WO1996027370A1 - Free radical scavenger molecules - Google Patents

Abstract

Use of a compound comprising one or more α-amino acid residues of general formula (I), wherein R is selected from the group consisting of hydrogen, optionally substituted C1-C20 alkyl, C1-C20 alkenyl, aryl and aralkyl groups and a group R5-OCO- where R5 is selected from C¿1?-C20, C1-C20 alkenyl, aryl and aralkyl groups; R?1 and R2¿ are independently selected from the group consisting of hydrogen and optionally substituted C¿1?-C6 alkyl, C1-C6 alkenyl, C7-C12 arylalkyl and aryl groups; or R?1 and R2¿ together represent a group =CR6R7, wherein R?6 and R7¿ are independently selected from hydrogen, halogen, and optionally substituted C¿1?-C6 alkyl and aryl groups; wherein the compound does not contain free thiol groups; or a pharmaceutically acceptable salt thereof in the preparation of a medicament suitable for the treatment of a disease state caused by production of free radical species or in which free radical species are produced is provided, together with formulations and methods for their preparation.

Description

FREE RADICAL SCAVENGER MOLECULES

The present invention relates to organic compounds which have been found to have free radical scavenging activity in biological systems. More specifically, the invention relates to free radical scavenging compounds, methods for their preparation, pharmaceutical formulations containing them and their use as free radical scavengers, in particular for scavenging radicals implicated as factors contributing to disease states of mammals.

Free radicals, such as reactive oxygen species (ROS) are thought to play a part in the occurrence and/or maintenance of several disease states such as cancerous, cardiovascular and inflammatory diseases and the like. Examples of disease states where at least ROS are present include atherosclerosis, ischemia/reperfusion myocardial injury and arthritis (see eg. Halliweli and Gutteridge (1989) Free Radicals in Biology and Medicine, Oxford, Clarendon Press, 86: 366 and Methods in Enzymology (1990), Vol 186, Part B, Eds L Pacher and AN Glazer, Academic Press, New York). Known free radical scavenging molecules include compounds such as Captopril, however, such molecules generally contain reactive thiol groups which may be toxic to mammalian cells or lead to the evolution of toxic compounds. There exists a need for the development of new free radical scavenging drugs which do not possess thiol groups and which are effective in combatting disease states in mammals where free radical molecules are implicated in the onset, maintenance and/or development of the disease state.

It is an object of the present invention to provide such new free radical scavenger compounds for use in methods of treating diseases wherein free radical molecules are implicated in the disease process in order to reduce levels of free radicals.

It is another object of the invention to provide free radical scavenging compounds for use as pharmaceuticals.

It is a further object to provide free radical scavenging compounds which do not possess thiol groups.

These and other objects of the invention will become apparent from the following description and examples. According to a first aspect of the present invention there is provided use of a thiol group free compound comprising one or more o-amino acid residues of general formula

(I):

R¹ R²

\ / C (I)

/ \ - NR CO - wherein R is selected from the group consisting of hydrogen, optionally substituted C_j-

C20 alkyl, C1-C20 alkenyl, aryl and aralkyl groups and a group R^-OCO- where R^ is selected from Ci -C20 alkyl. C1-C20 alkenyl, aryl and aralkyl groups;

R! and R² are independently selected from the group consisting of hydrogen and optionally substituted Cj-Cg alkyl, Cj-Cg alkenyl, C7-C12 arylalkyl and aryl groups; or R' and R² together represent a group =CR"R ', wherein R° and R ' are independently selected from hydrogen, halogen, and optionally substituted C_j-Cg alkyl and aryl groups; or a pharmaceutically acceptable salt or physiologically functional derivative thereof in the preparation of a medicament suitable for the treatment of a disease state caused by production of free radical species or in which free radical species are produced.

Preferably at least one of R^ and R² is a non-thiol group -(CH2)_n-L, where n is an integer from 1 to 3 and L is a leaving group or a group -O-I-A -S-L*. -CO-l or -CS-L^ where L^ is a leaving group wherein L and L* are selected from those groups, which leave under physiological conditions on interaction of the compound with a free radical.

In each case the optional substitution is preferably independently with CI, Br, F, I, OH, -S-alkyl where alkyl is Ci -Cg alkyl, -S-alkenyl where alkenyl is C2-Cg, - SC(O)C₆H₅; -SC(O)C₆H₅-CH₃, -O-SO₂C₆H₅, -O-SO₂C₆H₅-CH₃, -COOCH₂C₆H₅ or -COOCH₂CH₂C₆H₅.

The o-amino acid residues are preferably in their D-isomeric form, particularly when the compound comprises two or more such residues. A preferred use of the first aspect of the present invention provides use of a thiol group free compound of general formula (Ia)

Rl R² \ / C (I)

/ \

Z - (A ! )_O - NR CO - (A²)_p-Y

in the preparation of a medicament suitable for the treatment of a disease state caused by production of free radical species or in which free radical species are produced, wherein o and p are each 0 or an integer, and the sum of o + p + 1 is no more than 500; A^ is selected from the groups

Rl R² Rl R² Rl R² Rl R²

\ / \ / \ / \ / C ; C ; C ; C ;

/ \ / \ / \ / \ - NR CO- -NR CH₂- -CH₂ CO- -CH₂ CH₂-

Rl R² Rl R² Rl R² Rl R²

\ / \ / \ / \ / C ; C ; C and C

/ \ / \ / \ / \ CO- CH₂- -CH₂ -NR is selected from the groups

Rl R² Rl R² Rl R² Rl R²

\ / \ / \ / \ / C ; C ; C ; C ;

/ \ / \ / \ / \ ■ NR CO- -NR CH₂- CH₂ CO- -CH₂ CH₂-

Rl R² Rl R² Rl R² Rl R²

\ / \ / \ / \ / C C C and C

/ \ / \ / \ / \ -NR -CH₂ CH„-

— 2 RN-

Y is selected from hydroxy or optionally substituted, C _j-Cg alkyl, C_j-Cg alkenyl, Ci -Cg alkoxy, C_j-Cg alkenylyoxy, arylalkoxy (eg. benzyl), phenyl, phenyloxy, amino or alkylamino groups; alkylamino being particularly C _j -Cg alkylamino;

Z and R are independently selected from the group consisting of hydrogen, optionally substituted C1-C20 alkyl, C2-C20 alkenyl, aryl and arylalkyl groups, and a group R^-OCO- wherein R-> is selected from C_j-C20 alkyl, C2-C20 alkenyl, aryl and arylalkyl; or is XΗ2⁺ where X is halogen; or Z and Y together represent a single covalent bond; and

Rl and R², for each repeat unit of the o or p units, are independently selected from the group consisting of hydrogen and optionally substituted C_j-Cg alkyl, C_]-Cg alkenyl, C7-C 12 arylalkyl and aryl groups; or Rl and R² together represent a group

wherein R" and R ' are independently selected from hydrogen, halogen, and optionally substituted C _j -Cg alkyl and aryl groups;

or a pharmaceutically acceptable salt thereof.

Preferably at least one of Rland R² is a non-thiol group -(CH2)_n-L, where n is an integer from 1 to 3 and L is a leaving group or a group -0-L1 , -S-Ll, -CO-I-1 or -CS-L1 where I_l is a leaving group wherein L and Li are selected from those groups, which leave under physiological conditions on interaction of the compound with a free radical.

Again, in each case the optional substitution is preferably independently with CI, Br, F, I, OH, -S-alkyl where alkyl is Cj-Cg alkyl, -S-alkenyl where alkenyl is C2~Cg, - SC(O)CgH₅; -SC(O)CgH₅-CH₃, -O-SO₂CgH₅, -O-SO₂CgH₅-CH₃, -COOCH₂CgH₅ or -COOCH₂CH₂CgH5.

Preferably Z and R are independently selected from hydrogen, C_j-Cg alkyl, C2~Cg alkenyl, Cg aryl and C7-C12 arylalkyl. Conveniently Z and R are hydrogen.

A still, more preferred use of the present invention uses compounds of formula (lb):

in the preparation of a medicament suitable for the treatment of disease states caused by free radical species or in which free radical species are produced:

wherein:

m is an integer from 1 to 500;

Y, Z and R, R and R² are as defined above in connection with formula (Ia);

Preferably the compound of formula (I), (Ia) or (lb) is a free or protected o-amino acid, a peptide or a peptide analogue. Peptide analogues are covered by the broadest aspect of the invention and include those compounds corresponding to peptides with the exception that one or more peptide bonds have been replaced with, e.g. aliphatic carbon to carbon or carbon to nitrogen covalent bonds of non-amide character. Still more preferred are peptides consisting of 2 to 100 amino acid or analogue residues, more preferably 2 to 10 amino acid residues and most preferably 2 or 3 amino acid residues; that is where the sum of (o + p + 1) or m is an integer from 2 to 100, 2 to 10 and 2 to 3 respectively. Most preferred compounds include 2 or 3 amino acid or amino acid analogue residues.

Protected amino acids are those wherein one or both of the free amino or carboxyl terminal group are incorporated into a bond, eg. an amide or carboxylic acid ester bond, which provides a degree of resistance to metabolism or catabolism (eg. by polymerisation into peptides and proteins). It is also possible to incorporate one or more D-amino acids into the compound which is particularly advantageous in so far as it will result in resistance to enzymic degradation of any peptide bonds; such D-amino acid containing peptides and their analogues being preferred compounds for the use of the invention. Suitable protecing groups are those known to and employed by those skilled in the art of peptide synthesis and their hydrolysis products should preferably be physiologically acceptable in the amounts produced by the metabolism of therapeutic amounts of compound in vivo. The most preferred compounds for use in the present invention are cyclic peptides. particularly those comprising two amino acids joined to each other at both their carboxy and amino groups by way of peptide bonds ie. ketopiperazines.

For all subclasses of the compounds used for the present invention it is preferable that at least one of Rl and R² is a group -(CH2)_n-L, where n is an integer from 1 to 3 and L is a leaving group or a group -0-L 1 , -S-L\ -CO-LI or -CS-L,! where Li is a leaving group wherein L and Li are selected from those groups, which leave the compound under physiological conditions, i.e. in solution in a mammalian body, on interaction of the compound of formula (I) with a free radical species, eg. such as ROS. Most preferably L is a halogen atom, particularly chlorine or bromine, a hydroxy group or Li is an arylsulphonylalkyl group, e.g. tosyl or -COCgH5, an arylalkyl, eg.OCH2CgH5 or - OCH2CH2CgH5- It will be realised that where L is -S-L1 it is not -S-H and preferred compounds do not contain methionine residues.

