AP575A

AP575A - Anti viral and anti cancer agents.

Info

Publication number: AP575A
Application number: APAP/P/1995/000732A
Authority: AP
Inventors: Ayuko Washington Odur Dr
Original assignee: Radopath Ltd
Priority date: 1994-03-17
Filing date: 1995-03-17
Publication date: 1997-01-31
Also published as: WO1995024897A1; ATE198273T1; BR9507128A; EP0677292A1; EP0751768B1; OA10311A; AU1900195A; IL113025A0; AP9500732A0; CA2185817A1; ZA952191B; DE69519722D1; EP0751768A1

Abstract

2-chloro-5-nitrobenzoic acid and various other chloro

Description

ANTI-VIRAL & ANTI-CANCER AGENTS

The present invention relates to arylating agents, in particular phenylating agents, 5 which are suitable as therapeutic compounds, especially in the treatment of cancer and disease caused by viral infections (eg retrovirus infections such as HTV. As shown by studies reported herein, the compounds of the invention show marked anti-tumour activity and in AIDS and pre-AIDS therapy show the capacity to regress Kaposi’s sarcoma, improve CD4 count in humans, increase the level of various blood parameters in murine patient models and to achieve weight gain and general patient condition by action against HTV and/or its effects (eg opportunistic disease).

r , 15 i

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According to the invention, there is provided a compound of the following general formula for use in the treatment of prophylaxis or therapy of neoplasm or viral infection such as HTV-infection:-

wherein Xi and X₂ are, each independently, hydrogen or chloro and wherein X₃ and X4 are, each independently, hydrogen or Ditro, provided drat at least two of Xj, X₂, X₃and X4 are other than hydrogen, or a salt or ester thereof (eg a salt wherein -COOH is substituted by -COOR in which R is optionally substituted amino).

AP/P/ 95/00732

Conveniently, Xj is chloro, X₂ is chloro or hydrogen, X₃ is nitro and X+ is nitro or hydrogen. The following are preferred compounds:30

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

2-chicro-3. 5-dirtitrobenzoic acid

2-chloro-5-nitrobenzoic acid 2, 5-dichlorobenzoic acid 2, 4-dinitrobenzoic acid

3, 5-dinitrobenzoic acid

2, 5-dichloro-4-nitrobenzoic acid

2. 4-dichloro-5-nitrobenzoic acid

2, 6-dichloro-4-nitro benzoic acid

3, 5-dichloro-4-nitrobenzoic acid

4-chloro-3-nitrobenzoic acid

2-chloro-4-nitrobcnzoic acid 3, 4-dichlorobenzoic acid

Since Treatment in accordance with the invention is by what is believed to be an 15 arylating mechanism, use is typically at relatively high concentrations and, consequently, doses. Generally, the recommended dosage is 1 mg/kg body weight to

30mg/kg body weight per day for 5 days weekly.

The compounds of the invention may be prepared by known process techniques for 20 preparing benzene substituted compounds. Such techniques are described in various standard texts, for example, “Organic Syntheses” 1963 Collective Volume 4, pages

364 to 366, by Harry P Schultz and published by John Wiley and Sons Inc. Postpreparation sterilization may be required, at least following formulation of the compounds into pharmaceutical composition form.

The compounds of the invention may be formulated for use as pharmaceutical compositions (eg for iv, ip, oral or sc administration) comprising at least one active compound and a diluent or carrier. Thus, the invention includes a pharmaceutical composition, which composition comprises a compound according to the invention and a pharmaceutically-acceptable diluent or carrier (eg aqueous).

AP/P/ 95/00732

AP. Ο Ο 5 7 5

Such a composition may be in buIk form or, more preferably, unit dosage form. Thus, for example, the composition may be formulated as a tablet, capsule, powder, solution or suspension. Soft gel capsules may be especially convenient. The composition may be a liposomal formulation or administered in a slow sustained release delivery system.

