CZ307395A3 - Arylation agents, pharmaceutical compositions containing thereof and their use - 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

Arylating agents, pharmaceuticals. compositions containing them and their use ____ v-- ¹

U3 σ »

Cblast techniques σ

o řzx

OC

-n

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The invention relates to arylating agents, in particular phosphorylating agents, which are useful as compounds having a therapeutic effect, in particular in the treatment of cancer and diseases associated with cancer.

in the broadest sense, the invention relates to arylating agents which are useful 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 certain 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 do not include more than two nitrosubstitutes. The combination of a halogen atom (e.g., a chlorine atom) with a nitrosubstitute, especially in conjunction with a monocyclic aryl agent having a ring having a carboxylic acid substituent, is a particularly effective structure. An example of such a structure is a combination of mono-nitrosubstituted and mono-chlorosubstituted (for example, 2-chloro-nitrobenzoic acid and 2-chloro-4-nitrobenzic acid). , the invention

Accordingly, the present invention relates to a compound for use in the treatment of cancer or a disease caused by viral infection, in particular AIDS, which compound has an aromatic ring structure with at least one easily cleavable group substituent and at least one electrophilic group substituent provided that: Lam, wherein there are two ortho nitro groups and a para sulphonic group something three symmetrical nitroskupin.ya labile group at position one _with even such a group that is defined in international patent;

: nce

2 WO 91 / 152OC (International Specificatior.); it is used in a concentration higher than 1 x 10 ”mol / liter.

Generally speaking, the compound of the invention is of Formula I

ti) in which n is an integer and denotes at least 2 and X is the same or different and denotes a labile or electrophilic group, provided that at least two X groups which are different from the nitro group are at least one of which at least one is an electrophilic group.

In addition, it is contemplated that what is considered to be an arylation mechanism is the use of relatively high concentrations and hence high doses. In general, such concentrations of the compounds of the invention will be at least about 1x10. mol / liter, which in dosage terms is usually at least about 5 mg / kg.

When selecting the substituent grouping, the compound of the invention is an essential feature of providing a link between a particular aromatic ring and at least one substituent as a labile group and at least one electrophilic substituent. Moreover, a group to be classified as labile in connection with a particular ring may alternatively be classified as electrophilic in association with another ring. Further, where at least two nitro groups are substituents, the substituent may be labile. hydrogen atom groups in the ring ..

For the sake of clarity, preferred substituent groups may be defined as those wherein at least one X is selected from any one of the following groups, in particular a green electrophilic group, such as SO 3 H or SO 4 M, wherein ii is a metal atom such as potassium;

- 3 labile groups such as or a halogen atom and a nitro group; halogen atom, group

SO3H or SO4Si in which M represents a metal atom; amino, substituted amino, for example

NHR 2 R 2 ^and R 2 are the same or higher than the term "alkyl", "alkoxy" or "hydroxyalkyl"; carboxyl group,

CONH 8, substituted with CO ₂ NH ₂ , for example, CONHR 1, CO 2 NR ₂ R ₂ , R 1 and R ₂ mean the same as above) and also in group COORp kteréýRj

denotes a metal atom or an alkyl group.

Thus, general examples of the compounds of the invention are, in particular, the following compounds:

chlordinitrobensenesulfonic acids, chlorobenzenesulfonic acids, dichlorobenzenesulfonic acids, aminodinitrobensenesulfonic acids, nitromethylbenzenesulfonic acids, glutathionyldinitrobenzenesulfonic acids, nitrochlorobenzenesulfonic acids, dinitrobenzenesulfonic acids, dinitrobenzenesulfonic acids, dinitrofluorobenzenes, tricinitrobluorobenzenes, trichlorititrobenzenesulfonates

- 4 trinitroanilines, trinitrochlorobenzenes, trichloro-benzylic acid, chlorobenzoic acid, dichlorobenzoic acid, dinitrobenzoic acid, nitrochloranisols, aminodinitrobenzamides,

........ _> ....... — ______________ Dinitrochlorosnilins, chlornitroanilines, dinitrofluoranilins.

I ¹ !

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

wherein X 'is SO-JH or SQ-Al, where it is a metal atom; or a halogen atom such as chlorine, fluorine and the like; a CQQ group wherein Q is a hydroxyl group, an amino group or a substituted amino group, or an OR p group wherein IU is a metal atom or an alkyl group; amino, substituted amino, nitro or hydroxyl;

x represents a hydrogen atom, a halogen atom, a glutathione or a nitro group, the substituents 3 being the same or different and 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 have priority in these compounds:

X 1 ^- is SO 2 H or SO 2 M, wherein i; is a metal atom; a halo gene atom such as chlorine, fluorine and the like, amino, nitro or carboxyl, and

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

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

and

(a) a compound of formula (111)

wherein a represents a hydrogen atom, a halogen atom such as chlorine, fluorine and the like or glutathione;

B represents a hydrogen atom, a nitro group or a halogen atom such as chlorine 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.

The compounds of formula (III) are preferred, since the SO2H group is believed to be involved in the emulsifying effect which is beneficial, i.e. ¹ enhances the spread of the compound ( ³⁾ and contributes more to the improved bioavailability in cellular cells. walls.

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

4-chlorobenzenesulfonic acid, ____________ 2,5-dihydroxybenzene sulphonate = kya & l.i.na ------- = - - - -..... ....