Still more preferably Rl and R² together represent =CR"R', and conveniently =CH2 whereby there is provided an amino acid residue referred to herein as Δ-alanine or an analogue thereof. Preferred naturally occurring amino acid residues for incorporation into the compound of formula (I) include serine, threonine, tyrosine, phenylalanine, glycine and alanine, while 2-phenylglycine is also a preferred o-amino acid.

Preferred dipeptide compounds of formula (I) for the use of the invention are those of formula (II)

Rl R² \ /

C NH CO (II)

/ \ / \ / \

ZNH CO C Y

/ \ _{R3 R}4 wherein

Rl and R^ are independently selected from H, CgH5, C_j.g alkoxy-substituted phenyl, e.g. β-CH OCgH4 and optionally substituted C_j-Cg alkyl or C7.12 arylalkyl; wherein optional substituents are independently selected from CI, Br, F, I, OH, -S-alkyl where alkyl is Ci -Cg alkyl; -S-alkenyl where alkenyl is C2-Cg alkenyl, -SC(O)C₆H5, -OSO2-P- C₆H₅CH₃, -COOCH₂C₆H₅ , -COO(CH₂)₆C₆H₅ and -COOCH₂CH₂C₆H₅;

R² and R⁴ are independently selected from H and optionally substituted C_j-Cg alkyl wherein optional substituents are independently selected from CI, Br, F, I, OH, -SC(O)C₆H₅, -OSO₂p CgH₅-CH₃, -COOCH₂CgH₅, -COO(CH₂)gC₆H₅ and -COOCH₂CH2CgH₅; or

Rl and R² together and/or R^ and R⁴ respectively together represent =CH2;

Y represents an amino group or a group OR° where R° is independently selected from H or C_j-Cg alkyl;

Z represents R^OCO wherein R^ is independently selected from C1-C20 alkyl (preferably C1-C5 alkyl), phenylalkylcarbonyloxy, e.g. CgH5CH OCO, C9H19OCO; or a hydro-halo salt such as Cl"H->⁺; or Y and Z together represent a single covalent bond.

It is to be noted that where C H5 occurs in the text that it represents phenyl.

Preferably Rl and R² are not simultaneously optionally substituted alkyl or alkenyl and R^ and R^ are not simultaneously optionally substituted alkyl or alkenyl.

More preferably Rl is independently selected from H and CH3; still more preferably H, R² is independently selected from H, -CH₂OH, -CH₂C1 or -CH₂OSO₂-p-CgH5-CH3; R^ is independently selected from H or CH3; still more preferably H, R⁴ is independently selected from H, -CH₂OH, -CH₂C1, -CH2θSO₂-p-C H₅-CH3, and CH₃; or

R and R² together and/or R^ and R⁴ together respectively represent =CH2; and Y represents the group OR° and is independently selected from -OCH₃, OH and -OCH₂CH₃. Conveniently prepared compounds (represented by formula (I)) determined by the present inventors to be capable of being used in the preparation of a medicament of the present invention include members of the following groups: diprotected dipeptides, N-protected dipeptides, C-protected dipeptides. unprotected dipeptides and 2,5-diketopiperazine derivatives (i.e. cyclic peptides). Such groups are encompassed by general formula (I) and are further exemplified under the sub-generic formulae (Ha) to (VI) below and are preferred for reason that they are simplest to prepare whilst benefiting from excellent free radical scavenging properties. It will be realised that peptides incorporating greater numbers of amino acid residues or amino acid residue analogues will be suitable for use in the present invention but may involve increased numbers of synthesis steps in their provision and thus greater expense.

It will be realised by those skilled in the art that many amino acids and peptides have been used previously as medicament active ingredients for other purposes than scavenging of free radical species. However, many of peptides falling into the scope of the present formulae have not been so used , and thus a second aspect of the present invention provides such peptides for use as a medicament. Such compounds are those described as preferred herein for this use. Particularly compounds for use in the preparation of a medicament represented by formula (I) are compounds v) and B) as herein described.

Preferred dipeptide compounds:

(1) Diprotected dipeptides of Formulae Qla^ and (lib.

wherein R is as defined for formula (I) and particularly as for formula (II), preferably Rl being independently selected from H, C H5~, -pCgH4OCH₃, C_]-Cg alkyl, -CH₂SCOC₆H₅, -CH₂C1, and -CH₂OSO₂-p-C₆H₅-CH₃;

R² is as defined for formula (II), preferably R² being independently selected from H and -Cg alkyl;

R-* is as defined for formula (II), preferably R- being independently selected from H, CH3, -CH₂OH, -CH₂C1, -CH₂SC(O)CgH₅, -CH₂OSO₂-p-CgH5-CH₃, -COOCH₂C₆H₅, -COOCH₂CH₂CgH5 and C₆H₅; and

R⁴ is as defined for formula (II), preferably R⁴ being independently selected from H and C_j-Cg alkyl.

(2) N-protected dipeptides of Formula (HI.:

(III) wherein Rl is as defined for formula (II);

R is as defined for R^ or R⁴ of formula (II), preferably Rl being independently selected from CgH5, H and CH3; and R^ is independently selected from H. CgH5, -CH2OH, and CH3, or R ' represents =CH2-

(3) C-Protected dipeptides of Formula flW

(IV) wherein Rl is as defined for formula (II): R3 is as defined for R³ or R⁴ of formula (II) or R³' represents =CH2-

Preferably Rl is independently selected from CgH5 and CH₃; and R³' is independently selected from CgH₅, CH₃ and -CH₂OH.

(4) Unprotected dipeptides of Formula (VY.

(V) wherein

Rl is as defined for formula (II); and

R³ is as defined for R^J or R⁴ of formula (II) or R³' represents =CH2- Preferably R and R³ are independently selected from CgH5 and CH₃. The free acid/amine group form is also a preferred compound: i.e. without the zwitterion charge, as are pharmaceutically acceptable salts .

(5. 2.5-Diketopiperazines of Formula (VIi:

wherein

Rl is as defined for formula (I); and more conveniently II,

R³' is as defined for R³ or R⁴ of formula (II); and

Y and Z of formula (II) together represent a single covalent bond.

Preferably Rl and R³ are independently selected from CgH5, H, Ci -Cg alkyl,

C_j-Cg haloalkyl, or R^] and R³' is -CHR⁶R⁷, preferably =CH₂. Preferably R^] and R³ are independently selected from CgH₅, H, -CH OH, -CH₂C1,

H, CH₃, or Rl and R³' can represent =CH . Examples of preferred known compounds of formula (I) for use in the preparation of a medicament, and particularly a medicament suitable for the treatment of disease states involving free radical compounds include: A) 1HH: N-carbobenzyloxyglycylglycine ethyl ester B) IPO (where Z = CgH₅CH₂OCO): N-carbobenzyloxy-DL-2-phenylglycine-DL- serine methyl ester

C) 1MO: N-carbobenzyloxy-DL-alanine-DL-serine methyl ester

D) 1EH: N-carbobenzyloxy-Δ-alanineglycine ethyl ester

E) 2MM: N-carbobenzyloxy-DL-alanyl-DL-alanine F) 2PH: N-carbobenzyloxy-DL-2-phenylglycylglycine

G) 5PO: 3-hydroxymethyl-6-phenyl 2,5-piperazinedione

H) 5PH: DL-3-phenyl 2,5-piperazinedione

Preferably the compounds are selected from A), B), C), E), and F). The most preferred compound is B). Naturally, the skilled addressee will appreciate that the free acid addition salts

(e.g. hydro-halo salts,) of all the compounds referred to herein are encompassed within the ambit of the invention,

Suitable acid addition salts include those formed from hydrochloric, hydrobromic, nitric, perchloric, sulphuric, citric, tartaric, phosphoric, lactic, benzoic, glutamic, oxalic, aspartic, pyruvic. acetic, succinic, fumaric, maleic, oxaloacetic, isethionic, stearic, phthalic, methanesulphonic. p-toluene sulphonic, benzenesulphonic, lactobionic and glucuronic acids. Suitable base salts include inorganic base salts such as alkali metal (e.g. sodium and potassium salts) and alkaline earth metal (e.g. calcium) salts; organic base salts, e.g. phenylethylbenzylamine, dibenzylethylenediamine, ethanolamine and diethanolamine salts; and amino acid salts, e.g. lysine and arginine. Most preferably, the salts will be pharmaceutically acceptable.

In a further embodiment of the invention novel compounds of the invention are provided according to general formula (lb) as presented above wherein

R and R² together represent =CHR6R⁷, and particularly preferred examples of these are those where Rl and R² together represent =CH2; and/or where m is an integer from 2 to 10. Most preferred novel compounds of this type are cyclic peptides, or peptide analogues, e.g. ketopiperazines.

As another embodiment of the invention there are provided methods of manufacturing compounds of formula (I) and physiologically functional derivatives thereof, and medicament compositions comprising, these particularly those where Rl and R² together are =CR⁶R⁷ or =CH₂-

Further novel oligopeptide compounds of the invention are those of formula Ia wherein Rl is independently selected from CH3, H and CgH5; R² is independently selected from H, CH3, and CH2OH; R³ is independently selected from -CH₂OH, H, and -CH₂OSO2-p-CgH₅-CH3; or R³ and R⁴ together represent =CH2; and R⁴ is H.

Preferred novel compounds of the invention are those of formual (I), (Ia) and (lib) wherein at least one of Rl and R², or R³ and R⁴, is a group -(CH2)_n- , where n is an integer from 1 to 3 and L is a leaving group or a group -O-I-l, -S-1L.1, -CO-LI or -OS-Li where L 1 is a leaving group wherein L and Li are selected from those groups, which leave the compound under physiological conditions, i.e. in solution in a mammalian body, on interaction of the compound of formula (I) with a free radical species, eg. such as ROS. Most preferably L is a halogen atom, particularly chlorine or bromine, a hydroxy group or L 1 is hydrogen, an arylsulphonylalkyl group, e.g. tosyl or -COCgH5, an arylalkyl, eg.- OCH₂CgH₅ or -OCH2CH₂C H₅, or. -SCOCgH₅. Preferably n is 1. Preferably m is 2 to 10. Preferably at least one of the amino acid residues is a D-isomer.