Compositions in accordance with the invention may be prepared using the active compounds defined herein in accordance with conventional pharmaceutical practice. The diluents, excipients or carriers which may be used are well known in the formulation art and the form chosen for any particular regimen will depend on the given context and the physician’s choice.

Thus, for example, as illustrated below the compounds of the invention may be administered in solution in sterile deionised water. Also, if necessary, solution may be facilitated using dimethyl sulphoxide (DMSO) or alternatively an alcohol, a glycol or a vegetable oil. The compounds are most favourably administered in com oil or as a solution in DMSO/sterile water. In the case of the use according to the invention of sodium salts of the active compounds, the active ingredient is preferably administered as an aqueous solution.

In using a compound of the invention, dosage guidance can be taken from animal studies such as that described below. In such studies doses of from about 50mg/kg typically up to about lOOOmg/kg (eg up to about 400mg/kg,· conveniently about 200mg/kg or less). Thus it is to be expected that a typical dosage for humans will be from about 5mg/kg upwards (eg up to about 20mg/kg). The concentration and dose are to be sufficient lo bring an arylating mechanism into play. For intravenous administration the active compound will be dissolved or dispersed in an aqueous or other injectable liquid bearing in mind the aforementioned requirements of dosage. Typically, concentration will be 250mg/ml or less. Patient tolerance was found to be of a higher order for concentrations below that threshold although concentrations of more than 250mg/mI may be used. Typically, a preferred concentration is less than lOOmg/ml, with concentrations below 50mg/ml (eg 40mg/ml) being preferred. For >

co cn o

o ro oral administration, enteric coated tablets wall often be preferred. Maximum strength will be less than 500mg and preferably 400mg or less. Thus, for example, a range of strengths for oral administration (whether in tablet or other solid form) will be lOOmg, 200mg or 400mg. Patients who are subject to oral administration will generally fast for 8 hours prior to first dose with the fast continuing for 4 hours after first dose. Free access to fluid will generally be allowed during this fasting period. Intravenous treatment will generally take place by administration very slowly over a period, typically a period of approximately 20 minutes or more. Administration will typically be by catheter, for example a catheter left in siru after flashing with 2ml of saline. Of course, the catheter will in practice then be used for taking blood samples for pharmacokinetic measurements and removed prior to the patient’s departure from the clinic.

As shown by the results reported in Table 6 below, 2-chloro-5-nitrobenzoic acid show's considerable anti-tumour activity in vivo. This could not be supported in vitro and it appears some compounds according to the invention require activation in the patient’s liver. This and some other compounds may also be immunomodulators.

The following animal study illustrates the remarkable activity of compounds of the invention.

AP/P/ 9 5 / 0 0 7

ANIMAL STUDIES

The in vivo anti-tumour responses of various compounds according to the invention were assessed against the ascitic tumours, MAC15A murine colon adenocarcinoma and various solid tumour models. The MAC15A ascites tumour cells were transplanted into male NMR1 mice by ip innoculation at a cell density of 1 x 10^s cells in 200μ1 buffer (Table 1). The solid tumour models included the MAC13 and MAC16 murine colon adenocarcinomas, the B16 Fl murine melanoma and the M5076 reticulum cell sarcoma.

Treatment commenced 3 days after ip transplant or, in the case of solid tumours such as MAC13 and MAC16, treatment commenced when average tumour volumes reached 40mm³The animals were located in both cases into groups of 5 to 8 animals.

The animals were sacrificed after 12 days or when tumours ulcerated, tumour volume exceeded 1000mm³ or loss of body weight exceeded 50%.

Except where otherwise stated, the compounds used were dissolved in DMSO and diluted in sterile distilled water, at appropriate concentrations before administration in a solvent volume of 200μ1. Anti-tumour responses were obtained by comparing the median survival times or tumour growth inhibition against solvent controls. The results obtained are as shown in Tables 1 to 6 below.