4-amino-3, p-dinitrobenzenesulfonic acid,

3-nitro-4-methylbenzenesulfonic acid,

2-chloro-1,2-dinitrobenzenesulfonic acid,

2-glutathionyl-1,5-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-dinobenzobenzenesulfonic acid,

4-chlorobenzenesulfonic acid,

2.5-dichlorobenzenesulfonic acid,

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

2-chloro-3,5-dinitrobensenesulfonic acid,

b) Compound of formula IV

Hal

(B)

B (IV)

Wherein Hal represents a halogen atom, for example chlorine, fluorine and the like, and substituents 2 are the same or different and have the meaning given above. ^: ........... '· ~

Among the above compounds of formula IV, the most preferred are:

1-chloro-2,4-dinitrobenzene,

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

1,3-dichloro-4, b-dinitrobenzene.

the following compounds are particularly preferred:

1,3-aichloro-d, 6-dinitrobenzene, 1-chloro-2,4-dinitrobenzene,. 1-fluoro-2,4-dinitrobenzene.

c) A compound of formula V

in which substituents 2 denote a group SO4H or SQy2, wherein i ^: is a metal atom such as potassium; amino or substituted amino, halogen or hydroxyl,

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

- 8 2.4.6- trinitrophenol, ie picric acid,

2.4.6- trinitroaniline; . ____________

2.4.6- trinitrochlorobenzene,

2.4.6- trinitrobenzenesulfonic acid.

Of the aforementioned most preferred compounds, the first and third are particularly preferred.

d) General merger

COQ

G wherein substituents 3 are the same or different and have the meanings given above,

G is as defined above as C, except for the alkyl group a. 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 p group in which R 6 is a metal atom or an alkyl group.

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

2> 4-chloro-3,5-dinitrobenzoic acid, 4-chloro-3,5> -dinitrobenzoic acid, *

2,5-dichlorobenzoic acid,

2,4-dinitrobenzoic acid,

- 9 - 3,5-dinitrobenzic acid,

3-nitro-4-chloroanisole,

-4- [alpha] -3-, 7-trifluorobenzamide;

e) Compounds of formula VII

NK '. d

(VII),

wherein the substituents mentioned above, together with

Of the above compounds

Stejné, the same or different, have the meaning of their amino substituted derivatives.

of the formula VII are most preferred:

2,6-dinitroaniline,

2.4- dinitroaniline,

3.5- dinitroaniline,

2,4-dinitro-6-chloroaniline,

2, δ-dinitro - + - caloraniline,

2-chloro-4-nitroaniline,

2,4-dinitro-5-fluoroaniline. .

ihimoradiene is preferred

2, β-dinitroaniline.

As mentioned above, where there are at least two nitro compounds as substituents, the labile group represents a hydrogen atom in the ring. In this connection, the compound of formula VIII should be mentioned

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 conventional publications, such as, for example, U.S. Pat.

* 'Crganic' Syntheses 1363, Collective Volume 4, pp. 364-366, 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., for intravenous, intraperitoneal, oral or subcutaneous administration) containing at least one active agent and a diluent or carrier. Thus, the invention also includes a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable diluent, a 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, and 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.

- ir- ¹

The diluents, excipients or epode carriers 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 can be administered as a solution in sterile deionized water. Also, if possible, dissolution can be facilitated using dimethyl sulfoxide 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 the context of said applications, the same uses of a compound as characterized herein for the preparation of a medicament for the prophylaxis or therapy of cancer or viral infections, for example, to reduce or eliminate the growth of cancer.

When the compound of the invention is administered, the dosage can 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 be in the range of about s

from about 5 mg / kg to about 20 mg / kg and possibly generally up to about 40 mg / kg or above. The concentration and dose should be sufficient to activate the arylation mechanism.

.. 'iS * i' W

As can be seen from the above-mentioned particularly preferred compounds, those compounds of the invention which exhibit the highest activity are arranged, in particular, in any descending order:

4-chloro-J, p-dinitrobenzenesulfonic acid, 4-chlorobenzenesulfonic acid,

1,5-Dichloro-2,3-dinitrobenzene

2.4.5- trinitrophenol, ie picric acid,

_{2,4-dichloro-3-R} 5 dinitrobenzcová acid -dichlorber.zensulz ^ Z ^ sulfonic acid, 4-amino-3,5-dinitrobenzenesulphonic acid.

3-nitro-4-methylbenzenesulfonic acid,

4-chloro-3,5-dinitrobenzoic acid,

- 12 2,6-dinitroaniline,

2,4-dinitrochlorobenzene.

2, é-dinitrofluorobenzenes-- ~ “- ^L -

2.4.5- trinitrochlorobenzene,

2,5-dichlorobenzoic acid,

2-chloro-1,5-dinitrobenzenesulfonic acid, 2,4-dinitrobenzenesulfonic acid.

J2b. 3, at least one of the preferred compounds are such compounds, one X being selected from the group or groups in which labile substituents, such as electrophilic 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 30 or 30 -J, one or two nitro and / or 30-H or SO-J 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 the three symmetrical nitro groups as substituents or active ingredient are other than those mentioned in International Patent Application Ser.

WO 91/15200 (International Specification), as indicated above, the concentration of the active ingredient in any formulation must be greater than -2 -2 of 10 mol / liter and preferably at least 1 x 10 mol / liter.