Examples of novel compounds, particularly for pharmaceutical use, according to the above embodiments include: i) N-carbobenzyloxy-DL-alanine-Δ-alanine methyl ester [1ME]. ii) N-carbobenzyloxyglycyl-O-tosyl-L-serine methyl ester [1HT]. iii) N-carbobenzyloxyglycyl-Δ-alanine [2HE]. iv) N-carbobenzyloxy-DL-2-phenylglycyl-DL-serine [2PO]. v) 3,6-Bis-methylene 2,5-piperazinedione [5EE]. vi) N-carbobenzyloxyglycyl-Δ -alanine methyl ester [ 1 HE] . vii) N-carbobenzyloxy-DL-2-phenylglycyl-Δ -alanine methyl ester [ 1 PE] . viii) N-carbobenzyloxy-Δ -alanine methyl ester [1EE].

Preferred examples of novel compounds of the invention on the basis of their biological activity include i), ii), iii), iv) and v) and the corresponding unprotected dipeptides. Most preferred is the cyclic dipeptide (piperazine dione) compound v) and physiologically functional derivatives and analogues thereof.

Compounds of the present invention have been found to possess free radical scavenging activity in a biological system, as determined using luminol enhanced chemiluminescence generated by (i) porcine polymorphonuclear leukocytes (PMN's) activated by either phorbol myristate acetate (PMA) or ionomycin and (ii) xanthine-xanthine oxidase (X-XO). Thus, free radical scavenging activity of compounds of general formula (I) has been demonstrated in a number of X-XO and PMA-stimulated leukocytes tests in vitro. The results of such assay systems indicate that compounds of formula (I) possess a free radical scavenging activity with respect to at least ROS radicals such as superoxide radical, H2O2, hydroxyl radical, singlet oxygen, hypochlorous acid and the like. Preferred compounds of the present invention have been found to have good scavenging activity in lipid peroxidation assay, eg. compound 5EE. The neutrophil assay is sensitive to superoxide and hydrogen peroxide scavengers and myelo-peroxidase inhibitors but not lipid peroxidase scavengers and xanthine oxidase inhibitors. The xanthine-xanthine oxidase assay is sensitive to superoxide and hydrogen peroxide scavengers, xanthine oxidase inhibitors and lipid peroxidase scavengers but not myleoperoxidase inhibitors. The lipid peoxidase assay is sensitive to antioxidants but not superoxide and hydrogen peroxide scavengers.

It will be seen that biologically active compounds of the invention generally possess at least one captodative centre, i.e. a carbon substituted with both an electron-withdrawing and an electron-donating group. Thus where compounds of the invention do not posses a captodative centre as such, (e.g. IEE), they can be regarded as having at least two pro-captodative centres. Without the intention of being bound by theory it is thought that such compounds undergo radical addition to give a captodative radical which is capable of acting as a free radical scavenging molecule. As an embodiment of the invention are included compounds of formula (I) which contain at least two pro-captodative centres. The compounds of the present invention are useful for the treatment of diseases wherein free radicals are implicated in the disease process. They may be employed in treating various forms of cancer including leukaemias, lymphomas. sarcomas and solid tumours, atherosclerosis, ischaemia/ reperftision myocardial injury, inflammatory diseases of organs such as the lung, and inflammatory diseases of the joints such as arthritis and the like. The invention thus further provides a method for the treatment of free radical molecule generating and/or free radical molecule caused diseases such as those mentioned above in mammals, including humans, which comprises the administration of a clinically useful amount of compound of formula (I) or a pharmaceutically acceptable salt or physiologically functional derivative in a pharmaceutically useful form, once or several times a day or in any other appropriate schedule, orally, rectally, parentally, or applied topically.

In addition, there is provided as a further, or alternative, aspect of the invention, a compound of formula (I) or a pharmaceutically acceptable salt or physiologically functional derivative thereof for use in therapy, for example for treating atherosclerosis and the like, The amount of compound of formula (I) required to be effective as a free radical scavenging agent will, of course, vary and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being treated, the route of administration, and nature of the formulation, the mammal's body weight, surface area, age and general condition and the particular compound to be administered. A suitable effective dose of free radical scavenging agents of the invention generally lies in the range of about 0.01 to about 120 mg/kg bodyweight, e.g. 0.1 to about 120 mg kg body weight, preferably in the range of about 0.1 to 50 mg kg, for example 0.5 to 50 mg/kg.

The total daily dose may be given as a single dose, multiple doses, e.g. two to six times per day or by intravenous infusion for selected duration. For example, for a 75 kg mammal (e.g. a human) the dose range would be about 8 to 9000 mg per day, and a typical dose could be about 50 mg per day. If discrete multiple doses are indicated treatment might typically be 15 mg of a compound of formula (I) given up to 4 times per day.

Whilst it is possible for the active compound to be administered alone, it is preferable to present the active compound in a pharmaceutical formulation. Formulations of the present invention, for medical use, comprise a compound of formula (I) or a salt thereof together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) should be pharmaceutically acceptable in the sense of being compatible, e.g. physiologically and chemically compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The present invention, therefore, further provides a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt or physiologically functional derivative thereof together with a pharmaceutically acceptable carrier therefor.

There is also provided a method for the preparation of a pharmaceutical formulation comprising bringing into association a compound of formula (I) or a pharmaceutically acceptable salt or physiologically functional derivative thereof, and a pharmaceutically acceptable carrier therefor.

Formulations according to the present invention include those suitable for oral, topical, rectal or parenteral (including subcutaneous, intramuscular and intravenous) administration. Preferred formulations are those suitable for oral or parenteral administration.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product into desired formulations.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a solution or suspension in an aqueous or non-aqueous liquid such as a syrup, an elixir, an emulsion or a draught.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered active compound with any suitable carrier,

A syrup may be made by adding the active compound to a concentrated, aqueous solution of a sugar, for example sucrose to which may also be added any necessary ingredients. Such accessory ingredient(s) may include flavourings, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol for example glycerol or sorbitol,

Formulations for rectal administration may be presented as a suppository with a conventional carrier such as cocoa butter.

Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient. Such formulations suitably comprise a solution of a pharmaceutically and pharmacologically acceptable acid addition salt of a compound of the formula (I) that is isotonic with the blood of the recipient.

Useful formulations also comprise concentrated solutions or solids containing the compound of formula (I) which upon dilution with an appropriate solvent give a solution for parenteral administration as above.

In addition to the aforementioned ingredients, the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like.

In a preferred use of the present invention there is provided the use of a compound of formula (I) or pharmaceutically acceptable salt or physiologically functional derivative thereof for the manufacture of a medicament for the treatment of free radical generating disease, being a disease selected from leukaemia, lymphoma, sarcoma, solid tumours, atherosclerosis, ischaemia/reperfusion myocardial injury and inflammatory disease, particularly inflammatory disease of the lung and arthritis.

The present invention will now be illustrated by reference to the following non- limiting Examples. Further embodiments falling within the scope of the claims attached hereto will occur to those skilled in the art in the light of these.

EXAMPLE 1

N-CARBOBENZYLOXY-DL-2-PHENYLGLYCINE-DL-SERINE METHYL ESTER f IPO]

A suspension of N-carbobenzyloxy-DL-o-phenylglycine¹⁵ (2.054 gm, 7.199 mmol) and DL-serine methyl ester hydrochloride (1.183 gm, 7.604 mmol) in dichloromethane (50 ml, dry) was stirred for 15 minues and then triethylamine (3.5 ml) was added. The reaction mixture was cooled to (-5°C) and l-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HC1) (1.384 gm); 7.219 mmol) was added. Stirring was continued overnight and the temperature was left to rise to room temperature. The reaction mixture was diluted with ethylacetate (200 ml) and extracted with H2SO4 (5%, 50 ml), NaHCO3 (5%, 50 ml) and finally with water (50 ml). The organic layer was dried over MgSO4, and the solvent removed in vacuo at room temperature. The crude product was obtained as pale yellow oil (1.220 gm, 44% yield). It solidified on standing in the freezer (-20°C) overnight to give a white solid. M.P. 136-138^βC. vmax. [Nujol]: 3330, 1750, 1700, 1650 cm '. δ (CDCI3): 2.6 and 3.0 (1H, S, Broad OH, exchangeable); 3.7 and 3.8 (3H, S, OCH3); 3.9-4.0 (2H, m); 4.6 (lH, m); 5.1 (2H, m); 5.3 (lH, t); 6.1 (1H, t, NH exchangeable): 6.9 (1H, t, NH exchangeable); 7.3-7.5 (10H, m, ArH). Analysis for: C₂oH₂2 2θg found: C=61.95; H=5.69; N=7.01%; calculated: C=62.17; H=5.74; N=7.25%.

EXAMPLE 2

N-CARBOBENZYLOXYGLYCYLGLYCINE ETHYL ESTER HHH1

A suspension of N-carbobenzyloxyglycine (4.522 gm, 21.61 mmol) and glycine ethyl ester hydrochloride (3.023 gm, 21.66 mmol) in dichloromethane (50 ml dry) was stirred for 15 minutes and then triethylamine (3.5 ml) was added. The reaction mixture was cooled to (- 5 °C) and l-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HC1) (4.521 gm, 23.65 mmol) was added. Stirring was continued overnight and the temperature was left to rise to room temperature. The reaction mixture was diluted with ethylacetate (200 ml) and extracted with H₂SO₄ (5%, 50 ml), NaHCO₃ (5%, 50 ml) and finally with water (50 ml). The organic layer was dried over MgSO4, and the solvent removed in vacuo at room temperature. The crude product was obtained as colourless oil which crystallised out when left standing at room temperature (5.667 gm, 89% yield) to give a white solid. M.P. 78-80°C [cf Ref. 1,2 m.p. 79-81°C]. vmax. [Nujol]: 3336, 1750, 1704, 1666, 1546 cm ¹. δ (CDC1₃) 1.254-1.309 (3H, t, CH₂Cϋ₃) 3.918 and 3.941 (2H. d, CH₂) 4.022 and 4.043 (2H. d. CH₂); 4.167-4.253 (2H, 9, £H₂, CH3); 5.133 (2H. S, Ph-£H₂) 5.559 (1H, t, CONH exchangeable); 6.648 (1H, t, CONH exchangeable).

