4-methylbenzenesulfonic acid,

2-chloro-3,5-dinitrobenzenesulfonic acid,

2-Glutationyl-3,5-dinitrobenzenesulfonic acid,

4-glutathione-3,5-dinitrobenzenesulfonic acid,

3-nitro-4-methylbenzenesulfonic acid,

3-nitro-4-chlorobenzenesulfonic acid,

2,4- dinitrobenzenesulfonic acid.

The following compounds 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.

(b) Compound of formula IV *

Hal (IV) in which

Hal represents a halogen atom such as chlorine, fluorine and the like, and the substituents B are the same or different and have the meanings given above.

Of the above compounds of formula IV, the following are most preferred:

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.

The following compounds are particularly preferred:

1,3-dichloro-4,5-dinitrobenzene, 1-chloro-2,4-dinitrobenzene,

1-fluoro-2,4-dinitrobenzene.

wherein E is SO ₃ H or SO _and M, wherein M is a metal atom such as potassium; amino or substituted amino, halogen or hydroxyl.

Among the above compounds of formula V, the following are most preferred:

2.4.6- trinitrophenol, i. picric acid,

2.4.6- trinitroaniline,

2.4.6- trinitrochlorobenzene,

2.4.6- trinitrobenzenesulfonic acid.

Of these, the first and third compounds are preferred.

the compounds are in particular

d) Compounds of formula VI

(VI) in which:

the B substituents are the same or different and have the meanings given above,

G is as defined above as C, except alkyl and glutathione,

J represents a hydrogen atom or a halogen atom, and

Q represents a hydroxyl group, an amino group or a substituted amino group, or an OR ³ group in which R ³ represents a metal atom or an alkyl group. <

Among the above compounds of formula VI, the following are most preferred:

2,4-dichloro-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. e) Compounds of formula VII

E (VII) wherein the substituents B, same or different, are as defined above, together with their amino substituted derivatives.

Among the above compounds of formula VII, the following are most preferred:

2,6-dinitroaniline,

2.4- dinitroaniline,

3.5- dinitroaniline,

2,4-dinitro-6-chloroaniline,

2,6-dinitro-4-chloroaniline,

2-chloro-4-nitroaniline,

2,4-dinitro-5-fluoroaniline.

It is particularly preferred

2.6- dinitroaniline.

As mentioned above, where at least two nitro groups are present as substituents, it represents a labile group of hydrogen atoms in the ring. In this context, a compound of formula VIII (VIII) should be mentioned.

NO

NO ₂ which is

1,2- dinitrobenzene,

1.3- dinitrobenzene,

1,4- dinitrobenzene.

The compounds of the invention may be obtained by known procedures for the preparation of substituted benzene compounds. Such procedures are described in various common publications such as Organic Syntheses 1963, Collective Volume 4, p. 364-366, by Harry P. Schultz, issued by John Wiley and Sons Inc.

The compounds of the invention may be formulated for use as pharmaceutical compositions (e.g., intravenous, intraperitoneal, oral, or subcutaneous) containing at least one active agent and a diluent or carrier. Accordingly, the invention also includes a pharmaceutical composition comprising the compounds of the invention and a pharmaceutically acceptable diluent or carrier (e.g., aqueous).

Such a composition may be in the form of a bulk substance, or more preferably in the form of a dosage unit. Thus, for example, the composition may be formulated as a tablet, capsule, powder, solution or suspension. Soft gelatin capsules are particularly suitable. The composition may form a mixture with liposomes or may be applied by a sustained release system of small amounts.

The compositions of the invention may be prepared using the active compounds already described according to conventional pharmaceutical practice. The diluents, excipients or carriers and the like to be used are well known in the pharmaceutical art and the form chosen for the particular regimen will depend on the condition and the physician's choice.

For example, as explained below, the compounds of the invention may be administered as a solution in sterile deionized water. Also, as far as possible, dissolution can be facilitated by the use of dimethylsulfoxide or an alcohol, glycol or vegetable oil. The compounds are most preferably administered in corn oil or as a solution in a mixture of dimethylsulfoxide with sterile water.

The invention further encompasses, in connection with said applications, the use of a compound as described herein for the preparation of a medicament for the prophylaxis or therapy of cancer or viral infections, for example, to reduce or eliminate cancer growth.