PrcparatifiT]
Preparation of dosage solutions is exemplified as follows:-
Subjects:	No 10 animals Weight: 22g
Dosage:	50mg/kg weight per animal per day thus l,lmg per mouse per day
Total Mass Dosage:	55mg active ingredient (referred to 5 day treatment regime)
Total Formulation:	10ml solvent plus 55mg for division into 50 doses of l.lmg dissolves in 200μ1 solvent

AP/P/ 9 5 / 0 0 7 3 2

T/C% is determined as follows:5 Animal Survival Test T days

Control

C days

T/C% = IX 100 c

Example

Animal Survival Test

443 days

T/C% = 443 X 100 = 443

100

Control 100 days

Λ figure of 158 or above indicates performance justifying clinical trial.

The effect of a group of compounds on the growth rate of a number of experimental tumours has been evaluated in vivo and ihe following findings were noted :25

1. Structure-activity relationships against the MAC15A murine colon adenocarcinoma, in the female NMRI mice showed maximal activity on a split-dose schedule and when the halogen was maximally activated for nucleophilic attack.

2. Against the M5076 reticulum cell sarcoma, 2, 4-dichloro-3, 5-dinitroben7.oic acid showed activity on a split-dose schedule down to 25mg/kg body weight by both ip and sc routes. Both ±e amide and the methyl ester showed 10-fold increase in

AP/P/ 9 5 / 0 0 7 toxicity and were without anti-tumour activity. The acid also effectively inhibited growth of B16 murine melanoma and the MAC16 murine colon adenocarcinoma.

It is concluded that this group of compounds show a wide spectrum of activity against 5 murine models.

TABLE 1

Anti-tumour activity against MAC15A (murine adenocarcinoma colon). 5 animals 10 per group. Dose lOOmg kg*¹ ip per day.

Code	Compound	Schedule (days)	T/C%‘
C4	4-chloro-S, 5-dinitrobenzoic acid	1,2,3,4,5	271
Cl	2, 4-dichloro-3, 5-dinotrobenzoic acid	1,2	243
C12	2, 5-dichlorobenzoic acid	1, 2, 3, 4, 5	171
C28	2,4-dinitrobenzoic acid	1,2,3,4,5	100
C29	3, 5-dinitrobenzoic acid	1,2,3,4,5	100

a = median, T-test group, C-solvent control

TABLE 2

Anti-rumour activity against M5076-reticulum cell sarcoma 16 days after im transplant. 7 animals per group. Drugs dissolved in corn oil. Dosage is per day.

Compound	Dose	Route	Schedule		% Tumour Weight
	(mg/kg)		(days)		Inhibition
2, 4 BA	75¹	ip	1,4, 6,9		79, 88^b
	50	ip	1,4, 6,9		57
	25	’P	1, 2, 4, 6, 9		75
	75	sc	1,4.5,7,9		66
	50	sc	1,2, 4, 5,6, 7,9		76
	25	sc	1,2,4.5,6,7,9		63
2, 4 BM	1.0’	ip	1,2,3,4, 5, 6, 7,	3,9	41
	0.5	ip	1,2,3,4,5,6, 7,	3,9	39
	0.25	>P	1,2,3,4, 5,6, 7,	8,9	42

a = Maximum tolerated dose b = Two independent experiments; 4 animals had no tumour in the second experiment

2, 4 BA = 2,4-dichloro-3, 5-dinitrobenzoic acid (Cl)

2,4 BM = 2, 4-dichloro-3, 5-dinitrobenzoic acid methyl ester (C3) % Tumour Weight Inhibition:Treated Control

Agm Bgm Tumour weight % inhibition = B - A X 100

AP/P/ 9 5 / 0 0 7 3 2

Anti-tumour activity against B16F1-murine melanoma 12 days after sc transplant. 6 animals per group. Drugs dissolves in com oil. Dosage is per day.

TABLE?