EXAMPLE 3

N-CARBOBENZYLOXY-DL-ALANINE-DL-SERINE METHYL ESTER [IMP]

A suspension of N-carbobenzyloxy-DL-alanine (5.165 gm, 23.138 mmol) and DL-serine methyl ester hydrochloride (3.717 gm, 23.89 mmol) in dichloromethane (50 ml, dry) was stirred for 15 minutes and then triethylamine (3.5 ml) was added. The reaction mixture was cooled to (-5°C) and l-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HC1) (5.079 gm, 26.49 mmol) was added. Stirring was continued overnight and the temperature was left to rise to room temperature. The reaction mixture was diluted with ethylacetate (200 ml) and extracted with H2SO4 (5%, 50 ml), NaHCO₃ (5%, 50 ml) and finally with water (50 ml). The organic layer was dried over MgSO4, and the solvent removed in vacuo at room temperature. The crude product was obtained as pale yellow oil (5.735 gm, 70% yield). It solidified on standing in the freezer (-20°C) overnight to give a white solid. M.P. 137-139'C [cfRef 3 m.p. 138-140°C]. vmax. [Nujol]: 3494. 3293, 1755, 1694, 1650, 1545 cm ¹. δ (CDCI3): 1.384 1.391 and 1.412-1.419 (3H, d and d, Me); 3.061 and 3.345 (1H. broad, OH exchangeable); 3.764 and 3.773 (3H, S and S, OMe); 3.937 (2H, S, Cϋ₂-OH); 4.262-4.318 (IH, m, CH); 4.596-4.675 (IH, , CH); 5.058-5.148 (2H, m, Ph-£H₂);

5.51 1-5.596 (IH, m, CONH exchangeable); 7.098 -7.167 (IH, t, CONH exchangeable); 7.341 and 7.349 (5H, S and S, ArH).

EXAMPLE 4

N-CARBOBENZYL XYGLYCYL-L-SERINE METHYL ESTER .IHOj

A suspension of N-carbobenzyloxyglycine (4.180 gm , 20. 00 mmol) and L-serine methyl ester hydrochloride (3.190 gm, 20.50 mmol) in dichloromethane (50 ml, dry) was stirred for 15 minutes and then triethylamine (3.5 ml) was added. The reaction mixture was cooled to (-5^βC) and l-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) (4.000 gm, 21.00 mmol) was added. Stirring was continued overnight and the temperature was left to rise to room temperature. The reaction mixture was diluted with ethylacetate (200 ml) and extracted with H₂SO₄ (5%, 50 ml), NaHCO₃ (5%, 50 ml) and finally with water (50 ml). The organic layer was dried over MgSO4, and the solvent removed is vacuo at room temperature. The crude product was obtained as white solid (4.950 gm, 79% yield). M.P. 93-94^βC. [Ref. 4 m.p. 94-95^βC]. vmax. [Nujol]: 1727, 1698, 1669, 1553, 1524, 3364, 3306 cm"¹. δ (CDC1₃): 3.423 (IH, broad, OH exchangeable); 3.758 (3H, S, OMe); 3.758-3.979 (4H, m): 4.622-4.679 (IH, P); 5.111 (2H, S, Ph-CH₂); 5.765 (IH, t, CONH exchangeable); 7.175-7.205 (IH, d, CONH exchangeable); 7.342 (5H, S, ArH).

EXAMPLE g

[SI N-CARBOBENZYLOXV-L-SERINE-GLYCINE ETHYL ESTER . .0H1

A suspension of N-carbobenzyloxy-L-serine (4.170 gm, 17.43 mmol) and glycine ethyl ester hydrochloride (2.465 gm, 17.66 mmol) in dichloromethane (50 ml, dry) was stirred for 15 minutes and then triethylamine (3.5 ml) was added. The reaction mixture was cooled to (-5 ^βC) and l-e yl-3-dime yl--minopropylcarbodiimide hydrochloride (EDC.HCl) (3.472 gm, 18.1 1 mmol) was added. Stirring was continued overnight and the temperature was left to rise to room temperature. The reaction mixture was diluted with ethylacetate (200 ml) and extracted with H₂SO₄ (5%, 50 ml), NaHCO₃ (5%, 50 ml), and finally with water (50 ml). The organic layer was dried over MgSO4, and the solvent removed in vacuo at room temperature. The crude product was obtained as white solid (5.529 gm, 98% yield). M.P. 104-106°C [Ref 5 m.p. 106-107^βC]. vmax. [Nujol]: 3350, 1750, 1690, 1660, 1560, 1530 cm ¹. δ (CDC1₃): 1.237-1.313 (3H, t, CH₂Cϋ₃); 3.193-3.243 (IH, t, OH exchangeable); 3.649-3.745 (IH, p, CH); 4.024-4.141 (2H, m, CH₂); 4.168-4.254 (2H, q, £H₂CH₃); 4.304 (2H, t, CH); 5.139 (2H, S, ph-Cϋ₂); 5.876 and 5.904 (IH, d, CONH exchangeable); 7.038 (IH, broad, CONH exchangeable); 7.359 (5H, S, ArH).

EXAMPLE 6 N-CARBOBENZYLOXY-DL-2-PHENYLGLYCYLGLYCINE ETHYL ESTER flPHl

A suspension of N-carbobenzyloxy-DL-2-phenylglycine (6.003 gm, 21.04 mmol) and glycine ethyl ester hydrochloride (3.061 gm, 21.93 mmol) in dichloromethane (50 ml, dry) was stirred for 15 minutes and then triethylamine (3.5 ml) was added. The reaction mixture was cooled to (-5°C) and l-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) (4.260 gm, 22.22 mmol) was added. Stirring was continued overnight and the temperature was left to rise to room temperature. The reaction mixture was diluted with ethylacetate (200 ml) and extracted with H₂SO (5%, 50 ml), NaHCO₃ (5%, 50 ml) and finally with water (50 ml). The organic layer was dried over MgSO4, and the solvent removed in vacuo at room temperature. The crude product was obtained as white solid (6.987 gm, 90% yield). M.P. 136-138^βC [Ref. 6 m.p. 146-148"C]. vmax. [Nujol]: 3300, 1730, 1685, 1650, 1554 cm ¹. δ (CDCI3): 1.193-1.251 (3H, t, CH₂Cϋ₃); 3.848-3.986 (2H, m, CH₂); 4.038-4.102 (2H, q, Cϋ₂CH₃); 5.055 and 5.069 (2H, S and S, ph-£H₂); 5.310 and 5.336 (IH, d, CH); 6.156 and 6.181 (IH, d, CONH exchangeable); 6.523 (IH, broad, CONH exchangeable); 7.255-7.373 (10H, m, ArH).

EXAMPLE 7

[7] N-CARBOBENZYLOXY-DL-ALANINE-DL-β-CHLOROALANINE METHYL ESTER [lMCll N-Carbobenzyloxy-DL-alanine-DL-serine methyl ester (0.503 gm. 1.551 mmol) was dissolved in chloroform (50 ml, dry), then PCI5 (0.354 gm, 1.699 mmol) was added at room temperature with stirring. The stirring was continued at room temperature overnight. The mixture was then extracted with water (100 ml) , and the organic layer dried over MgSO4, and the solvent removed in vacuo at room temperature. The product was obtained as pale yellow solid (0.530 gm, 99% yield), M.P. 144-146^βC. vmax [Nujol]: 3310, 1747, 1694, 1650 cm '. δ (CDCI3): 1.42 and 1.45 (3H, S, Me); 3.82 (3H, S, OMe); 3.90-4.05 (2H, m); 4.32-4.38 (lH, t); 4.94-4.98 (lH; m); 5.14 (2H, S); 5.27 (IH, broad NH exchangeable); 6.89 (IH, broad, NH exchangeable); 7.30-7.40 (5H, S, ArH).

Mass for C₁₅H₁₉N2θ5Cl: Found: 342.0989; Expected: 342.0983.

EXAMPLE 8

N-CARBOBENZYLOXY-L-β-CHLOR ALANTNEGLYCINE ETHYL ESTER flCHl N-carbobenzyloxy-L-serineglycine ethyl ester (2.413 gm, 7.439 mmol) was dissolved in chloroform (50 ml, dry), then PCI5 (2.010 gm; 9.652 mmol) was added at room temperature with stirring. The stirring was continued at room temperature overnight. The reaction was then extracted with water (100 ml), and the organic layer dried over MgSO4, and the solvent removed in vacuo at room temperature. The product was obtained as pale yellow solid which was then chromatographed on neutral silica gel ( 1 : 1 ethyl acetate: pet. ether) to give the product as white solid (1.441 gm, 56% yield). M.P. 128-130°C (cf Ref.

7 m.p. 132°C; Ref. 8, m.p. 138°C). vmax. [Nujol]: 3311, 1750, 1694, 1657, 1546 cm-¹. δ (CDCI3); 1.264-1.321 (3H, t, CH₂Cϋ₃); 3.718-3.784.(1H, q, CH); 3.952-4.046 (2H, P, CH₂); 4.185-4.271 (2H, q, £H₂ ^CH3)? ⁴-616-4.648 (IH, m);

5.160 (2H, S, ph-£H₂); 5.664 and 5,695 (IH, d, CONH exchangeable);

6.815 (IH, t, CONH exchangeable); 7.374 (5H, S, ArH).

EXAMPLE 9

N-CARBOBENZYLOXYGLYCYL-L-β-CHLOROALANINE METHYL ESTER I1HC1

N-carbobenzyloxyglycyl-L-serine methyl ester (0.513 gm, 1.653 mmol) was dissolved in dichloromethane (10 ml, dry), then PCI5 (0.440 gm, 2.1 1 mmol) was added at room temperature with stirring. The stirring was continued at room temperature overnight. The reaction was then extracted with water (100 ml), and the organic layer dried over MgSθ4, and the solvent removed in vacuo at room temperature. The product was obtained as white solid (0.530 gm, 97% yield). M.P. 120-122°C [cf Ref. 7 m.p. 126°C]. vmax. [Nujol]: 3320, 1750, 1690. 1650, 1530 cm-¹. δ (CDCI3): 3.848 (3H, S, OMe); 3.860-3.925 (4H, m); 4.968-5.022 (IH, p, CH); 5.093 (2H, S, Ph-Cϋ₂): 5.460 (IH, t, CONH exchangeable); 6.904 and 6.930 (IH, d, CONH exchangeable); 7.332 (5H, S, ArH).