When the compound of the invention is administered, the dosage may be adjusted according to the animal studies described below. Doses of from about 50 mg / kg to about 200 mg / kg and up to about 400 mg / kg and above have proven effective in these studies. Thus, suitable human doses can be expected to range from about 5 mg / kg to about 20 mg / kg, and generally up to about 40 mg / kg or more. The concentration and dose should be sufficient to initiate the arylation mechanism.

As is evident from the particularly preferred compounds mentioned above, those compounds of the invention which exhibit the highest potency are arranged, in particular, in any descending order:

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

1,5-dichloro-2,3-dinitrobenzene,

2,4,6-trinitrophenol, i. picric acid,

2,4-dichloro-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,

2,5-dichlorobenzoic acid,

2-chloro-3,5-dinitrobenzenesulfonic acid,

2,4-dinitrobenzoic acid.

Particularly preferred compounds are those wherein at least one X is selected from the group or groups:

labile substituents such as one or two halogen atoms, and / or amino or substituted amino and / or carboxyl or substituted carboxyl and / or alkyl and / or SO H or SO M.

^F 3 3 electrophilic substituents such as one or two nitro groups and / or SO ₃ H group or SO ₃ M group and / or one or two halogen atoms.

In addition, while the compounds of the invention may be used within the dosage regimen illustrated above, where three symmetrical nitro groups as substituents or the active substance is different from that mentioned in WO 91/15200 (International Specification), as indicated above, to be the concentration of active compound in the preparation of any more than 1 x 10 ^"3 MPL /, and is preferably at least 1 x 10" ² mol / l.

As shown in Table 8 below, 2-chloro-5-nitrobenzoic acid exhibits significant antitumor activity in vivo. However, this could not be confirmed in vitro and some compounds of the invention appear to require activation in the patient's liver. Thus, this compound and some other compounds may be immunomodulators.

The following animal studies illustrate the remarkable efficacy of the compounds of the invention.

Animal studies

The purpose of these studies was to evaluate the antitumor properties of a group of structurally similar compounds that may act as arylating agents. Their in vivo anti-tumor responses were determined in two ascites tumors, in MAC15A mouse colon adenocarcinoma and P388 mouse leukemia, and in various solid tumor models. MAC15A ascites tumor cells were transplanted into male NMRI mice by intraperitoneal inoculation at 1 x 10 ^s cells in 200 μΐ buffer (Table 1). P388 leukemia was transplanted intraperitoneally to male BDF mice at a density of 1 x 10 * cells in 200 μΐ buffer (Table 2). Solid tumor models included murine MAC13 and MAC16 column adenocarcinomas, murine B16 F1 melanoma and reticular cell sarcoma M5076.

Treatment was initiated three days after intrapertoneal transplantation or, in the case of solid tumors such as MAC13 and MAC16, treatment was started when the average tumor volume reached 40 mm ³ .

Animals were divided into groups of 5 to 8 in both cases.

Animals were sacrificed after 12 days, or when tumors began to ulcerate, or when tumor volume exceeded 1000 mm ³ , or when weight loss exceeded 50%. <

Except in the above cases, the compounds used were dissolved in dimethylsulfoxide and diluted with sterile distilled water to a suitable concentration prior to administration in a solvent volume of 200 μΐ. Anti-tumor response was obtained by comparing mean survival times or inhibiting tumor growth versus solvent control.

The results obtained are shown in Tables 1 to 8 below. The preparation of the dosing solutions is exemplified below:

Subjects: number: 10 animals weight: 22 g

Dosage mg / kg body weight / animal / day, i.e. 1.1 mg / mouse / day

Total dose:

Total mixture:

mg of active ingredient (based on five-day treatment regimen) ml of solvent + 55 mg to be divided into 50 doses of 1.1 mg, dissolved in 200 μΐ of solvent

The T / C% is determined as follows:

Animal survival test

inspection

T days

C days

T / C% = Tx100

Example

Animal survival test

443 days

100 days

443

T / C% = -X100 = 443

100

Number 158 or above of clinical trials.

-t points to the legitimacy of execution

conclusions

The effect of the primary halogenated arylating compound group on the growth rate of the experimental tumor group was evaluated in vivo and the following results were recorded:

The structure-activity relationships against the adenocarcinoma of mouse MAC15A in male NMRI mice showed the highest efficacy in the divided dose schedule and when the halogen was maximally activated for nucleophilic attack.