AP.00575 c

Compound	Dose (mg/kg)	Route	Schedule (days)	% Tumour Weight Inhibition
2,4 BA	75’	ip	1,5	71, Sl^b
	50	ip	1,5	45, 56^b
	25	ip	1,5	13
	75	sc	1, 3, 5	30
	50	sc	1,3, 5	9
	25	sc	1, 3, 5	22
4 BA	100	’Ρ	1,5	39
	75	ip	1,5	41
	50	ip	1,5	10
4 BM	2.5*		1,3	67
	1.25	’Ρ	1,2,3	43

a - Maximum tolerated dose b · Two independent experiments

AP/P/ 95/00732

2,4 BA = 2,4>dichloro-3, 5-dinitrobenzoic acid (Cl)

4BA = 4-chloro-3,5-dinitrobenzoic acid (C4)

BM = 4-chloro-3,5-dinitrobenzoic acid methyl ester (C6)

ΤΆΒΙ.ΐ 4

Anti-tumour activity against M.AC13 murine colon adenocarcinoma 12 days after im transplant. Drugs dissolved in com oil. Dosage is per day.

Compound	Dose (me/kg)	Route	Schedule (days)	% Tumour Weight inhibition
2. 4 BA	75’	ip	1,4,5	45
2, 4 BA	50	ip	1,2,3,4..5.	6,7,8,9 39
2,4 BA	graph¹	’Ρ	graph¹	graph¹
2 BA	graph²	’Ρ	graph²	graph²

a = Maximum tolerated dose

2, 4 BA = 2, 4-dichloro-3, 5-dinitrobenzoic acid¹ (Cl)

BA = 2-chloro-S-nitrobenzoic acid (Cl7) (1: see Figure 2 of the drawings; 2: see figure 3 of the drawings) lASL£i

Anti-tumour activity against MAC 16, murine colon adenocarcinoma sc transplanted on day 11 after the beginning of treatment with 2, 4-dichloro-3, 5-dinitrobenzoic acid (2, 4 BA; Cl). Drug dissolved in com oil. The tumour volume? were at least 40mm³at the beginning of the treatment 6 animals per group. Dosage is per day.

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Compound	Dose (mg/kg)	Route	Schedule (days)	% Tumour Weight Inhibition
2, 4 BA	75’	ip	1.2,5,8	88
	50	ip	1,2, 4.5,8	91

a = Maximum tolerated dose

AP.00575

TABLE 6

Anti-tumour activity of Cl7 against B16 murine melanoma 12 days after sc transplant 5 on female C57/black mice. 6 animals per group. Dosage is per day and is ip.

Code	Compound	Dose (mg/kg)	Schedule (days)	% Tumour Weight Inhibition
10 C17 r	2-chloro-5- nitrobenzoic acid	700	1,2, 3,4, 5, 6	62

Anti-tumour activity and toxicity studies have additionally been completed for the following compounds with broadly satisfactory results:C22 2, 5-dichloro-4-mtrobenzoic acid

C23 2,4-dichloro-5-nitrobenzoic acid

C24 2,6-dichloro-4-nitrobenzoic acid

C26 2-hydroxy-5-nitrobenzoic acid

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

In addition, the following primary assay was used to investigate the anti-viral activity of compounds in accordance with the invention.

Primary Assay (i) Acute Infection Assay. High titre virus stocks of the human immunodeficiency virus HIV-1 were grown in H9 cells with RM1 1640 (Flow laboratories) supplemented with 10% fetal calf serum, penicillin (lOOIU/ml). Cell debris was removed by low speed centrifugation, and the supernatant stored at -70°C until required. In a typical assay C8166 T-lymphoblastoid CD4+ cells were incubated with 10xTCID50 HIV-1_rf at 37°C for 90 minutes and then washed three times with