EXAMPLE 10

N-CARBOBENZYLOXY-DL-ALANINE-A-ALANINE METHYL ESTER HME1 N-Carbobenzyloxy-DL-alanine-p-chloroalanine methyl ester (0.496 gm, 1.447 mmol) was dissolved in ethyl acetate (25 ml), triethylamine (0.5 ml) was added at room temperature with stirring. The stirring was continued overnight at room temperature. The reaction mixture was then filtered, and the filtrate extracted with water and ethyl acetate. The organic layer was dried over MgSO4, then filtered. The solvent was then removed in vacuo at room temperature to give light brown oil (0.360 gm), 81% yield). Trituration with ether, followed by chilling gave the product as pale yellow solid. M.P. 240°C (decomp). vmax. [Nujol]: 3300, 1730, 1700, 1660 cm ¹. δ (CDCl₃); 1.419 and 1.448 (3H, S and S, Me); 3.849 (3H. S. OMe); 4.315 (lH, t, CH) 5.138 and 5.149 (2H, S and S, ph-Cϋ₂); 5.306 and 5.331 (IH, d, CONH exchangeable) 5.925 and 5.930 (IH, d, olefinic H); 6.607 (IH, S, olefmic H); 7.358 (5H, S, ArH) 8.335 (IH, broad. CONH).

EXAMPLE 11

N-rARB BENZYl-OXY-A-ALANINEGLYCINE ETHYL ESTER flEHJ N-Carbobenzyloxy-L-β-chloroalanineglycine ethyl ester (0.301 gm, 0.878 mmol) was dissolved in DCM (10 ml, dry). To this was added l,4-diazobicyclo[2,2,2]octane (0.163 gm, 1.453 mmol) at room temperature with stirring. The reaction was then left stirring at room temperature overnight. The reaction mixture was diluted with DCM and then extracted with 2N HCI (7 ml). The water layer was extracted with DCM (14 ml) and the combined organic layers were extracted with water (7 ml) then dried over Na2SU4. The solvent was removed in vacuo to give pale yellow oil, which was dissolved in DCM and charcoal, then filtered over cotton. Solvent was removed in vacuo at room temperature to give colourless thick oil which was crystallized from dry ether/dry pet. ether at 0°C. The white crystalline product was filtered to afford product (0.192 gm, 71 % yield). M.P. 76-78°C [cf Ref. 4 m.p. 76-78°C]. vmax. [Neat]: 3387, 1735, 1677, 1635, 1510, cm ¹. δ (CDC1₃); 1.267-1.383 (3H, t, CH₂Cϋ₃); 4.095 and 4.115 (2H, d, CH₂) 4.214-4.242 (2H, q, Cϋ₂CH₃); 5.169 (^2H _> ^s- Ph-£H₂); 5.210-5.224 (IH, t, olefinic H) 6.148-6.156 (IH, d, olefinic H); 6.631 (IH, t, CONH); 7.357 (5H, S, ArH) 7.523 (IH, S, CONH).

EXAMPLE 12

N-CARBOBENZYLOXYGLYCYL-A-ALANINE METHYL ESTER [THE]

N-Carbobenzyloxyglycyl-L-β-chloroalanine methyl ester (0.500 gm, 1.521 mmol) was dissolved in CHCI3 and applied to a neutral alumina column using pet. ether/ethyl acetate

(1/3) as eluant. The product was obtained as white crystalline material

(0.300 gm. 68% yield). M.P. 71-74°C [cf Ref 9, m.p. 71-74 -C]. vmax. [Nujol]: 3350, 1730, 1680, 1570 cm ¹. δ (CDCl₃); 3.857 (3H, S, OMe); 3.976 and 3.999 (2H, d, CH₂); 5.167 (2H, S, Ph-£H₂); 5.389 (IH. broad. CONH); 5.939 (IH, S, olefinic H); 6.616 (IH, S, olefinic H);

7.371 (5H, S, ArH); 8.198 (IH, broad, CONH).

EXAMPLE 13

N-ΓARB BENZVT. XYGLYCYL-O-TOSYL-L-SERINE METHYL ESTER [1HT]

Tosyl chloride (0.436 gm, 2.287 mmol) was added to a solution of N-carbobenzyloxyglycyl-L-serine methyl ester (0.342 gm, 1.102 mmol) in pyridine (3 ml, dry) precooled to -5°C, and the mixture was stirred at -5°C for 2 hours). Cold water (30 ml) was added to the reaction mixture with stirring. Upon seeding and scratching, the crystalline product separated out. Recrystallization from acetone water afforded (0.249 gm, 70% yield). M.P. 86-88'C. vmax. [Nujol]: 3420, 3330, 1760, 1730, 1670 cm ¹. δ (CDC1₃): 2.44 (3H, S); 3.72 (3H, S, OMe); 3.9 (2H, d); 4.30-4.44 (2H, dq); 4.76-4.80 (lH, q); 5.15 (2H, S); 5.42 (IH, t, NH, exchangeable); 6.91-6.93 (IH, d, NH, exchangeable); 7.40-7.50 (7H, m, ArH); 7.73-7.77 (2H, d, ArH). Mass: (M-Ts): C₁₄HιgN2θ₅; Found: 292.1059, required 292.1038.

EXAMPLE 14 N-CARBOBENZYLOXYGLYCYL-A-ALANINE (2HEJ

N-Carbobenzyloxyglycyl-Δ-alanine methyl ester (0.250 gm, 0.855 mmol) was dissolved in methanol (15 ml). Sodium hydroxide solution (10%, 3 ml) was added to it then diluted with water (7 ml). The reaction mixture was left at room temperature for 2 hours, then the reaction mixture was warmed up to 40°C for a few minutes. Methanol was removed in vacuo at 40^βC, then the misture was allowed to cool to room temperature. It was then diluted with water and extracted with ether. The water layer was acidified with concentrated HCI. The product (as white solid material) was collected by filtration and washed with water, then dried in vacuo to give CO.148 gm. 62% yield .. M.P. 186-188^βC. [cf Ref. 4, m.p. 188-189°C]. vmax. [Nujol]: 3300, 1650, 1540cm ¹. δ (DMSO-dg): 3.776 and 3.801 (2H, d, CH₂); 5.045 (2H, S, ph-£H ₂); 5.708 (IH, S, olefinic H); 6.313 (IH, S, olefinic H); 7.352 (5H, S, ArH); 7. 619-7.668 (IH, t, CONH exchangeable); 9.107 (1 H, S, CONH exchangeable).

EXAMPLE 15 N-CARBOBENZYLOXY-PL-2-PHENYLGLYCYLGLYCINE (2PHl

N-Carbobenzyloxy-DL-2-phenylglycylglycine ethyl ester (0.501 gm, 1.353 mmol) was suspended in methanol (15 ml). Sodium hydroxide solution (10%, 1 ml) was added to the mixture then that was diluted with water (7 ml). The reaction mixture was left at room temperature for 2 hours, then it was warmed up to 40 °C for a few minutes. Methanol was removed in vacuo at 40 °C, then the mixture was allowed to cool to room temperature. It was then diluted with water and extracted with ether. The water layer was cooled to 0^βC then acidified with diluted HCI. The product as white solid material was collected by filtration and washed with water, then dried in vacuo to give (0.235 gm, 51% yield). M.P. 154-156 °C [cf Ref. 14 m.p. 152-153^βC]. vmax. [Nujol]: 3300. 1650, 1700 cm ¹. δ (DMSO-dg): 3.739-3.753 (2H, dd, CH₂); 5.035 (2H, S, Ph£H₂); 5.305 and 5.340 (IH, d, CH); 7.265-7.451 (10H, m, ArH); 7.952 and 7.987 (IH, d, CONH); 8.511-8.557 (lH, t, CONH).

EXAMPLE 16 N-CARBOBENZYLOXY-DL-2-PHENYLGLYCYL-DL-SERINE [2PO]

N-Carbobenzyloxy-DL-2-phenylglycyl-DL-serine methyl ester (0.267 gm, 0.691 mmol) was dissolved in methanol ( 10 ml). Sodium hydroxide solution ( 10%, 3 ml) was added to it then diluted with water (7 ml). The reaction mixture was left at room temperature for 1 hour, then it was warmed up to 40°C for a few minutes. Methanol was removed in vacuo at 40°C, then it was allowed to cool to 0°C. It was then diluted with water and extracted with ether. The water layer was acidified with diluted HCI. The product pale yellow solid material was collected by filtration and washed with water, then dried in vacuo to give (0.113 gm, 47% yield). M.P. 154-156^βC. vmax. [Nujol]: 3319, 1732, 1652, 1537 cm^"1. δ (DMSO-dg): 3.50-3.70 (2H, m); 4.19-4.27 (lH, m); 5.04 (2H, S); 5.38-5.44 (IH, q);

7.26-7.46 (loH, m, ArH); 7.93-7.97 (IH, d, NH.exchangeable); 8.43-8.51 (IH, q, NH, exchangeable).

Mass: (M-H₂0): C₁₉H₁₈N₂05; Found: 354.1204; Expected: 354.1216.

EXAMPLE 17

N-CARB BENZYL XY-DL-ALANYL-DL-ALANfNE f2MMl °

DL-Alanyl-DL-alanine (0.456 gm, 2.847 mmol) was dissolved in (2N NaOH, 1.6 ml). It was cooled to 0°C, then benzylchloroformate (0.5 ml, 3.488 mmol) and sodium hydroxide (2N, 1.7 ml) were added dropwise simultaneously with vigorous stirring. When the addition was finished, it was left stirring at room temperature overnight. The pH of the reaction mixture was adjusted to pH 11 by adding sodium hydroxide with stirring. It was extracted with ethyl acetate after adding water. The water layer was acidified at 0°C with concentrated HCI (pH 2). It was then extracted with ethyl acetate, dried over Na2SO4, and removed in vacuo. to give white material (0.650 gm, 78% yield). M.P. 140-144°C. [cf Ref. 10 m.p. 144-145^βC]. vmax. [Nujol]: 3320, 1700. 1660. 1550 cm-¹. δ (CD₃OD): 1.239-1.412 (6H, m. 2Me); 4.137-4.192 (lH. m, CH); 4.367-4.395 (IH, m, CH); 5.079 and 5.093 (2H, d, ph-£H₂); 7.261-7.356 (5H, m, ArH).