As shown in Table 3 below, 2-chloro-5-nitrobenzoic acid has significant antitumor efficacy 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 immuno-immunotranslators.

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

Animal studies have been known to have been a group of compounds with structural similarity in nature. which may act as arylating agents. Their anti-tumor responses in vivo were determined in two ascitic tumors, the MAC1pA mouse colon adenocarcinoma and the P388 mouse leukemia, and in various solid tumor models. IIAC1pA ascites tumor cells were transplanted into male NdR1 mice by intraperitoneal inoculation at 1 x 10 5 cells in 200 µl of buffer (Table 1). Leukemia 55OS was transplanted intraperitoneally to male BDF strains at a density of 1 x 1q6 cells in 200 µl of buffer (Table 2). Solid tumor species included murine adenocarcinomas of the MAC13 and ϋΑΟΙό column, murine melanoma, B16 F1, and reticular cell sarcoma jfi5G75.

Treatment was initiated three days after or, in the case of solid tumors, it started when the average intraperitoneal transplant, such as. is iDAClj and ilAClo, the tumor volume was 40 nmr ⁵ .

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 1CCC mm ^J , 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 an appropriate concentration prior to administration in a solvent volume of 2 CO 2. The tumor tumor response was obtained by comparing mean survival times or inhibiting tumor growth versus solvent control.

The results obtained are listed in Tables 1 to 8 below.

The preparation of dispensing solutions is given below as an example:

Subjects:

number: 10 animals weight: 22 g

Dosage:

mg / kg body weight / animal / day, -fcedy- ^ v ^^ / ^ yš / deir- ··· —---------------- Total dose of the substance:

The total amount of a mixture of mg of active ingredient (based on the five-day treatment regimen) ml of solvent + 55 mg to be divided into 50 doses of 1.1 mg dissolved in 200 µl of solvent.

T / C $ is determined as follows:

Animal survival test

Control

T days

C days

T / C = T x 1C-C

Example

Animal survival test

443 days

100 days

T / C2 = 443 x 100 - 443 ICO number 153 or higher indicates the justification for clinical trials.

- 15 Conclusions of the effects of the primary halogenated> arylaline group on the growth rate of the group of excerptive tumors were evaluated in vivo and the following results were recorded:

Structure-activity relationships against adenocarcinoma of the mouse ttiAC15A column in male NxKI mice showed the highest efficacy in a split 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 10 mg / kg body weight according to a five-day schedule.

3. Against 345076 reticular cell sarcoma, 2,4-dichloro-3,5-dinitroacetic acid showed efficacy at 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 S16 celanoma and MAC16 mouse colon adenocarcinoma.

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

Table 1 '

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

Compound

Scheme φ / piz ⁸ (days) ^{I / C} *

4-chlorobenzenesulfonic acid 1,2,3,4,5 443 4-chloro-3,5-dinitrobenzenesulfonic acid 1,2,3,4,5 414 1,5-dichloro-2,5-dinitrobenzene 1,2,3 , 4.5 386

2.4.6- trinitrophenol

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

2,4-dichloro-1,5-dinitrobenzoic acid

2-Glutathionyl-3,5-dinitrobenzenesulfonic acid j-nitro-4-methylbenzenesulfonic acid

2.6- dinitroaniline

2,5'-dichlorobenzenesulfonate-6-l-ina

1,4-dinitrobenzene l-c´nlor-3,4-dinitrobenzene

1-chloro-2,4-dinitrobenzene 2,4,4-trinitrobenzenesulfonic acid

2-chloro-4-nitroaniline

2,5-dichlorobenzoic acid

2,4- dinitrobenzenesulonic acid

1,2-dichloro-4, p-dinitrobenzene 4-chloro-3-nitrobenzenesulfonic acid

2-chloro-3,5-dinitrobenzenesulfonic acid 1-chloro-2,4,6-trinitrobenzene 4-glutathicnyl-3,5-dinitrobenzene

2.4- dinitroaniline.

2,4- dinitrobenzoic acid

3.5-dinitrobenzic acid

4-amino-3,5-dinitrobenzaid

4-chloro-3-nitroanisole

4-chloro-2,6-dinitroaniline

6-chloro-2,4-dinitroaniline 1-fluoro-2,4-dinitroaniline 1-fluoro-2,4-dinitrobenzene

1,2,3 300

1,2,3,4,5 236

1,2,3,4,5 · 271 1,2 243 1,2,3,4,5 242 1,2,3,4,5 229 1,2,3,4,5 214 '£ T2 ~ 1,2 200 1,2,3,4,5 . 200 1,2.3,4,5 13S 1,2,3,4,5 183 1,2,3,4,5 171 1.2, j, τ, j 171 1,2,3,4,5 171 1,2,3,4,5 171 1,2, j, 4,5 140 1,2,3,4,5 137 1,2,3 113 1,2,3,4 ¹ '113 1,2 100 ALIGN! 1,2,3,4,5 100 ALIGN! 1,2,3,4,5 100 ALIGN! 1 100 ALIGN! 1,2,3,4,5 100 ALIGN! 1,2,3,4,5 '37 1,2,3,4,5 37 1 75 1 62,

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

- 17 Table 2

Against Tumor Additives against P3Sc (murine leukemia). 8 animals per group. Intraperitoneal application from day 1 to 5. Dosage daily.