AP/P/ 9 5 / 0 0 7 3 2 phosphate buffered saline (PBS). Cell aliquots (2 x 10 j were resuspended m 1.5ml growth medium in 6m! tubes, and active compounds in log dilutions [200μΜ to 0.2μΜ] were added immediately. 20mM stock solutions of each compound were made up in 70% alcohol. The compounds were stored as a powder and made up freshly in distilled water before each experiment or were stored as a 90 mM stock solution in 70% alcohol. The final concentration of alcohol in the tissue culture medium was 1%. The cells were then incubated at 37°C in 5% CO₂. At 72 hours post-infection 200μ1 of supernatant was taken from each culture and assayed for HTV (Kingchington et al, 1989. Robert et al 1990) using an antigen capture ELISA which recognizes all the core proteins equally (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 inhibitor of HIV proteinase. The IC_i0 activities of 8959, AZT and ddC in infected cells were 1, 10, 20 nM and 200nM respectively (accompanying

Figure 2). The ELISA plates were read with a spectrophotometer. Compounds were tested in duplicate at each concentration, and the data shown is the average of at least two assays. This assay assesses the activity of compounds by measuring their inhibition of HIV core antigen levels.

(ii) Chronically Infected Cell Assay. Chronically infected cells (H9rf) were washed three times to remove extracellular virus and incubated, with the active compounds (200-0.2 μΜ) for four days. HTV-1 antigen in the supernatant was then measured using an ELISA.

To test for compound toxicity, uninfected H9 cells were incubated with the compounds for four days. Supernatants were discarded and tire cells resuspended in 200μ1 pg growth medium containing ^,4C protein hydrolysate. After 6 hours the cells were harvested and the ¹⁴C incorporation measured.

(iii) Toxicity Assay. To test for compound toxicity, aliquots of 2 x 10⁵ of uninfected cells were cultured ’with the compounds in the same dilutions for 72 hours. The cells were then washed with PBS A and resuspended in 200μ1 of growth medium

AP/P/ 95/0073 2

AP.00575 containing *⁴C protein hydrolysate. .Alter 12, hours the cells were harvested and the ^!4C incorporation measured. Uninfected, untreated cells were used as controls.

Toxicity is expressed as inhibition of uptake of ^I4C protein hydrolysate.

The results of these assays are summarized in Table 7 below. The IC₅₀ is the drug concentration that causes a 50% reduction in HTV core antigen levels as detected by the Coulter P24 antigen assay and is determined by doubling dilutions of supernatant taken from tubes containing untreated acutely infected cells. The CD_J0 is the concentration of drug that causes a 50% inhibition of cells as measured by ^l4C protein hydrolysate uptake. The therapeutic index (TT) is determined by dividing the CD_f0bv the IC_S0.

c

TABLE!

Code Compound I£$_o CDso II

C4 4-chloro-3,5-dinitrobenzoic acid 30pm 70 pm 2.33

Following the same methodology, more extensive assays were performed as reported 20 in Tables 8 below.

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AP/P/ 9 5 / 0 0 7 3 2

TABLEU

STRUCTURE ACTIVITY RELATIONSHIP AGAINST HIV VIRUS

CODE	COMPOUNDS
Cl	2, 4-dichloro-3, 5-dinitrobenzoic acid
C3	2,4-dichloro-3, 5-dinitrobenzoic acid methyl ester
C4	4-chIoro-3, 5-dinitrobenzoic acid
C6	4-chIoro-3, 5-dinitrobenzoic acid methyl ester
C7	2-chloro-3, 5-dinitrobenzoic acid
CS	2-chloro-3,5-dinitrobenzoic acid methyl ester
C9	4-chIoro-3-nitrobenzoic acid
CIO	2-chloro-4-nitrobenzoic acid
Cll	3. 4-dichlorobenzoic acid
C12	2, 5-dichlorobenzoic acid
C17	2-chIoro-5-nitrobenzoic acid

AP/P/ 9 5 / 0 0 7 32

20	TABLE 8.2
		IC50	CC50	SI
		(Antiviral)	(Toxicity)	(Selectivity Index)
	Against. HTV-IΠΙΒ			-
25	CI	5	70	14
		36	70	2
		33	70	2
		35	60	2
	Average	27	70	3
30	Cl	10	30	3
		2.5	20	8