EXAMPLE 18

PL-3-PHENYL 3.5-PiPERAZIIVEPIQ E [5PH1" N-Carbobenzyloxy-DL-2-phenylglycylglycine ethyl ester [15] (1.018 gm, 2.748 mmol) was suspended in methanol (20 ml), to it was added Pd/C (10%. 87 mg). It was hydrogenated at (1 atm.) for 5 hours with stirring, then filtered over cotton and the solvent removed in vacuo to give colourless oil. It was heated under reflux in toluene for 48 hours. The solid precipitate was filtered, then triturated with acetone to give white crystalline material (0.300 gm, 57% yield).

M.P. 232-234°C. [cf Ref. 11 m.p. 241-243^βC]. vmax. [Nujol]: 3330, 1685, 1677 cm ¹. δ (DMSO-dg): [(3.681 and 3.701, d); (3.759 and 3.772, d);

(3.914, S); (3.986, S); 6 signals represent CONH-CH₂-CONH]; 4.864 and 4.876 (IH, d, CH); 7.322-7.389 (5H, m, ArH); 8.150 (IH, S, CONH exchangeable); 8.634 (IH, d, CONH exchangeable).

EXAMPLE 19

3-HYnROXYMETHYL-6-PHENYL 2.5-PIPERAZINEDΪONE fSP l

N-Carbobenzyloxy-DL-2-phenylglycyl-DL-serine methyl ester [1] (1.078 gm, 2.789 mmol) was dissolved in a mixture of methanol (10 ml) and ethyl acetate (10 ml). After adding Pd/C (10%, 78 mg), the resultant mixture was hydrogenated at (1 atm.) overnight, then filtered and solvent removed in vacuo at 50°C. The product was obtained as thick oil, which afforded a white solid material upon trituration with methanol and ether, (0.565 gm, 92% yield). M.P. 218-220°C. vmax. [Nujol]: 1687 cm ¹. δ (DMSO-dg): 3.5-3.9 (2H, m; 4.90 and 4.93 (IH, S); 5.15 and 5.20 (IH, t, OH exchangeable);

7.3-7.5 (5H, m, ArH); 8.05 and 8.12 (IH, S, NH, exchangeable); 8.51 and 8.53 (IH, S, NH exchangeable).

Analysis for: C_{1 1},H₁₂N₂O₃: found: C=59.13; H=5.38; N=12.53% calculated: C=59.54; H=5.49; N= 12.72%.

EXAMPLE 20a

DL-SERINE METHYL ESTER" " DL-serine methyl ester hydrochloride (5.021 gm, 32.273 mmol) was suspended in chloroform (50 ml, dry), and ammonia gas was passed through for 1 hour. The reaction mixture was filtered and washed with chloroform. Solvent was removed in vacuo to give the product as colourless oil (3.840 gm, 100% yield), vmax. [Neat]: 3400, 1750. 1600, 1450 cm ¹. δ (CDC1₃); 2.091 (2H, S, NH₂); 3.705 (3H, S, OMe); 4.633 (IH, S, CH); 7.264-7.401 (5H. m, ArH).

EXAMPLE 20b

3.6-BTS-HYDROXYMETHYL- 2.5-PTPERAZINEDIONE 15001" " The solvent freeDL-serine methyl ester (3.840 gm, 32.236 mmol) from Example 20a was left standing at room temperature for 48 hours. The product obtained as white solid was filtered and washed with a small volume of cooled methanol to give (2.749gm, 96% yield).

M.P. 260°C (decomp.) [lit^{12 13} m.p. >265^βC (decomp.)]. vmax. [Nujol]: 3260, 3190, 1675 cm ¹. δ (DMSO-dg): 3.547-3.747 (6H, m); 4.987-5.049 (2H. 2xt, 2 (OH) ) ; 7.904 and 8.030 (2H, d, 2(NH)).

EXAMPLE 20c

3.6-BTS-CHLOR METHYL 2.5-PIPERAZINEDΪNE 15CC]" " 3.6-Bis-hydroxymethyl 2,5-piperazinedine^{12 13} (1.00 gm, 5.742 mmol) was added portion wise on a period of 10 minutes, to a cooled solution of phosphorus pentachloride (2.610 gm, 12.534 mmol) in chloroform (50 ml, dry) with stirring. The stirring continued overnight at room temperature. The yellow solid material was filtered and washed with dry chloroform to give the required product (0.525 gm, 62% yield). M.P. 220^βC (decomp.) [ lit^{12 13} m.p. >220°C (decomp.)]. vmax. [Nujol]: 3200, 3100, 1700 cm ¹. δ (DMSO-dg): 3.422-3.750 (3H, ); 4.013-4.066 (IH, dd); 4.418 (IH, S); 5.020 (IH, S exchangeable); 8.024 (IH, S exchangeable); 8.529 (IH, S exchangeable).

EXAMPLE 20d 3.6-BISMETHYLENE 2.5-PIPERAZINEDIONE [5EE)'²-"

3,6-Bis-chloromethyl 2,5-piperazinedione^{12 13} (100 mg, 0.680 mmol) was dissolved in methanol (30 ml. dry), to it was added diethylamine (1 ml). The reaction mixture was left standing at room temperature overnight, and the solid product was filtered and washed with small amount of cooled methanol to give (60 mg, 64% yield). M.P. >300°C (decomp) [lit^{12 13} m.p. 300°C (decomp)]. vmax. [Nujol]: 3200, 1700, 1650. δ (DMSO-dg: 4.907 (2H, S, olefinic H); 5.249 (2H, S, olefinic H);

8.039 (2H, S, 2 (NH) exchangeable).

EXAMPLE 21 N-CARBOBENZYLOXY-L-SERINE-L-SERINE METHYL ESTER HOP]

To a stirred suspension of N-carbobenzyloxy-L-serine (1.050 gm, 4.389 mmol) and L-serine methyl ester hydrochloride (0.802 gm, 5.154 mmol) in dichloromethane (10 ml, dry) was added triethylamine (0.780 ml). After 15 minutes the reaction mixture was cooled to -10°C and l-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (EDC.HCl) (1.005 gm) was added. Stirring was continued overnight and the temperature was left to rise to room temperature. The reaction mixture was diluted with ethyl acetate (200 ml) and extracted with H SO₄ (5%, 50 ml), NaHCO₃ (5%. 50 ml) and finally with water (50 ml). The product was obtained as colourless oil which crystallised out when left standing at room temperature (0.975 gm, 56% yield). M.P. 141-142^βC. [Ref. 16 m.p. 138-141°C]. vmax. [KBr]: 3438. 1750, 1654, 1567 cm ¹. β (CDC1₃): 3.884 (3H, s, OMe), 3.945-4.072 (3H, m). 4.116-4.290 (IH, m), 4.621-4.682 (IH, m), 5.138 (2H, s, Ph-£H₂), 5.832 and 5.857 (IH, d, CONH exchangeable). 7.366 (5H, s, ArH).

EXAMPLE 22

N-CARBOBENZYLOXY-L-p-CHLOROALANINE-L-p-CHLOROALANINE METHYL ESTER [1CC1 N-Carbobenzyloxy-L-serine-L-serine methyl ester (example 21 ) (0.940 gm, 2.772 mmol) was dissolved in dichloromethane (DCM) (20 ml, dry), then PCI5 (1.369 gm) was added with stirring at room temperature. The reaction mixture was then left standing for 3 hours, at room temperature, then it was diluted with DCM and extracted with water (50 ml) . The water layer was again extracted with DCM. The combined organic layers were dried over Na2SO4, then removed in vacuo to give a semi-solid material. Trituration with ether followed by recrystallisation from ether/ethyl acetate gave the product as a white solid material (136 mg, 13% yield). M.P. 140-145^βC. vmax. [Br]: 3313, 1741, 1695, 1660, 1557, 1534 cm ¹. δ (CDCI3): 3.732-4.113 (4H, m), 3.831 (3H, s, OMe), 4.664-4.781 (IH, m), 4.934-4.991 (IH, m), 5.180 (2H, s, Ph-£H.₂), 5.638 and 5.668 (IH, d, CONH), 7.08-7.205 (IH, q, CONH), 7.385 (5H, s, ArH). Mass: for C₁₅H₁₈N₂05Cl2; found: 376.0583; required: 376.0593.

EXAMPLE 23 N-CARBOBENZYLOXY-A-ALANINE METHYL ESTER flEE]

N-Carbobenzyloxy-L-p-chloroalanine-L-p-chloroalanine methyl ester (example 22) (0.059 mg, 0.157 mmol) and 1.4-diazobicyclo[2,2,2] octane (DABCO) (0.042 mg) were dissolved in DCM (10 ml, dry) at room temperature with stirring, then left standing for 48 hours at room temperature. The reaction mixture was diluted with DCM (100 ml) then 2N HCI (7 ml) was added and extracted. The organic layer was washed with brine (5 ml) and then dried over Na2SO4- The solvent was removed in vacuo at room temperature to give pale yellow oil which was stirred with charcoal then filtered over cotton. DCM was removed at room temperature to give colourless oil (0.032 gm, 69%). vmax. [Neat]: 3393, 1739, 1684, 1509 cm ¹. δ (CDC1₃): 3.889 (3H, s, OMe); 5.119 (2H, s, Ph-£H₂); 5.270-5.286 (IH, t); 5.981-5.985 (IH, d); 6.220-6.229 (IH, d); 6.622 (IH, s); 7.381 (5H, s, ArH); 7.444 (IH, s, CONH exchangeable); 8.486 (IH, s, CONH exchangeable).

