This invention relates to immunosuppressive agents .

The purine derivative azathioprine interferes with cell replication, and has ^' immunosuppressive and antileukemic properties. It is used extensively as a drug to treat a wide range of chronic inflammatory diseases in which immune mechanisms are involved such as rheumatoid arthritis ; chronic active hepatitis ; kidney disease; skin disease; and multiple sclerosis. It is also used to inhibit the rejection of transplanted organs such as kidneys. However, its beneficial therapeutic effects are compromised by the fact that it is toxic to bone marrow. It is the object of the present invention , to provide an immunosuppressive agent which does not have this disadvantage.

According to one aspect of the invention there is provided a compound with immunosuppressive properties , said compound having the formula:

V

in which

R comprises a heterocyclic ring system, a homocyclic ring system, a combined heterocyclic and homocyclic ring system, alkoxycarbonylalkyl or aryloxycarbonylalkyl group

2

R is alkyl, aryl or hydrogen and

R is hydrogen, alkyl, aryl or nitroaryl

Preferably

2

R is hydrogen, methyl, ethyl or propyl; and

R is hydrogen, methyl or 4-nitrophenyl.

According to a preferred embodiment of the invention said compounds with immunosuppressive properties have the following formula:

in which

2

R is hydrogen, or methyl or ethyl or propyl; and

3 R is hydrogen or methyl or 4-nitrophenyl,

1 and in which R may be one of the following:

1.

in which

4 R is hydrogen or methyl or ethyl or propyl or phenyl or 1-naphthyl

in which

5 R is hydrogen or trifluoromethyl or phenyl or

4-chlorophenyl or 4-methoxypheny1 or ^' 3-pyridyl

H ^y' X.

in which

R is hydrogen or phenyl or chlorophenyl or

4-methoxypheny1

in which

R is hydrogen or an alkoxy group; and

R is hydrogen, alkyl, nitro, halide or an alkoxy group; and o

R is hydrogen, halide or an alkoxy group; and

9 R is hydrogen or an alkoxy group

- A - in which

R is hydrogen, methyl, or hydroxyl; and R 12 is hydrogen or methyl; and

X is nitrogen or C-H

6.

in which

2 is oxygen or sulphur or N-H; and

Y is C-H or nitrogen

in which R 13 is hydrogen or an amino or carboxyl group; and R 14 is hydrogen or methyl; and R 15 is hydrogen or methyl

8.

in which R 16 is alkyl or aryl

9. _s

in which R 17 is alkyl or aryl

11.

12 .

in which R 18 is alkyl or aryl

Specific illustrative examples of the first embodiment of the invention are as follows:

(3)

(1) (2)

(5) (6)

(4)

(9)

(7) (S)

(14)

(12) (13)

(15) (16) (17)

(20)

(18) (13)

23) (24)

(21) (22) (

Some of the compounds, as referred to above are previously unknown.

Therefore, according to a further aspect of the invention there are provided compounds comprising the following formula:

wherein the molecular groups R 1 , R2, and R3 are defined as one of the following:

1. R is defined as the following:

in which

4 R is hydrogen or methyl;

₂ R is ethyl; and

3 R is methyl

OR

R 2 is methyl and R3 is hydrogen; and

4 R is phenyl or 1-naphthyl

OR

2. R is defined as the following:

CHa

in which R 4 is methyl; and R 2,R3, and R5 are one of the following combinations:

R 2 is methyl, R3 is 4-nitrophenyl, and R5 is hydrogen; or

2 3 5

R is methyl, R is hydrogen, and R is trifluoromethyl; or

2 3 5

R is ethyl, R is methyl, and R is hydrogen; or

R 2 is methyl, R3 is hydrogen, and R5 is phenyl; or

2 3 5

R is methyl, R is hydrogen, and R is 4-chlorophenyl; or

R 2 is methyl, R3 is hydrogen, and R5 i ^• s 4-methoxypheny1; or

2 3 - 5

R is methyl, R is hydrogen, and R is 3-pyridyl

OR

3. R is defined as the following:

H N-N

R • ¹ A - in which

2

R is methyl; and

3 R is hydrogen; and

R is hydrogen or phenyl or 4-chlorophenyl or

4-methoxyphen 1

OR

4. R is defined as the following:

in which

2 R is methyl; and

3 R is hydrogen, and

R 7, R8, R9, R10 are one of the following combinations:

R 8, R9, R10 are all hydrogen and R7 is methyl; or

R 8, R9, R10 are all hydrogen and R7 is a nitro group; or

R 9 and R10 are both hydrogen, R7 is fluorine, and R8 is chlorine; or

R 9 and R10 are both hydrogen, and R7 and R8 are both methoxy groups; or

R and R are both hydrogen and R and R are both methoxy groups

OR

5. R is defined as the following:

in which

2

R is methyl; and

3 R is hydrogen; and R 11,R12 and X are defined as one of the following combinations: R 11 and R12 are both hydrogen, and X is C-H; or

R 11 is a hydroxy group, R12 is hydrogen, and X is nitrogen

OR

2 R is ethyl; and

R , R and R are all methyl; and

X is nitrogen OR

6. R is defined as the following:

in which 03-

2 R is methyl; and

R is hydrogen; and

Y and Z are defined as one of the following combinations :

Y is C-H and X is oxygen; or

Y is sulphur and Z is N-H; or

Y is nitrogen and Z is N-H OR

7. R is defined as the following:

in which

2

R is methyl; and

3 R is hydrogen; and

R 'R , and R are defined in either of the following combinations:

R is hydrogen, and R and R are both methyl; or is carboxyl, and R and R are both hydrogen

OR

8. R is defined as the following:

R is hydrogen; and

16 R is methyl or phenyl

OR

9. R is defined as the following:

OR

10. R is defined as the following:

11. R is defined as the following;

Referring now to Reaction Scheme 1, azathioprine I was originally designed as a "pro-drug" for 6-mercaptopuxine II, to which it is rapidly converted by red blood cells. 6-mercaptopurine II has immunosuppressive properties, but the mechanism of its action is unclear. REACTION SCHEME 1

cell membran

N

IMMUNOSUPPREΞΞION AND TOXZCITY

- 15 -

It would appear that the immunosuppressive action of azathioprine I is due not solely to prior conversion of 6-mercaptopurine II in the body, but to the immunosuppressive action of azathioprine I itself. The mechanism of action of azathioprine I differs from that of 6-mercaptopurine II. There is strong evidence to suggest that 6-mercaptopurine II is converted in vivo into a nucleotide metabolite that is associated with bone marrow toxicity. There is no evidence to suggest that metabolites derived from the l-methyl-4-nitroimid- azole moiety of azathioprine give rise to toxicity.

It is thought that azathioprine I alkylates thiol groups in the lymphocyte cell membrane, most probably by a process of addition-elimination with the consequent release of β-mercaptopurine II. It is further supposed that the alkylation of the lymphocyte results in immunosuppression, whilst the 6-mercaptopur- ine II gives rise, as stated above, via its nucleotide metabolite, to bone marrow toxicity. Support for this hypothesis is provided by the fact that lymphocyte cell membranes are richly endowed with thiol groups, and by the known propensity of azathioprine to react with thiols both in vitro and in vivo.

5-(l-alkyl-4-nitroimidazole) derivatives VI of relatively non toxic alkyl thiols or aryl thiols V are therefore suitable candidates for evaluation as good.

relatively non toxic immunosuppressive agents , as ⁱ s shown in Reaction Scheme 2 . REACTION SCHEME 2

Pv = alkyl, aryl, _ or heterocyclic

R = Me or Et R ³ = H or Me

Examples of immunosuppressive agents of this kind are shown previously in examples 1-43. It is not intended that the invention be limited. to these illustrative examples.