AP.00575

Against T-TTV-IRf

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
Agaip^±[I-V-l-mB
C2		2	70	35
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
			>200	>10
	Average	22	>175	8
G7		>1	35	>35
		10	30	• J
C8		10	60	5
C9		>200	>200	-
CIO		>200	>200	-
Cll		>200	>200	-
C12		>200	>200	-
C17		>1	>1000	>1000
		5	>1000	>200

AP/P/ 9 5 / 0 0 7 3 2

Patient Tests

A pilot study was conducted for safety, pharmacokinetics and preliminary activity of Cl7 in patients with HIV-1 infection and HIV-related Kaposi’s sarcoma. In the pilot study, 10 patients were enrolled but only 6 were eligible for evaluation. These patients were positive for serum antibody to HIV-1 as determined by both enzymelinked immunosorbent assay (ELISA) and Western Blot. These patients had WHO Clinical Stage 2 to 3 for HTV infection and disease and fulfilled the inclusion and exclusion criteria as per the Clinical Trial Protocol. Their CD4 ranged from 0.072 to 0.812 χ 10³ cells/mm³ (normal range 1.0 x 1.3 x 10³ cells/mm³). Patient 6 had AIDSrelated Kaposi’s sarcoma confirmed on histology.

C17 was prepared for intravenous administration under GMP (good manufacturing practice) in strength of 40 mg/ml following the method set forth below' for producing a 250mg/ml solution, additional water being added at Step 7:Method of Preparation

1. To 275ml water for injection add 266.4ml 4N sodium hydroxide solution and mix.

2. Add with stirring 182.5g of active compound.

: Add additional 4N sodium hydroxide if needed to obtain solution.

4. Filter solution through a Sterivex GV 0.22pm filter.

5. Dilute to approximately 680ml with water for injection.

6. Adjust to pH 7.2-7.6 with hydrochloric acid.

7. Make-up to 730ml water for injection. Adjust pH if necessary.

8. Transfer solution to aseptic room and filter through Sterivex GV 0.22pm filter into injection rials. Seal with rubber stoppers and aluminium closures.

9. Effect terminal sterilization by autoclave.

AP/P/ 9 5 / 0 0 7 3 2

AP. Ο Ο 5 7 5

The patients received 10mg/kg body weight/day of Cl7 by deep intramuscular injection daily for 5 days weekly. The CD4 counts were estimated before and at about weeks after the beginning of the therapy.

The CD4 counts in all 6 patients showed an increase which was associated with clinical improvements in patient general condition including weight gain and a marked decrease in opportunistic infections and diaorrhea. Patient observations are shown in Tables 9 below.

In the case of Patient 6, the sarcoma lesions disappeared.

Murine Haematology

Preliminary data on the administration of Cl 7 in mice and rats showed increases in 15 red blood cell count, haemoglobin concentration, macrophage count, white blood cell count (WBC), lymphocyte count and haematocrit (HTC) on day 7 when maximum tolerated dose (MTD) was given by 5 consecutive daily intravenous injections. These values returned to normal on day 14. The MTD values were 950mg and 900mg/kg body weight/day respectively in mouse and rat The data are reported in Figures 4 to

11.

AP/P/ 9 5 / 0 0 7 3 2

PATIENT 1 IMMUNOLOGY:lABLEJU.

AP.OOJ75

DAY 1 20

	Lymphocytes x 10³mm³	2.1	2.0
	CD4 %	19	38
10	CD4 ABS/mm³	0.399	0.760
	CD8 ABS/mm*'	0.567	0.840
15	CD4/CD8	0.70	0.90
	PATIENT 2 IMMUNOLOGY:-	TABLE 92
20	DAY	1	14
	Lymphocytes x 10 mm’¹	2.0	1.9’
25	CD4 %	18	27
	CD4 ABS/mm³	0.360	0.513
	CD8 ABS/mm³	0.940	0.874
30	CD4/CDS	0.38	0.59