EXAMPLE 24 N-CARBOBENZYLOXY-DL~2-PHENYLGLYCINE-DL-p-CHLOROALANINE METHYL ESTER flPCl

N-carbobenzyloxy-DL-2-phenylglycine-DL-serine methyl ester (example 1) (0.263 gm, 0.681 mmol) was dissolved in chloroform (50 ml, dry), then PCI5 (0.277 gm, 1.329 mmol) was added with stirring at room temperature. The reaction mixture was left standing for 24 hours at room temperature, then diluted with chloroform and extracted with water. The organic layer was dried over MgSO4, filtered, and the solvent removed in vacuo at room temperature. The product was triturated with ether to give white solid material (0.270 gm, 98%). M.P. 128-130^βC. vmax. [Nujol]: 3300, 1750, 1690, 1650, 1530 cm ¹. δ (CDCI3): 3.352-3.683 (2H, m, Cϋ₂Cl); 3.737 and 3.809 (3H, two singlets, OMe); 3.834-3.967 (2H, dt); 5.003-5.876 (IH, d); 5.105 and 5.103 (2H, two singlets, Ph-CH₂); 5.308 (IH, broad. Ph-£H); 5.961 (IH, broad CONH exchangeable); 6.581 and 6.606 (IH, d, CONH exchangeable); 7.270-7.396 (10H, m, ArH). Mass: for C₂oH2iN₂0₅Cl; found: 404.1135 required: 404.1139 EXAMPLE 25

N-CAl^OBENZYLOXY-DI^2-PHEN^GLYCINE.A-ALANINE METHYL ESTER

UEE1

N-Carbobenzyloxy-DL-2-phenylglycine-p-chloroalaninemethyl ester (0.160 gm, 0.0395 mmol) was dissolved in ethyl acetate (12 ml), then triethylamine

(200 μl, 1.076 mmol) was added at room temperature with stirring. The stirring was continued overnight at room temperature. The salt was filtered and the organic layer was extracted with water, then dried over MgSO4. Ethyl acetate was removed in vacuo at room temperature, to give pale yellow oil. The crude product was purified by column chromatography using neutral alumina and eluted with (1/2 pet. ether and ethyl acetate) to afford a colourless oil which was crystallised out when triturated with ether to give a white crystalline material (130 mg, 89%), m.p. 130-132°C. vmax. [Nujol]: 3400, 3360, 1730, 1680 cm-'. δ (CDCl₃): 3.804 (3H, s, OMe); 5.097 and 5.113 (2H, d, Ph-Cϋ₂); 5.271-5.295 (IH, d); 5.898 and 5.903 (IH, d); 6.026 (IH, d); 6.591 (IH, s); 7.344-7.407 (5H, m, ArH);

7.952 (lH, s, CONH).

Analysis for: C20H20N2O5: Found: C, 65.13; H, 5.56; N, 7.42

Calculated: C, 65.21; H. 5.47; N, 7.60.

Experimental protocol for assays of Example 26 Chemiluminescence was measured from PMA- or ionomycin-stimulated neutrophils (i.e. included in the leucocyte fraction) or by X-XO. The effects of various concentrations of scavengers or standard compounds were examined by addition just prior to the stimulant. Results are calculated as peak chemiluminescence or a percent of the peak chemiluminescence and expressed as mean + SEM. The effects of inhibitors are expressed as percent changes of the peak chemiluminescence by reference to peak chemiluminescence obtained in control aliquots from each cell suspension. Effects of pH on Captopril response were determined using Student's unpaired t-test. A p< 0.05 was considered statistically significant. Materials

PMA (Sigma) and ionomycin (Calbiochem) were dissolved in ethyl alcohol and DMSO respectively, and fresh dilutions were made daily with saline before use. Luminol (Sigma) was prepared daily in 2M NH4OH (2.5%) and diluted with PBS. Xanthine (sodium salt) and xanthine oxidase (Grade I, from buttermilk; 0.69 units/mg) were obtained from Sigma and dissolved in distilled water. L-Ascorbic acid (sodium salt), superoxide dismutase (from bovine erythrocytes; 4200 units/mg), catalase (2000 units/mg). sodium azide, D-mannitol (all from Sigma), glutathione (reduced form. BDH) and captopril (Squibb) were dissolved in either distilled water or PBS before use.

EXAMPLE 26

Susceptibility Of ROS generated from neutrophils to a range of (i) model compounds and (ii) free radical scavengers of formula (I).

(i . Isolation and Separation of Leukocytes f neutrophils.

Cells obtained from Large White/Welsh Landrace cross breed pigs (25-40 kg) were prepared on a daily basis. Sedation of the pigs prior to anaesthesia was achieved by intramuscular injection of azaperone (Stresnil, Janssen, 5 mg/kg), and anaesthesia induced by halothane (4% in oxygen). The animals were then intubated and maintained on halothane anaesthesia (1% in air). Following femoral vein catheterisation, porcine blood

(18 ml) was taken into a tube containing 3.8% sodium citrate (2 ml). Histopaque-1077 (4 ml, Sigma) was added and allowed to sediment at 37^βC for 45 minutes. The leukocyte-rich (i.e. including neutrophils) upper layer was removed and centrifuged at 900 rpm for 10 minutes. The resulting supernatant was centrifuged at 3000 rpm for 5 minutes to prepare platelet poor plasma. Erythrocyte lysis was performed by washing cells with distilled water for 30 seconds and mixing immediately with a double volume of 1.8% NaCl. The leukocytes (neutrophils) were then centrifuged at 900 rpm for 10 minutes and the pellet was resuspended in platelet-poor plasma.

After leukocyte count in a cell counter (Medonic, Cellanalyzer CA 460, Sweden), cell yield was adjusted to 10⁷ cells/ml (stock cell suspension), by using platelet poor plasma. Luminol-enhanced chemiluminescence (CL) was measured at 37^βC using a chemiluminometer (Lumi-vette, Chrono-log Co ., Havertown, PA, USA) as described below. Cell viability was assessed by trypan blue exclusion. More than 98% of the cells were found to be viable leukocytes (n=9). Differential cell counts were also performed using Leishman stain and 39.3% and 40.8% of PMN leukocytes (including neutrophils) were found in porcine blood and leukocyte cell suspension respectively (n=2). The stock cell suspension was stored at room temperature until use.

(ϋ) PhQlfrQl mvristate (PMA) SOU ionomvcin-induced chemiluminescence

Cneutrophil assay . Stock leukocyte cell suspension (0.45 ml) was diluted with 0.45 ml phosphate buffered saline (PBS, 10 mM KH2PO4 and 150 mM NaCl, pH 7.4) in a cuvette containing a stir bar. Following preincubation at 36 °C for 5 minutes, the cuvette was transferred to measuring chamber (37^βC) and 0.1 ml luminol (225 μM; final cuvette concentration) was added, producing a final cell yield of 4.5 x 10⁶ cells/ml. Then stimulant (PMA or ionomycin) was added to yield final cuvette concentrations of 0.8 μM and 3 μM respectively. Chemiluminescence from essentially the neutrophil fraction of the leukocytes was measured continuously for 15 minutes. Approximately 30 aliquots were obtained per 10 ml of blood. Results are shown in Tables 1 to 4.

(iii) X-XO-induced chemiluminescence In order to characterize the X-XO induced chemiluminescence, 0.9 ml phosphate buffered saline was mixed with 0.1 ml luminol (225 μM; final cuvette concentration) in a cuvette containing a stir bar. Following the addition of 10 μl xanthine (final concentration lO^M), and 6.25 μl xanthine oxidase (final concentration 2.5 mU/m) to the cuvette, the chemiluminescence produced was measured continuously for 5 minutes. Duplicate assays were performed in all experiments. Results are shown in Tables 1 to 4. TABLE 1 - DIPROTECTED DIPEPTIDES

| Substance Neutrophil Assay# X-XO

-/+% -/+%

BTG1HH -14 (10°) -34 (lO^"3) ooo-) 8C10-⁴)

BTG IPO -34+ (lO^"3) -32(10°)

-12 (10-⁴) -18 (lO^"4)

8 (lO⁶) 6(10^"6)

BTG 1MO -21 (10°) -26(10°) -23 (lO^"4)

-8 (10°) -3 (10-⁶)

BTG 1ME -54(10'³⁾ -22(10°)

-37 (lO^"4) -19 (lO^"4)

-27(10-°) -2 (10°)

BTG 1HT -51 (lO^"3) -7(10°)

3 (lO^"4) 38 (lO"⁴)

6(10-°)

BTG 1EH -28 (lO^'3) -4 (10°) 27 (lO^"4)

#Cl_max values. (Chemiluminesence max values)

*M concentration is shown in ().

+ The net effect, i.e. drug-solvent effect is indicated for the neutrophil assay. TABLE 2 - N PROTECTED DIPEPTIDES

Substance Neutrophil Assay# X-XO -/+% -/+%

BTG 2HE -28⁺ (iσv -35 (10-⁴)

-16 (lO ⁶) 14 (10 °)

BTG 2MM -10 (lO^"4) -19 (lO^"4) 0 (10^"°)

BTG 2PH -32 (lO^"4) -14 (10-⁴)

BTG 2PO l^ -4 O0-⁴) 2 (10-°)

#Cl_max values.

*M concentration is shown in ().

+ The net effect, i.e. drug-solvent effect is indicated for the neutrophil assay.

TABLE 3 - 2,5-DIKETOPIPERAZINES

Substance Neutrophil Assay# X-XO -/+% -/+%

BTG 5EE -57⁺ (10°)* -49 (10°) -25 (10-⁴) -18 (10-⁴) 3 (10 ) 29 (lO^"4)

BTG 5PO -5 (10°) -12 (10°) -1 (lO^"4)

BTG 5PH -12 (10°) -1 (10°)

-9 O0-⁴)

1 (lO^"6)

#Cl_max values.

*M concentration is shown in ().

+ The net effect, i.e. drug-solvent effect is indicated for the neutrophil assay.

TABLE 4 - MODEL COMPOUNDS TESTED

Substance Neutrophil Assay# x-xo 1 -/+% -/+%

Captopril -12⁺ (10°)* -75:-56^x (10°) -6 (10"°) -28 (10*)

Glutathione 1 (10°) -98 (10-⁴)

SOD -87 (500 U/ml) -98 (10 U/ml)

Catalase -52 (3000 U/ml) -97 (1000 U/ml)

L-Ascorbate -5 (10°) -100 (104³)

Sodium azide -86 (10°) 13 (10°)

Mannitol -14 (10-') -9 (10°)

x pH: 2.3 and 7.4.

#Cl_maχ values.

*M concentration is shown in ().

+ The net effect, i.e. drug-solvent effect is indicated for the neutrophil assay.

EXAMPLE 27

Lipid peroxidation of rat liver microsomes

The ability of known free radical scavengers/anti-oxidants and BTG compounds 5-E-E,

2-H-E, 1-M-E, 1-P-O, 3-M-M and 1-nona-P-O to inhibit the peroxidation of membrane lipids was determined as follows.

Membrane lipids were supplied in the form of rat liver microsomes. Lipid peroxidation was initiated by the addition of ascorbic acid and ferrous sulphate or ascorbic acid only to the microsomal suspension. An oxygen electrode was used to measure lipid peroxidation as lipid peroxidation is accompanied by an uptake of oxygen [17]

METHODS: Preparation of rat liver microsomes

1-2 rats' livers were extracted, weighed and immersed into ice-cold 0.15M KC1. Livers were minced into small pieces and washed repeatedly with ice-cold 0.15M KG to remove haemoglobin. An equal volume of ice-cold 0.12M KC1, 0.02M Na HPO₄ (pH 6.0) buffer was added to the liver tissue and the tissue was homogenized (to break up cells) using a Polytron homogenizer.