2. The most active compound was 4-chlorobenzenesulfonic acid (T / C% 443), administered in doses of 100 mg / kg body weight according to a five-day schedule.

3. Against sarcoma M5076 reticular cells, 2,4-dichloro-3,5-dinitrobenzoic acid showed efficacy on a divided dose schedule of up to 25 mg / kg body weight, both intraperitoneally and subcutaneously. Both its amide and methyl ester showed a ten-fold increase in toxicity and no anti-tumor efficacy. The acid also significantly inhibited the growth of murine B16 melanoma and MAC15A mouse colon adenocarcinoma.

It can be concluded that this class of compounds exhibits a broad spectrum of activity against mouse models.

Table 1

Anti-tumor activity against MAC15A (mouse colon adenocarcinoma). Structure-efficiency relationship. Groups of 5 animals. Dose 100 mg / kg intraperitoneally daily.

compound

Scheme (days)

T / C%

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

1,2,3,4,5 443

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 l-chloro-3,4-dinitrobenzene 1,2,3,4,5 200 l-chloro-2,4-dinitrobenzene 1,2,3,4,5 188 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 l-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

a = mean, T = test group, C = solvent control, b = poisoning

Table 2

Anti-tumor activity against P388 (mouse leukemia), 8 animals per group. Intraperitoneal application from day 1 to 5. Dosage daily.

compound dose T / C 4-Chloro-3,5-dinitrobenzenesulfonic acid 100 mg / kg 203 4-chloro-3,5-dinitrobenzén- sulfonic acid 50 mg / kg 259 a = mean, T = experimental group, C = solvent control

Table 3

Anti-tumor activity against P388 (mouse leukemia) treated with intraperitoneal administration of 4-chloro-3,5-dinitrobenzenesulfonic acid (CDNSA). 8 animals per group. Dosage daily.

compound Dose (mg / kg) Scheme (days) T / C% " CDNs 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 in reticular cell sarcoma M5076, 16 days after intramuscular transplantation. 7 animals per group. Compounds dissolved in corn oil. Dosage daily.

compound dose mg / kg Application % Inhibition Scheme days tumor 2,4 BA 75 ' ip 1,4,6,9 79.88 ^it 50 ip 1,4,6,9 57 25 ip 1,2,, 4,6,9 75 75 sc 1,4,, 5,7,9 66 50 s .c. 1,2,4,5,6,7,9 76 25 sc 1,2,4,5,6,7,9 63 2.4 BZ 2,5 ' 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 BM 1.0 ' 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 = highest tolerated dose b = two independent experiments; 4 animals had no tumor at the second experiment 2.4 BA = 2,4-dichloro -3,5-dinitrobenzoic acid, 2.4 BZ = 2,4-dichloro -3,5-dinitrobenzamide. 2.4 BM = methyl ester 2,4 dichloro-3,5-dinitrobenzoic 4

% tumor weight inhibition:

Treatment group Control

A g B g Tumor weight

B - A% inhibition »x 100

B

Table 5

Antitumor activity of mouse B16F1 melanoma 12 days after subcutaneous transplantation. 6 animals per group. Compounds dissolved in corn oil. Dosage daily.

compound dose mg / kg Application scheme days With inhibition materials. tumor 2,4 BA 75 " ip 1/5 71.81 " 50 ip 1.5 45.56 " 25 ip 1.5 13 75 sc 1,3,530 50 sc 1,3,59 25 sc 1,3,522, 2.4 BZ 2,5 ' ip 1.2 39 1.25 ip 1.2 17 4 BA 100 ip 1.5 39 75 ip 1.5 41 50 ip 1.5 10 4 BZ 5 ' ip 1,3,518 2.5 ip 1,3,518 1.25 ip 1,3,527 4 BM 2,5 ' ip 1.3 67 1.25 ip 1.2, 343

a = highest tolerated dose b = two independent experiments

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

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

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

ΒΖ = 4-chloro-3,5-dinitrobenzamide,

ΒΜ = 4-chloro-3,5-dinitrobenzoic acid methyl ester

Table 6

Antitumor activity in MAC13 adenoacrcinoma mouse column 12 days after intramuscular transplantation. Compounds dissolved in corn oil. Dosage daily.