AP/P/ 9 5 / 0 0 7 3 2

TABLE 9.3

PATIENT 3 IMMUNOLOGY:-

DAY	1	20
Lymphocytes x 10³mm'³	0.9	1.1
CD4 %	S	17
CD4 ABS/mm³	0.072	0.187
CDS .ABS/mm³	0.270	0.682
CD4/CD8	0.27	0.27
PATIENT 4 IMMUNOLOGY:-	TABLE 9..4
DAY	1	14
Lymphocytes x 10³mm'³	1.8	2.0
CD4 %	6	22
CD4 ABS/mm³	0.108	0.440
CD8 ABS/mm³	1.350	1.260
CD4/CD8	o.os	0.35

?/P/ 9 5 / 0 0 7 3 2

AP . 0 0 5 >5

TABLE 9.5

PATIENT 5 IMMUNOLOGY:-

5	DAY	1	21
	Lymphocytes x 10³mm’³	2.S	3.1
	CD4 %	29	33
10	CD4 ABS/mm³	0.812	1.023
	CD 8 ABS/mm^J	1.456	1.550
15	CD4/CDS	0.56	0.66
	PATIENT 6 IMMUNOLOGY:-	IABLEJLS
20	DAY	1	34
	Lymphocytes x 10³mm'³	2.1	1.9
25	CD4 %	18	23
	CD4 ABS/mm³	0.378	0.437
	CD8 ABS/mm³	1.218	0.931
30	CD4/CD8	0.31	0.47

AP/P/ 9 5 / 0 0 7 3 2

Claims

1. A compound of the following general formula for use in the treatment of prophylaxis or therapy of neoplasm or viral infection: - f wherein X; and X₂ are, each independently, hydrogen or chloro and wherein X₃ and

X₄ are, each independently, hydrogen or nitro, provided that at least two of X_b X₂, X₃and Χ^ are other than hydrogen, or a salt or ester thereof.

15 2. A compound as claimed in Claim 1 wherein X, is chloro, X₂ is chloro or hydrogen, X₃ is nitro and X4 is nitro or hydrogen.

2-chloro-5-rutrobenzoic acid and various other chloro and nitro substitute benzoic acid

2-chloro-, (ii) when X₃ and X4 together represent 5-nitro-, X[ and X₂ together represent 2chloro- or 2,4-dichloro-, (iii) when X₃ and X, together represent two hydrogen atoms, X, and X₂ together

30 represent

3,

3. A compound as claimed in Claim 2 wherein X_t and X₂ arc other than 4-chloro.

20 4. A compound as claimed in Claim 1 or Claim 2 wherein Xi and X₂ together represent 2-chloro-, 4-chloro-, 2,5-dichloro-, 2, 4-dichloro-, 2, 6-dichloro-, 3, 5dichloro- or 3, 4-dichloro- and wherein X₃ and X₄ together represent two hydrogen atoms, 3-nitro-, 4-nitro-, 5-nitro-, 2,4-dinitro- or 3, 5-dinitro-, provided that:25 (i) when X_? and X, together represent 3, 5-dinitro-, Xj and X₂ together represent

4-dichloro-, (iv) when X₃ and X₄ together represent 4-nitro-, X_t and X₂ together represent 2, 5dichloro-, 3, 5-dichloro-2-chloro- or 2, 6-dichloro-,

AP/P/ 9 5 / 0 0 7 3 ?.

AP.00575 (v) when X-. and X4 together represent 3-nitro-, Xj and X; ’together represent 4chloro-.

5. A compound as claimed in Claim 4 wherein X_L X₂, X₃ and X4 are as there 5 defined subject to provisos (I), (ii), (iv) and (v) and subject to the further proviso that when X₃ and X₄ together represent 5-nitro-, X, and X₂ together represent 2, 4dichloro-.

6. 2-chloro-5-nitro-benzoic acid.