Compound

Dose T / C% ^a

4-chloro-3,5-uinitrobenzanesulfonic acid

4-chloro-3,5-dinitrobenzenesulfonic acid

ICO mg / kg 203 mg / kg 259 a-mean, T-test group, C = solvent control

Table 3

Anti-tumor activity against P383 (mouse leuksmis), treated by intraperitreal administration of 4-chloro-3,5-dinitrobenzenesulfonic acid (COHSA). 8 animals per group. Dosage daily.

Compound

Dose (mg / kg)

Diagram (days) T / C% ^a

CDJÍSA. ICO 1,2,3,4,5 225

1,2,3,4,5 300 a = diameter, T = test group, C = solvent control

- 18 Table 4

Antitumor activity in M5O76 reticular cell sarcoma 16 days after intramuscular transplantation. 7 animals per group. Compounds dissolved in corn oil. Dosage daily.

Compound Dose Application Diagram % inhibition mg / kg days ------------------ · '·· - --- = --- - tti-c-rti - 2.4 SA 75 ^a i.p. 1,4,6,9 79,68 ^p 50 i.p. 1,4,6,9 57 '· 25 i.p. 1,2,4,6,9 75 75 s .c. 1,4,5,7,9 66 50 s, c. 1,2,4,5,0,7,9 76 .. 25 o · ΰ » 1, 4, 5, 6, 7.9 63 2.4 3Z · 2.5 ^a i.pi 1,2, c, 4,5,6,7, c, 9 51 1,25 i.p. 1, 2,3,4,5, o, 7, o, 9 34 2.4 Bii 1.0, ^and i.p. 1,2,3,4,5,0,7,6,9 41 ' 0.5 i.p. 1,2,5,4,5,6,7,6,9 39 0.25 i.p. 1,2,3,4,5,6,7,3,5 42 a = highest tolerated dose b = two independent experiments; 4 animals had no tumor at the second

kuse

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

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

2.4 Bwi = 2,4-Dichloro-3,5-dinitrobenzoic acid methyl ester fc Inhibition of tumor weight: Treatment group Control

A g B g% inhibition = B-A x 100

Tumor weight

-19-Table 5 Cancer activity in mouse melanoma ΚΓΪ5ΚΓΪ2άηί after subcutaneous transplantation. 6 in a group. Compounds dissolved in corn oil. Dosage daily. ,

Compound Dose Application Scheme% Inhibition of Mass

Mg / kg days. the nose of the tumor

2,4 ΞΑ 75 ^a i .p. 1.5 71.61 50 i.p. 1,5 =. 45.56 25 i.p. 1,5 ' 13 75 s.c. 1,3,530, 50 s.c. 1,3,5 9 25 s.c. 1,3,522 2,4 32 2,5 ^a i.p. 1,2 39 1,25 í · p · 1,2 17 4 3a 100 ALIGN! X · p. 1.5 39 75 i.p · 1.5 41 50 Lep * 1.5 10 4 3Z 5 ^a i.p. 1,3,513 2.5 i.p. 1,3,513 1,25 and·?· 1,3,527 4 3M 2,5 *. i.p * 1.3 67 1,25 i.p. 1,2,343

a = the highest tolerated dose b = two non-continuous experiments

2,4 3A = 2,4-dichloro-1,5-dinitrobenzoic acid,

2,4 32 = 2,4-dichloro-1,5-dinitrobenzamide,

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

4 ^l BS - 4-chloro-3,5-dinitrobenzaniid ____

3M ~~~ 4-chloro-3,5-dinitrobehzoic acid methyl ester

Table 6 frotin tumor activity in wiAC13 mouse column adenocarcinoma 12 days after intra-muscular transplantation. Compounds dissolved in corn oil. Dosage daily.

Compound Dose mg / kg and Application Diagram days % inhibition of tumor tumor 2,4 SA 75 ^a i.p. 1.4,5 45 2.4 ba 50 i.p. 1,2,3, 4.5,6.7, S, 9 39 2,4 BA giant. i.p. giant. and 0 « 2.4 BZ - e2 4,5 i .p. 1.2,3, 4.5, o, 7, S, 9 51 2 ^ 4 POI 1,25 i .p. 1.2,3, 4,5.6,7,3,9 17 2 3A Giant. ⁴ i.p. Fig. ⁴ Fig. ⁴

and - the highest tolerated dose

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

2,4-BZ = 2,4-dichloro-1,5-dinitrobenzamide,

BA - 2-chloro-5-nitrobenzoic acid, (3: see Fig. 3 in the attached drawing; 4: see Fig. 4 in the attached drawing)

Table 7 Tumor adenocarcinone MAC 16 activity of a mouse column transplanted subcutaneously on the eleventh day after initiation of 2,4-dichloro-3,5-dinitrobenzoic acid (2.4 3A) therapy. Compound dissolved in corn oil. Tumor volume was at least 40 mm} at initiation of therapy. 5 animals per group. Dosage daily,

Compound Dose Application Scheme mg / kg days% tumor weight inhibition 2.4 2.4 75 75 ® · 1,2 · 1.2.5.3 83

i.j. 1,2,4,5,3 91 a = highest tolerated dose

Table 3: Tumor activity in mouse melanoma 316 12 rises after subcutaneous transplantation to black male C 57 strain. 6 animals per group. Dosage daily, intraperitoneally.