compound dose mg / kg Application Scbéma days % inhibition materials. tumor 2,4 BA 75 ^a ip 1,4,5 45 2,4 BA 50 ip 1,2,3,4,5,6,7,8,9 39 2,4 BA Fig. ³ ip Fig. ³ Fig. ³ 2.4 BZ 2,5 ' ip 1,2,3,4,5,6,7,8,9 51 2.4 BZ 1.25 i .p. 1,2,3,4,5,6,7,8,9 17 2 BA FIG. * ip FIG. * Fig. *

a = highest tolerated dose

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

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

BA = 2-chloro-5-nitrobenzoic acid, ( ³ : see figure 3 in the attached drawing, see figure 4 in the attached drawing)

Table 7

Antitumor activity in adenocarcinoma of a MAC16 mouse column, transplanted subcutaneously, on the 11th day after initiation of 2,4-dichloro-3,5-dinitrobenzoic acid (2.4 BA) therapy. Compound dissolved in corn oil. Tumor volume was at least 40 mm ³ at initiation of therapy. 6 animals per group. Dosage daily.

compound dose mg / kg Application scheme days % inhibition materials. tumor 2,4 BA 75 " ip 1,2,5,8 88 50 ip 1,2,4,5,8 91

a = highest tolerated dose

Table 8

Antitumor activity in murine melanoma BI 6 12 days after subcutaneous transplantation in black male C 57 mice. 6 animals per group. Dosage daily, intraperitoneally.

compound dose mg / kg scheme days % inhibition materials. tumor 2-chloro-5-nitro- benzoic acid 700 1,2,3,4,5,6 and 62

Additionally, antitumor activity and toxicity studies for the following compounds have been added, with significantly satisfactory results:

C 22

C 23

C 24

C 25

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

2,6-dichloro-4-nitrobenzoic acid, 2-amino-5-nitrobenzoic acid,

C 26

C 27

2-hydroxy-5-nitrobenzoic acid,

3,5-dichloro-4-nitrobenzoic acid.

Primary test

1) Acute infection test

High-titer human immunodeficiency virus strains of HIV-1 were cultured in H9 cells with RPMI 1640 (Flow Laboratories) supplemented with 10% fetal calf serum and penicillin (100 IU / ml). Damaged cells were removed by slow centrifugation and the supernatant was stored at -70 ° C if necessary. In a typical C 8166 assay, CD4 + T-lymphoblastoid cells were incubated with 10 x TCID 50 HIV-1 _rjp at 37 ° C for 90 minutes and then washed three times with phosphate buffered saline (PBS). Aliquots of cells (2 x 10 ^B ) were resuspended in 1.5 ml growth medium in 6 ml tubes and immediately thereafter test compounds were added in logarithmic dilution (200 μΜ to 0.2 μΜ). Stock solutions of 20 mM of each compound were prepared in 70% ethanol. Compounds were stored as a powder and freshly dissolved in distilled water prior to each experiment, or stored as a 20 mM stock solution in 70% ethanol. The final concentration of ethanol in the tissue culture medium was 1%. The cells were then incubated at 37 ° C in an atmosphere of 5% carbon dioxide. 72 hours after infection, 200 μΐ of the supernatant was collected from each culture and tested for HIV (Kingchington et al., 1989, Robert et al., 1990) using an antigenic ELISA method that also recognizes all core proteins (Coulter Electronics, Luton, UK). The following controls were used: supernatants taken from uninfected and infected cells, infected cells treated with AZT (Roche Products UK, Ltd.) and ddC (Roche) and RO31-8959 (Roche), an HIV proteinase inhibitor. Conditioning agents _having the IC 8659, AZT and ddC in infected cells were 1, 10, 20 nM and 200 nM (FIG. 2). ELISA plates were read using a spectrophotometer. Compounds were tested in duplicate at each concentration and the data presented is the average of at least two assays. This test confirmed the efficacy of the compounds by measuring their levels of HIV antigen in the nucleus.

2) Chronically infected cell assay

Chronically infected cells (H9rf) were washed three times to remove the extracellular virus and incubated with the active compounds (200 to 0.2 μΜ) for 4 days. The presence of HIV-1 antigen was then measured by ELISA.

To test the toxicity of the compounds, unmodified H9 cells were incubated with the compounds for 4 days. The supernatants were discarded and the cells were resuspended in 200 µg of growth medium containing ¹ 'C-protein hydrolyzate. After 6 hours, cells were harvested and ^X4 C incorporation was measured.