Compounds 1 to 17, 19 to 22, 25 to 32, 34 and 36 to 43 may be synthesised, for example, by reaction of 5-chloro-l- methyl-4-nitroimidazole IV with the appropriate thiol V and potassium carbonate in a suitable solvent such as, for example, tetrahydrof ra , dimeth lformamide, or water. Compounds 18, 23, 24 and 35 may be synthesised, for example from 5-chloro-l-ethyl-2-methyl-4-nitroimidazole Vll and the appropriate thiol under suitable conditions, such as, for example, in acetone in the presence of potassium carbonate.

Sixteen of these compounds were screened for immunosuppressive activity by means of the human mixed lymphocyte reaction, which is well known and need not be further described here, the results of which are shown in Table 1.

TABLE 1 Com _p ound Concentration % Inhibition of Solution (μM) of ³ M-Thymidine

79

I (azathioprine) 25

10 89

21 82 22 10 32 25 98

36 98

1

25 86 2

50 59

4 25 94

5 25 41

6 76

15 25 47

16 25 29 19 ²⁵

25 47

13

8 25 87

9 25 27 34 13 25 65 30 ²⁵

The individual figures in the right hand column of the above table are directly proportioned to immunosuppressive activity.

As can be seen from Table 1 all of the compounds screened for immunosuppressive activity displayed significant immunosuppression, and six were substantially more active than azathioprine at the same concentration (25μM) .

Table 2 shows the measure of the immunosuppressive activity and toxicity of compounds 21,22,32, and 11.

TABLE 2

ED 50 = concentration (μM) which brings about 50% inhibition of ³ H-thymidine incorporation in the human mixed lymphocyte reaction: lower figures indicate increased reactivity

b ^LD 25 ⁼ concentration (μM) at which 25% of the cells are killed: lower figures indicate increased toxicity c No LD_ ₅ because only 19% of cells killed at 100 μM d No LE>25 because only 15% of cells killed at 215 μM e No D_ ₅ because only 16% of cells killed at 25 μM (For c, d and e these are maximum values: no more cells were killed at higher concentrations) vh very high Referring now to Table 2 , imidazole derivatives 21 and 22 and the triazole derivative 32 are shown to be substantially more immunosuppressive than azathioprine and also considerably less toxic to lymphocytes. Therapeutic indices for these compounds are therefore at least an order of magnitude greater than for azathioprine.

The compound 11 in which the 6-mercaptopurine moiety of azathioprine is replaced by 8-hydroxy-6-merc- aptopurine moiety, is less immunosuppressive than azathioprine according to the mixed human lymphocyte reaction, as is shown in Table 2, but is much less toxic to the lymphocytes . The therapeutic index of compound 11 is therefore much greater than that of azathioprine, according to these tests. The significance of this result resides in the fact that

8-hydroxy-6-mercaptopurine VIII is known to be a non

toxic metabolite of azathioprine, and its metabolic fate is known.

Compound 32 showed pronounced immunosuppressive activity in vivo. It was tested in CBA mice transplanted with skin from Balb/c mice, according to the skin grafting technique described by Billingham and Medawar (J.Exp. Biol, 28, 385-405 (1951). The effect compound 32 had on skin graft survival was compared with a control of saline, and with the standard immunosuppressive agent azathioprine. All drugs were injected into the peritoneum starting three hours before surgery and then daily until the graft had fully rejected. Mice were placed in groups at random, and rejection of the graft was evaluated by visual inspection by an independent observer. The results are recorded in Table 3.

TABLE 3

Dosage* Graft survival Treatment Number of mice (mg/ g/day) (Mean±SEM days)

Control 28 NA 12.5 + 0.4

Compound 32 24 45 14.2 ± 0.4 athloprine 29 52 12.4 ± 0.3

*Compound 32 and azathioprine were adminstered in equimolar amounts

The prolongation of graft survival caused by compound

32 (1.7 days) compared to a saline control is significant

_3 statistically (p=5.9 x 10 ) . Similarly, the prolongation

of graft survival caused by compound 32 (1.8 days) compared to azathioprine is also highly significant (p=2.84 x 10~ ³ ) .

It is to be understood that the above described examples are for illustration only and that many modifications and variations can be made within the scope of the invention.