Compound

Dose Scheme mg / kg days% tumor weight inhibition

2-chloro-5-nitro-700 1,2,3,4,5,6 62 benzoic acid

Additionally, studies of antitumor activity and toxicity of the following compounds and supplementary results have been added with the following results:

C 22 2,5-dichloro-4-nitrobenzoic acid,

C 23 2,4-dichloro-5-nitrobenzoic acid;

C 24 2,6-dichloro-4-nitrobenzoic acid,

C 25 2 - anino - 5 - nitrobenzoic acid,.

C 26 2-hydroxy-5-nitrobenzoic acid,

C 27 3,5-dichloro-4-nitrobenzoic acid.

Primary test

1) Acute infection test. High titer HIV-L · - human immunodeficiency virus strains were cultured in H9 cells with EPiril 1640 (Flow Laboratories) supplemented with 10 µl fatal calf serum and penicillin (100 IU / ml). The damaged cells were removed by slow centrifugation and the supernatant was stored at -70 ° C if necessary. In a typical 0 3166 assay, CD4 + T-lymphoblastoid cells were incubated with 10 x TCID 50 EIY-L, - at 37 ° C for 90 min and then washed three times with phosphate buffered saline (PBS). Aliquot. aliquots of cells (2 x 10 6) were re-suspended in 1.5 ml growth medium in 6 ml tubes and test compounds were immediately added in logarithmic dilutions (200 µl to 0.2 µl). Stock solutions of 20 ml of each compound were prepared in 70% ethanol. The compounds were stored in powder form and freshly dissolved in distilled water before each experiment, or were stored as a 20 mass stock solution in 7C ethanol. The final ethanol concentration in the tissue culture medium was ltfc. The cells were then incubated at 37 ° C in an atmosphere of 5% ‘carbon dioxide. After 72 h after infection, 200 µl of supernatant was collected from each culture and tested for HIV (Kingchington et al., 1939, Robert 3e et al., 1990) using the antigen ELISA method, which recognizes all core proteins in the same way (Coulter Electronics, Luton, UK). The following controls were used: 3upernatants taken from uninfected and infected cells, infected AZT-treated cells (Roche Products UK, Ltd) and ddC (Roche) and R031-3959 (Roche), a proteinase inhibitor

HIV. IC50 activities of 5959, A2T, and ddC in infected cells were 1, 13, 20 µM and 200 nM (see 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 compounds by measuring their levels of HIV sntigene in the nucleus.

2) Chronically infected cell assay. Chronically infected cells, which were washed and washed with 7 ³³ removed the extracellular virus (0.2 / 1.4) after d, were incubated with 3 active compounds cbu for 4 days. The presence of HIV-1 antigen was (200 to then measured by ΞΙΙΞΑ). To test the toxicity of the compounds, uninfected H9 cells were incubated with the compounds for 4 days. The supernatants were discarded and resuspended in 200 µl pg of growth medium containing C-protease hydrolvzate. After 6 h, the cells were glazed and C. incorporation was measured.

3) Toxicity test. For compound toxicity testing, aliquots of 2 x ⁷ uninfected cells were cultured with ^tk compounds at the same dilution for 72 h. The cells were then washed with phosphate buffered saline and resuspended in 20C / µl growth medium with containing C-protein hydrolyzate. After 12 h, cells were harvested and C. incorporation measured. X-infected and non-cultured cells were used as con-4.

-.ΐ ΐ

trola. Toxicity is expressed as an inhibition of incorporation of a tein hydrolyzate.

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

Table 9 Compound" ^IC 50 CD 50 TI. 4-chloro-3,5-di-nitro- benzenesulfonic acid 3) iM eo them 23.6 The IC-θ value indicates the concentration effectively Substances which raises

decrease in core antigen levels as determined by the Coulter F24 antigen assay and as determined by doubling the supernatant taken from tubes containing untreated acutely infected cells. The CD50 indicates the concentration of drug that causes 50% cell inhibition when measuring ¹⁴ C-pro tein hydrolyzate uptake. Therapeutic index (TI) can be determined as a ratio of CD50 ^IC 50 ·

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

Table 10

Compound

2-chloro-1,5-dinitrobenzenesulfonic acid

4-Amino-3,5-dinitrobenzenesulfonic acid

2,4,6-trinitrophenol

4-chloro-3,5-dinitrobenzoic acid

IC

0, them ^0D 5ú

200 jim

TI s

them them

100/1 µm

475 jim 70 jun 2.33

Initial tests carried out at approximately the same time showed that, if not more than any of the compounds, 2-chloro-5-nitrobenzoyl-quin-2-amino-2-amino-1-amino-1-amino-1-aminophenol-2-chloro-inarnamine whose tests are listed here. According to the methodology described previously for the HIV test, more detailed tests were performed as shown in Table 11 below.

- 26 Table 11.1

Relationship between structure and activity against HIV

CODE Compound

Oh

IC

Tox

CC

Group A

Pl pikrylehlorid,

P2 picric acid,

F3 picrylsulfonic acid (sodium salt).