7. A compound as claimed in Claim 1 and as set forth by name below or an ester or salt thereof:7.1 2,4-dichloro-3. 5-dinitrobenzoic acid

15 7.2 4-chloro-3. 5-dinitrobenzoic acid

7.3 2-chloro-3, 5-dinitrobenzoic acid

7.4 2, 5-dichlorobenzoic acid

7.5 2.4-dinitrobenzoic acid

7.6 3,5-dinitrobenzoic acid

20 7.7 2,5-dichloro-4-nitrobcnzoic acid

7.8 2,4-dichIoro-5-nitrobenzoic acid

7.9 2,6-dichloro-4-nitrobenzoic acid

7.10 3, 5-dichloro-4-nitrobenzoic acid

7.10 4-chloro-3-nitrobenzoic acid

25 7.12 2-chloro-4-nitrobenzoic acid

7.13 3,4-dichlorobenzoic acid

8. A pharmaceutical composition for use in the treatment by prophylaxis or therapy of neoplasm or viral infection, the composition comprising a compound as

30 claimed in any preceding claim together with a pharm3ceutically-acceptable carrier or diluent

AP/P/ 9 5 / 0 0 7 3 2

9.

A composition as claimed in Claim 8 wherein the carrier or diluent is aqueous.

10. A composition as claimed in Claim 8 wherein the carrier is a solid excipient.

11. A composition as claimed in Claim 8 which is in tab^ capsule, powder, solution or suspension form.

12. A composition as claimed in any one of Claims 8 to 11 which is in unit dosage form.

13. A composition as claimed in any one of Claims 8 to 12 and comprising 2chioro-5-nttrobenzoic acid, or a salt or ester thereof, encapsulated by a soft gel capsule or contained by a sustained release drug delivery system.

14. A method of preparing a pharmaceutical composition as claimed in any one of Claims 8 to 13 which method comprises combining a compound as claimed in any one of Claims 1 to 7 with aqueous sodium hydroxide solution to form a solution, adjusting the pH of the solution to an alkaline pH of not more than 8 by addition to the solution of an acidic material, optionally diluting the pH-adjusted solution with physiologically injectable water and sterilizing the solution.

15. A method of treatment by prophylaxis or therapy of neoplasm or viral injection in mammalian subjects, which method comprises· administering to a mammalian subject an effective dose or dosage regime of a compound as claimed in any one of Claims 1 to 7.

16. Use of a compound as claimed in any one of Claims 1 to 7 for the preparation of a medicament for the treatment by prophylaxis or therapy of neoplasm or viral infection in mammalian subjects.

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AP. Ο Ο 3 / 5

17. Use of a compound as claimed in any one of Claims 1 to 7 for the treatment of Kaposi’s sarcoma.

18. Use of a compound as claimed in any one of Claims I to 7 for the preparation of a medicament for the treatment of Kaposi’s sarcoma.

19. A method of treatment of mammals for the prophylaxis of Kaposi’s sarcoma 10 which method comprises administering to the patient an effective dose of a compound as claimed in any one of Claims 1 lo 7.

20. A pharmaceutical composition in the form of enteric coated tablets comprising a compound as claimed in any one of Claims 1 to 7 in strength 100 to 400mg, the

15 compound being formulated as a mixture comprising the compound together with an inert pharmaceutically-acceptable solid tabletting carrier.

21. A pharmaceutical composition in the form of an injectable solution or dispersion of a compound as claimed in any one of Claims 1 to 7 in an aqueous or

20 other injectable liquid carrier in a concentration of less than 250mg/ml.

DATED THIS 17th DAY OF March 1995

Patent Agents for the Applicants

AP/P/ 9 5 / 0 0 7 3 2

5 derivatives are disclosed for administration in tablet or injectable solution form for the prophylaxis and therapy of neoplasm or viral infection. The active compounds have surprising efficacy in various treatment regimes and have particular application to

HTV-infection and demonstrate various beneficial effects, including regression of lesions in HIV-related Kaposi’s sarcoma.