3) Toxicity test

In testing the toxicity of the compounds of the aliquots of 2 x 10 cells cultured ^with uninfected with the compounds in the same dilutions for 72 hours. The cells were then washed with sodium chloride phosphate buffer solution and resuspended in 200 μΐ of growth medium containing ¹⁴ C-protein hydrolyzate. After 12 hours, cells were harvested and measured at ¹⁴ C. Uninfected and uncultivated incorporation was used as cell incorporation.

-t control. Toxicity is expressed as inhibition of ⁴ C-protein hydrolyzate.

The results of these 4-chloro-3,5-dinitrobenzenesulfonic acid tests are shown in the attached Figure 1, where RC is Radopath C, i.e. 4-chloro-3,5-dinitrobenzenesulfonic acid. The results are also summarized in Table 9 below.

Table 9

compound IC CD TI 50 50 4-chloro-3,5-dinitro- benzenesulfonic acid 3 μΜ 80 μΜ 28.6

The IC _bq value indicates the concentration of the drug that causes a 50% reduction in HIV antigen levels in the nucleus, as determined by the Coulter P24 antigen assay and as determined by doubling the supernatant taken from tubes containing untreated acute infected cells. The CD _b0 value indicates the concentration of drug that causes 50% cell inhibition when measuring the uptake of ¹ * C-protein hydrolyzate. The therapeutic index (TI) is determined as a quotient

CD: IC. so so

Further results with other compounds of the invention are summarized in Table 10 below.

Table 10

compound IC CD ΤΙ SO 50 2-chloro-3,5-dinitro- benzenesulfonic 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 showed that 2-chloro-5-nitrobenzoic acid indicated at least as much efficacy as no greater than some of the compounds whose tests are listed herein. Following the methodology described for the HIV test first, more detailed tests were performed as shown in Table 11 below.

Table 11.1

Structure-activity relationship against HIV

Code Compound

Ag

IC.

Tox

CC.

with

Group A

Pl picryl chloride,

P2 picric acid,

P3 picrylsulfonic acid (sodium salt),

Group B

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

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

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

C4 4-chloro-3,5-dinitrobenzoic acid,

C5 4-chloro-3,5-dinitrobenzamide

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

C7 2-chloro-3,5-dinitrobenzoic acid,

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

C9 4-chloro-3-nitrobenzoic acid,

Duty 2-chloro-4-nitrobenzoic acid,

Cl1 3,4-dichlorobenzoic acid,

C12 2,5-dichlorobenzoic acid,

C13 4-chlorobenzoic acid.

Group C

4-chloro-3,5-dinitrobenzenesulfonic acid,

2-chloro-3,5-dinitrobenzenesulfonic acid,

4-amino-3,5-dinitrobenzenesulfonic acid,

4-chloro-3-nitrobenzenesulfonic acid,

4-chlorobenzenesulfonic acid,

4-nitrotoluenesulfonic acid,

2,5-dichlorobenzenesulfonic acid,

2,4-dinitrobenzenesulfonic acid.

Group D

EI 1-chloro-3,4-dinitrobenzene,

E2 1-chloro-2,4-dinitrobenzene,

E3 1,2-dichloro-4,5-dinitrobenzene

E4 2,3-dichloronitrobenzene,

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

Eli 1,2,4-trichloro-5-nitrobenzene,

E12 2,4,6-trichlorobenzene,

E13 2,3,4,6-tetrachloronitrobenzene,

E14 pentachloronitrobenzene.

-t

Table 11.2

P-compounds IC with CC 50 ARE YOU* Against HIV-1RF Pl 0.6 7 10 - 5 - 0.4 - - average 0.5 6 12 P2 38 67 2 P3 > 200> 200 - Against HIV-11IB Pl 0.6 7 11.6 1 7 7 average 0.8 7 9 Against chronically infected cells Pl 0.9 7 8 2 12 6 average 1.5 9.5 7 Table 11. 3 Against HIV-IIIB Cl 5 70 14 36 70 2 33 70 2 35 60 2 average 27 70 5

x = selectivity index

Against HIV-1RF

Cl 7 60 8.5 - - 56 16 56 3.5 average 11.5 57 5

Against chronically infected cells Cl 16 30 2 16 95 6 Average 16 63 4

Against HIV-11IB 2 70 35 C2 C3 0.3 7 23 C4 40 100 2.5 30 70 2.3 average 35 85 2.4 C5 5 50 10 C6 5 60 12 C7 23 150 6 5 > 200 > 10 average 22 > 175 8 C8 10 60 5 C9 > 200 > 200 - C-10 > 200 > 200 - C-II > 200 > 200 - C-12 > 200 > 200 -