Group 3

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

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

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

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

C5 4-chloro-3,5-dinitrobenzamide,

6 4-chloro-3,5-dinitrooenzoové acid methyl _t

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

CS 2-chloro-3,5-dinitrobenzoic acid methyl ester,

Cy 4-chloro-3-nitro'oenzoic acid,

CIO 2-chloro-4-nitrobenzoic acid,

Cil 3,4-dichlorobenzanoic acid,

C12 2,? - dicfilorben3OO7á acid,

C13 4-chlorobenzoic acid.

Group C14-4-chloro-3,5-dinitrobenzenesulfonic acid,

2-chloro-3,5-dinitrobenzenesulfonic acid,

4-aainO'3,5-dinitrob3nzensulfonic acid,

Sal-___ 4-Cec-3-nitrobenzenesulfonic acid, ____

Sp 4-chlorobenzenesulfonic acid, ε 6 4-nitrotoluenesulfonic acid,

2,5-dichlorobenzenesulfonic acid,

Sg 2,4-dinitrobenzenesulfonic acid.

Sknoina 2'-chloro-3 _of 4-dinitrobenzene l-chloro-2,4-dinitrobenzene,

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

2,3-dichlomi trobenzene, **

2,4-dichloromethylbenzene,

2,5-dichloromethylbenzene,

3,4-dichloromethylbenzene,

3,5-dichlomitrobenzene,

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

210 1,2,3-trichloro-4-nitrobenzene

211 l, 2,4-trichloro-5-nitrobenzene,

E12 2,4,6-trichlorobenzene

313 2,3,4,6-tetrachloronitrobenzene,

314 pentachlomitrobenzene. '' -reTffr · ·

- 28 Table 11.2

P-Compounds Against HIV-1R - ^IC 50 CC- _p Ου SI ^X Pl 0.6 7 ~ ..... 5 10 0.4 Diameter 0.5 / · O 12 P2 j 38 67 2 P3 > 200 > 20C - Against HIV-11IB Pl 0.6 7 11.6 1 7 7 Diameter 0.8 7 y

_buňkám

Pl 0.9 7 8 2 12 6 Diameter 1.5 9.5 6 Table 11.3 C-Compounds Against HIV-IIIB Cl 5 70 14 36 70 2 33 70 O 35 60 2 Diameter 27 Mar: 70 3

x = selectivity index

- 29 Against

Cl 7 60 8.5 - - 56 16 56 3.5 Diameter U, 5 57 5 Fro ti _ synchronic _ cells ----——-—..... -------------------- Cl ló 30 2 15 Dec 95 6 Diameter 16 63 4 Against ΞΙΥ-1ΙΣΧΒ C2 2 70 35 C3 0.3 7 23 C4 40 100 ALIGN! 2.5 30 70 2.3 Diameter 35 65 2.4 C5 « 5 50 10 C6 and*· 0 60 12 C7 23 , 150 6 0 > 200 > 10 Diameter 22nd > 175 8 C8 10 60 5 C9 > 200 > 200 - C-10 > 200 > 200 - > 200 > 200 - C-12 > 200 > 200

Table 11.4

S-Compounds

Against _3IV _of 1H?

SI 20 100 5

Diameter 20 May ⁸⁰ 4 S2 NH ' S3 NH 1 S4 > 200 > 200 - S5 > 200 > 200 - So > 200 > 200 - S7 > 200 > 200 - ss 40 100 ALIGN! 2.5 30 70 2 Diameter 35 75 2.4

Table 11.5

E-Compounds Against HIV-1HP El 4. 10 2.5 Ξ2 4 13 »3 E3 4 7 . 1.5 E4 80 > 200 1.5 E5 180 > 200 1 E6 110 > 200 2

E7 > 200 > 200 £ 8 --Ϊ20- > 200 1.5 S9 ND 310 > 200 90 - Eli > 200 > 200 - E12 > 200 > 200 in

£ 13 --—-------- 60 ~~ ........-.....—

Ε14> 200> 2C0

While the invention has been described above with various special details, it is to be understood that many different modifications may be practiced within the spirit and within the scope of the claims that follow.

For example, the functional groups may be in various other positions, of which those specifically mentioned above serve only as examples.

Description of the picture

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

Figure 2 shows the internal controls of AZT and ddCyd.

Figure 3 (see also Table 6) shows the effects of 2,4-dichloro-3,5-dinitrobenzoic acid applied intraperitoneally on adenocarcinoma growth of a MCICI3 mouse column transplanted subcutaneously, using a divided dose schedule, 6 animals in the 3-group . The individual curves, corresponding to certain doses of the active compound (in mg / kg animal body weight), express the tumor size in mm 2 versus time in days and compare it to the control. Abbreviation t.h. means body weight.

- 31 a

FIG. shows the effects of 2-chloro-5-nitrobenzoic acid (see also Table 6), administered daily intraperitoneally, on the growth of the adenocarcinoma of a 3UC13 mouse column transplanted subcutaneously in animals in the group. The individual curves, corresponding to certain doses of active substance, in terms of weight, are dependent on the tumor size in mm versus time. in days and compare it to control. Abbreviation t.h. means body weight.

- 52 .Industry3usability

The compounds of the invention, all known benzene derivatives, show remarkable preventive and therapeutic efficacy in precancerous and cancerous conditions as well as in diseases caused by viral infections, for example HIV. After processing and processing into a suitable dosage form in accordance with conventional practice in the pharmaceutical industry, it is possible to envisage a potential drug of the aforementioned disease state.

J. KUBAT Patent Attoaey

- 33 • o <ΌC *>> o CO Ϊ * λ c σ ** X patent nLbjT. ^ Y

LO σι

O c, -x o

CC

Claims (32)

Arylating agent useful as a medicament comprising an aromatic ring structure and having at least one labile easily cleavable substituent and at least one electrophilic substituent. A compound according to claim 1 having the general formula I X (1), wherein one of X 1 to X 1 is labile readily cleavable. the substituent with which the electrophilic substituent balances and the remaining X are the same or different and represent a hydrogen atom or a substituent. A compound of formula I according to claim 2, wherein X ¹ is a labile easily cleavable substituent, one of X ² to X 3 is an electrophilic substituent and the remaining X, independently of one another, is a hydrogen atom or a substituent, provided that when X ² and X 1 are nitro, X * is neither nitro, sulfonic acid or sulfonate, or X- is not labile as defined below, in particular hydroxyl, amino, sulphos, carboxyl, methoxy, a halogen atom or a hydrazyl group of the formula Z - N - N I I Y A in which A denotes a hydrogen atom or an unpaired electron of a nitrogen atom, Y denotes a hydrogen atom or an organic group and Z denotes an organic atom - 34, or Ϊ and Z, together with the adjacent nitrogen atom, form a nitrogenous heterocycle._ ________ A compound of formula I according to claim 2, wherein one of X 1 to X 0 is 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, that at least two of them are other than nitro groups, at least one of which is labile. in the name of j-edno ™ j-e-electrophilic substituent. A compound of formula (X) according to any one of claims 2 to 4, wherein at least one of X 1 to X 1 is an electrophilic substituent or a labile substituent selected from the group of electrophilic sostituents, such as SO 2 H or SO 2. wherein M represents a metal atom; or a halogen atom and a nitro group; or a labile substituent, such as a halogen atom, a group SO3E or SO4M, in which M is a metal atom; or optionally substituted amino, carb oxyl, optionally substituted. CONHg and COORj in which Rj represents a metal atom or an alkyl group. A compound according to any one of the preceding claims having the general formula II wherein; denotes:; </ 7 ‘ X 'group. SO 3 H or SO 4 M, wherein U is a metal atom; or a halogen atom, a COQ group wherein Q is a hydroxyl group, an amino group or a substituted amino group, or an OR6 group wherein a metal atom or an alkyl group; or amino, substituted amino, nitro or hydroxyl; p is hydrogen, halogen, glutathione or nitro; Χ-θ _and x ^, independently of one another, a hydrogen atom, a halogen atom or a nitro group; and X is hydrogen, nitro, optionally substituted amino, halogen, alkyl or glutathione. A compound of formula (II) according to claim 6, wherein: X? a SQyl group; Q X is hydrogen, halogen or glutathione; '. <6 · 9 10 X and X, independently of one another, are hydrogen, halogen or nitro; X is hydrogen; and - 36-2 X is hydrogen, nitro / optionally substituted amino, -halo-halo-alkyl-or -g-lutathione. The compound of claim 7, named specifically below: 4-chloro-3,5 ^- dinitrobenzene-3-sulfonic acid, 4-chlorobenzene-sulfonic acid, Sichlorobenzenesulfonic acid. acid ,, ......................... ....... ~~ “4-am'ino-3y5dl-nl ~ troben3-ensui-phon ~ k´y3elÁ-nay —----------- r 3-nitro-4'-methylbenzenesulfonic acid, 2-chloro-3,5 "dinitrobenzenesulfonic acid, 2-glutathionyl-3,5-dinitrobenzenesulfonic acid, 4-glutathionyl-3,5-dinitrobenzenesulfonic acid, 3-nitro-4-methylbenzenesulfonic acid, 3-nitro-4-chloroenzenesulfonic acid, 2,4-dinitrobensenesulfonic acid, 4-chloro-3,5-dinitrobenzenesulfonic acid, a salt of any of the foregoing. A compound of formula (II) according to claim 6, wherein it is X is halogen; . X ⁸ , Χθ, and X ¹² , independently of one another, a hydrogen atom, a halogen atom or a nitro group; and X ¹¹ is hydrogen. A compound according to claim 9, named specifically below: 2,4- dinitrochlorobenzene, ' 3.4- dinitrocblorobenzene, 2.4- dinitrofluorobenzene, 1,2-dichloro-4,5-dinitrobenzene, 1,3-dichloro-4,5-dinitrob enz en, 1,5-dichloro-2,3-dinitrobenzene. Compound of formula (II) according to claim 6, characterized in that it represents: - 7: - - - - - X is SQ 4 H or SO 4 M, wherein M is a metal atom; or amino or substituted amino, halogen or hydroxyl; ^X8 nitro; __________ ________________________ ·. · .......................... ......... X is hydrogen; X ¹⁰ is hydrogen; X 1 nitro; and t ¹ 2 X · nitro. A compound according to claim 11, named specifically below: 2.4.6- trinitrophenol or picric acid, 2,4,6-trinitroaniline, 2.4.6- trinitrochlorobenzene. A compound of formula II according to claim 6, wherein: X a group of the formula -COQ, wherein Q is a hydroxyl group, an optionally substituted amino group, or has the formula OR 6, wherein R 6 is an alkyl group or a metal atom; X ⁸ is hydrogen or halogen; Q IQ X