Table 11.4

S-compounds

Against HIV-IRF

ARE YOU average 20 19 20 100 60 80 5 3 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 35 75 2.4

Table 11.5

Compound E

Against HIV-IRF

E1 4 10 2.5 E2 4 13 3 E3 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 - Eli > 200 > 200 - E12 > 200 > 200 - E13 > 200 80 - E14 > 200 > 200 -

While the invention has been described above with various specific details, it is to be understood that many different ranges of claims have to be stated that modifications are possible within the meaning and limits of the following. For example, the functional groups may be in various other positions, of which those specifically mentioned above serve only as examples.

Description of pictures

Figure 1 shows HIV antigen and toxicity tests, where RC is 4-chloro-3,5-dinitrobenzenesulfonic acid.

Figure 2 shows internal controls and AZT and ddCyd.

Figure 3 (see also Table 6) shows the effects of 2,4-dichloro-3,5-dinitrobenzoic acid, administered intraperitoneally, on the growth of MAC13 mouse subcutaneously transplanted adenocarcinoma using a divided dose schedule, 6 animals per group . The individual curves pertaining to certain doses of active compound (in mg / kg animal body weight) express the tumor size in mm ³ versus time in days and compare it to the control. The abbreviation stands for body weight.

In FIG. 4 shows the effects of 2-chloro-5-nitrobenzoic acid (see also Table 6), applied daily intraperitoneally, on the growth of adenocarcinoma of a mouse column transplanted subcutaneously, 6 animals per group. The individual curves pertaining to certain doses of active compound (in mg / kg animal body weight) express the tumor size in mm ³ versus time in days and compare it to the control. The abbreviation stands for body weight.

Industrial usability

The compounds according to the invention, together with the benzene derivatives which are known, show remarkable preventive and therapeutic efficacy in precancerous and cancerous conditions as well as in diseases caused by viral infections, for example HIV. Once processed and formulated in a suitable dosage form according to the conventional practice of the pharmaceutical industry, the potential drugs of the aforementioned disease states are potential.

-t

Claims (5)

A compound useful as a medicament comprising an aromatic ring structure and having at least one labile readily cleavable substituent and at least one electrophilic substituent. A compound according to claim 1 having the formula I x (I) in which one of X ¹ to X ® is a labile, easily cleavable substituent with which an electrophilic substituent balances and the remaining X are the same or different and represent a hydrogen atom or substituent. A compound according to claim 2, wherein X ¹ represents a readily displaceable labile substituent, one of x ² and X ® is an electrophilic substituent, and the remaining X, independently of one another, are H or a substituent, with the proviso that when ^X and X ® is nitro, X * is not a nitro, sulfonic acid or sulfonate group, or X ¹ is not a labile group as defined below, in particular a hydroxyl group, an amino group, a sulfo group, a carboxyl group, a methoxy group, a halogen atom or a hydrazyl group of formula wherein A is a hydrogen atom or an unpaired electron of a nitrogen atom, Y is a hydrogen atom or an organic group and Z is an organic group, or Y and Z, together with the adjacent nitrogen atom, form a nitrogen heterocycle. A compound of formula (I) according to claim 2, wherein one of X ¹ to X ^e represents a labile, easily cleavable substituent with which an electrophilic substituent balances, and the remaining Xs are the same or different and represent a hydrogen atom or a substituent, respectively. wherein at least two of them are other than nitro, at least one is a labile substituent and at least one is an electrophilic substituent. A compound of formula I according to any one of claims 2 to 4, wherein at least one of X ^x to X ^e is an electrophilic substituent or a labile substituent selected from the group of electrophilic substituents, such as SO ₃ H or SO ₃ M, wherein: M is a metal atom; or a halogen atom and a nitro group; or labile substituents such as halogen, SO ₃ H or SO ₃ M, in which M is a metal atom; optionally substituted amino, carboxyl, optionally substituted CONH _, and further a COOR ³ group wherein R ³ is a metal atom or an alkyl group. A compound according to any one of the preceding claims having the general formula II (II) in which X- ⁷ represents SO ₃ H or SO ₃ M, where M is an atom