and X, independently of one another, are hydrogen, halogen or nitro; X is hydrogen; and * X is hydrogen, nitro, optionally substituted amino or halogen. A compound according to claim 13, named specifically below: 2-chloro-5-nitrobenzoic acid, 2,4-Dichloro-3,5-dinitrobenzoic acid or its alkyl ester, 4-Chloro-1,5-naphthyl or its alkyl ester 2,5-dichlorobenzoic acid, 2,4- dinitrobenzoic acid, 3.5-dinitrobenzoic acid, 3-nitro-4-chloroanisole · 4-amino-3,5-dinitrobenzamide, 4-chloro-3,5-dinitrobenzamide, 2,4-dichloro-3,5-dinitro'o enz amide. A compound of formula II according to claim 6, wherein: X optionally substituted amino; and Χθ to X * 2, independently of one another, a hydrogen atom, a halogen atom or a nitro group. A compound according to claim 15, named specifically below: 2.6- dinitroani.lin, 2.4- dinitroaniline, 3.5- dinitroaniline, 2,4-dinitro-S-chloroaniline 2,6-dinitro-4-chloroaniline 2-chloro-4-nitroaniline, 2,4-dinitro-5-fluoroaniline. ΐ _Λ1 A compound according to any one of claims 1 to 5, wherein the ----------- hydrogen-hydrogen-carbon boundary forms a labile sub-formula. A compound according to claim 17, named specifically below: 1,2- dinitrobenzene, 1.3- dinitrobenzene, ... 1,4-dinitrob enzen, A compound of formula (I) according to any one of claims 2 to 5, wherein at least one of X 1 to X 1 is selected from the group of labile substituents such as one or two halogen atoms and / or amino or substituted amino and / or carboyl groups. a group or substituted cardioyl group and / or an alkyl group and / or a SO1 group or electrophilic sulfides such as one or two nitro groups and / or a group. And / or one or two halogen atoms. A compound for use in the treatment or prevention of cancerous or precancerous conditions or diseases caused by viral infections, which compound comprises an aromatic ring structure with at least one readily replaceable substituent and at least one electrophilic substituent. A compound useful in the treatment or prevention of cancerous or precancerous conditions or diseases caused by viral infection. _f .př.idemž "tytQ_S-loučenlny selected from the following groups of organic compounds: chlordinitrobenzenesulfonic acid, _f chlorbenzeasulfonic acid, * »> dichlorobenzenesulfonic acid aminodinitrobenzensulfonová acid nitromethylbenzensulfonová acid glutathionyldinitrobenzensulfonová acid nitrochloroenzensulfonová acid dinitrobenzenesulphonic acid ,, dinitrochlorbsnzen, dinitrofluorobenzene, dichlordinitrobenzen, trinitrophenol or picric acid, trinitroanilin, trinitrochlorbenzen, trinitrobenzene sulfonic acid, chlornitrobenzoová acid chlordinitrobenzoová acid dichloroenzoová acid dichlornitrobenzoová acid dichlordinitrobenzoová acid, dinitrobenzoic acid, nitrochloranisole, aminodinitrobenzamide, dinitroaniline, dinitrochloraniline, - 41 chlornitroaniline, - - ..dlnltrofluoranllln .--. · - ‘----- - ··· - A compound useful in the treatment or prevention of boar boars. or precancerous conditions or diseases caused by a viral infection, the compound of which is specifically listed below: 2,4,6-trinitrophenol, ___ 2,4-dichloro-3,5-dinitrobenzoic acid, 4-chloro-3,5-dinitrobenzoic acid. A compound useful in the treatment or prevention of a cancerous * * = or precancerous condition, which compound is specifically listed below: 1,4-dichloro-2,3-dinitrobenzene, 2-chloro-5-nitrobenzoic acid, 4-chlorobenzenesulfonic acid, 4-chloro-3,5-dinobenzenesulfonic acid. A compound useful in the treatment or prevention of a disease caused by a viral infection, which compound is specifically listed below: 4-chloro-3,5-dinitrobenzamide, 2,4-dichloro-3,5-dinitrobenzamide. A pharmaceutical composition comprising a compound according to any one of the preceding claims and a pharmaceutically acceptable diluent or carrier. 26. The composition of claim 25 wherein the diluent or carrier is aqueous. A composition according to claim 25 or 26, characterized in that it is in the form of dosage units. - 42 28. The composition of claim 27, wherein the composition is formulated to form a powder, solution or suspension. Use of a compound according to any one of claims 1 to 24 for the preparation of a medicament for the prophylaxis or treatment of cancerous or precancerous conditions or viral infections, 307 Use according to claim 297 which has been performed at a concentration and dose that allows the arylation mechanism to take effect. .i i - * A compound according to any one of claims 1 to 24 or a composition according to any one of claims 25 to 28 for the treatment of a disease caused by a viral infection. l A compound according to any one of claims 1 to 24 or a composition according to any one of claims 25 to 28 for the treatment of a cancerous or precancerous condition. 33. A compound or chloro- nitrobenzene sulfonic acid, _I4 or chloro- derivative or a nitrobenzoic acid derivative nitrobenzamide chloro- or for use as a medicament.