The homogenate was centrifuged 3 times at 10 350g, 4°C for 13 minutes. The resultant supernatant was respun after each centrifugation whilst the remaining pellet (containing cell nuclei, mitochondria and debris) was discarded.

The supernatant after the last centrifugation was then centrifuged twice at 105 OOOg, 4°C for 40 minutes. The supernatant was removed after each centrifugation and discarded.

The remaining pellet (containing microsomes) was resuspended in 0.12M KG,

0.02M Na₂HPO₄ (pH 6.0) buffer.

The microsome suspension was then assayed for protein concentration by the Lowry method [18] and stored at -20°C.

Measurement of Oxygen Uptake bv Rat Liver Microsomes

Pre-warmed microsomes (at 0.25 or 0.5 mg protein/ml in 0.12M KG, 0.02M Na2HPO4 (pH 6.0) buffer) were placed in an equilibrated Clarke oxygen electrode at 37^βC. After equilibration, the test compound or vehicle (DMSO or buffer) was added and incubated with the microsomes for 10 minutes. Lipid peroxidation was initiated with freshly prepared 20 IM FeSO4 and 0.5 mM ascorbic acid or 0.5 mM ascorbic acid only. Oxygen consumption was measured continuously throughout the above period for 25-30 minutes. Oxygen consumption was expressed as nmol O2 consumed/mg protein. Test compounds probucol, vitamin E and BTG compounds were added to the microsomal suspension in DMSO. Glutathione, superoxide dismutase and catalase were added to the microsomal suspension in buffer.

BTG compounds were at 1 mM concentration for 2-H-E, 1-H-C, 1-P-O, 3-M-M, 5-E-E and 0.1 mM concentration for 1-M-E and 1-nona-P-O (as these two compounds were insoluble at higher concentrations). Results were calculated as percentage of the control response and expressed as mean ± SEM.

Statistical analysis of results was performed, where appropriate, using students' paired t-test.

RESULTS: Addition of 20 μm FeSO4 enhanced the effect of ascorbic acid on oxygen uptake by the microsomes. Results obtained were similar to those obtained in identical experiments by Beswick et α/. (1981). [19]

Effect of ve icle PMSQ

Addition of vehicle 25 μl DMSO per ml microsomal suspension caused a slight reduction in oxygen uptake in the presence of 0.5 mM ascorbic acid either with or without 20 μM FeSO4- 12.5 μl DMSO per ml microsomal suspension did not affect oxygen uptake in the presenec of 0.5 mM ascorbic acid and 20 μM FeSO4- Vitamin E, probucol and the BTG compounds were dissolved in DMSO before they were added to the microsomal suspension. The amount of DMSO in the microsomal suspension was 10 μl per ml suspension where lipid peroxidation was initiated by 0.5 mM ascorbic acid and 20 μM FeSO (Tables 5 & 6).

Probucol and 1-nona-P-O would not dissolve in the microsomal suspension unless they were contained in a minimum volume of 25 μl DMSO per ml suspension. Probucol and 1-nona-P-O were tested alongside control samples containing 25 μl DMSO per ml microsomal suspension.

Microsomal Oxygen Untake - Ascorbic acid and FeSO^

Effect of known free radical scavengers/anti-oxidants (Table 5.

1 mM desferrioxamine completely abolished oxygen uptake, probably by chelating the propagating iron species.

10 mM glutathione (GSH) was only observed to inhibit oxygen uptake in microsomes stored at -20°C for less than 48 hours after their preparation.

Table 5: Effect of known free radical scavengers/anti-oxidants (expressed as % control response) on oxygen uptake by rat liver microsomes (0.25 mg protein/ml) by 0.5 mM ascorbic acid and 20 μM FeSO4-

Compound n Integral±SEM Peak±SEM

Superoxide dismutase (100 μg/ml) 2 104.35 108.43

Catalase (100 μg/ml) 2 149.50 170.68

1 mM desferrioxamine* 2 0 0

1 mM vitamin E# 3 85.67± 14.83 85.67±14.73

10 mM GSH+ 2 3.79 5.78

20 mM p-nitrophenol 2 47.37±13.96 52.88±15.25 |

Literature values:-

* 4% control response (for 1 mM) ref: Willis ED (1969) Biochem J 113;325-32

# 79% control response (for 10 μM) ref: Battioni JP, Fontecave M, Jaouen M, Mansay D (1991) Biochem Biophys Res Commun 174(3);1103-08

+ 15% control response (for 10 mM) ref: McCay PB, Brueggeman G, Lai EK,

Powell SR (1989) Ann NY Acad Sci 570;32-45. Effect of BTG compounds

Table 6: Effect of BTG compounds expressed as % control response) on oxygen uptake by rat liver microsomes (0.25 mg protein ml by 0.5 mM ascorbic acid and 20 μM FeSO4

1 Compound n Integral±SEM Peak±SEM |

1 2-H-E 5 98.85 ± 3.26 103.79 ± 3.31

1-H-C 5 90.93 ± 3.79 93.25 ± 3.24

5-E-E 5 92.59 ± 4.27 97.75 ± 4.07

1-M-E 5 106.86 ±18.86 99.45 ±20.01

1-P-O 5 100.79 ±14.91 99.02 ±14.80 Note: 1-M-E tested at 0.1 mM; other compounds tested at 1 mM.

The slight apparent inhibitory effect of 1-H-C was not found to be statistically significant by students' paired t-test using the 'Peak' values.

Vitamin E did not cause much inhibition of lipid peroxidation in our assay (Table 5) although it is one of the main protective agents against lipid peroxidation in v vo.[17] It was therefore decided to retest the effect of vitamin E on microsomal lipid peroxidation in the presence of ascorbic acid without ferrous sulphate. Probucol. shown to prevent lipid peroxidation in vivo [20] and p-nitrophenol were also tested.

Table 7: Effect of vitaman E, probucol and p-nitrophenol (expressed as % control response) on oxygen uptake by rat liver microsomes (0.5 mg protein/ml) by 0.5 mM ascorbic acid.

Compound n Integral±SEM Peak (at l0 min)±SEM

1 mM vitamin E 4 44.47 ± 6.30 44.75± 10.66 50 μM probucol 4 70.74± 10.06 71.52 ± 9.81 20 mM p-nitrophenol 2 38.17 ± 2.90 37.93 ± 2.89

50 μM probucol concentration was chosen as probucol was insoluble at above this concentration

The omission of ferrous sulphate from the assay enhanced the inhibitory effect of vitamin E and p-nitrophenol (Tables 5 and 7) on microsomal oxygen uptake. It was then decided to retest the effect of BTG compounds on microsomal oxygen uptake initiated by ascorbic acid only (Table 8). 3-M-M and 1-nona-P-O were tested in addition to the BTG compounds previously tested. Table 8: Effect of BTG compounds (expressed as % control response) on oxygen uptake by rat liver microsomes (0.5 mg protein ml) by 0.5 mM ascorbic acid.

tested at 0.1 mM: other compounds tested at 1.0 mM.

Microsomal oxygen uptake initiated by 0.5 mM ascorbic acid was inhibited in the presence of 1 mM 5-E-E in two out of three experiments.

CONCLUSION:

Microsomal lipid peroxidation (as measured by oxygen uptake) initiated by 0.5 mM ascorbic acid and 20 μM FeSO4 was unaffected by BTG compounds 2-H-E. 1-H-C, 5-E-E, l-P-O (at 1.0 mM) and 1-M-E and l-nona-P-O (at 0.1 mM) but was inhibited by 1 mM vitamin E and 20 mM p-nitrophenol to 86% and 50% of the control response, respectively.

Omission of 20 μM FeSO4 from the above assay increased the inhibitory effect of 1 mM vitamin E and 20 mM p-nitrophenol to 45% and 38% of the control response, respectively. Microsomal lipid peroxidation initiated by 0.5 mM ascorbic acid only was then measured in the presence of individual BTG compounds 2-H-E, 1-H-C, l-P-O, 3-M-M, 5-E-E (at 1.0 mM), 1-M-E, l-nona-P-O (at 0.1 mM) and 50 μM probucol. Inhibition of lipid peroxidation only occurred in the presence of 1 mM 5-E-E and 50 μM probucol to 76% and 71% of the control response, respectively. LIST OF REFERENCES

[I] J. Honzl and J. Rudinger, Coll. Czechoslov. Chem. Communs. 1961, 26, 2333-2344. [2] S. Goldschmidt and M. Wick, Ann.. 1952, 5 , 217

[3] H. Zahn, E. Schanabel, Liebigs Annalen der Chemie. 1957, 6J& 212-232. [4] A. Srinivasan, R.W. Stephenson and R.K. Olsen, J. Org. Chem.. 1977, 42(131 2253-2256.

[5] S. Yamada and Y. Takeuchi, Tet. letters. 1971, 3595-3598.

[6] E. Schnabel, Liebigs Annalen der Chemie. 1965, ££8, 238-249.

[7] I. Photaki and V. Baradakos, Chem. Communications. 1966, 818. [8] M. Wilchek, C. Zioudrov and A. Patchomik, J. Org. Chem.. 1966, H, 3865-3867.

[9] A. Carlstrom and T. Frejd. Acta Chemica Scandinavica. 1992, 46, 163-171.

[10] M. Szekerke, R. Wade. Acta Chim. Acad. Sci. Hung. 1969,_$20--, 87-93.

[I I] K.D. Kopple and M. Ohnishi, J. Amer. Chem. Soc. 1969, 91(4), 962-970. [12] C.S. Marvel and W.A. Noyes, J. Amer. Chem. Soc. 1920, 42, 2259-2278. [13] G. Losse, R. Wagner, P. Nevland and J. Rateitschak, Chem. Ber. 1958, 9_L

2410-2417. [14] G.A. Maslvaand l.T. Strukov. J. General Chemistry of the USSR 1967, 37(1), 76-78. [15] F.P. Doyle, G.R. Fosker, J.H.C. Nayler and H. Smith, J. Chem. Soc. 1962,

1440-1444. [16] S. Yamada. S. Terashima and M. Wagatsuma, Tet. Letters. 1970, IS, 1501 -1504. [17] B. Halliweli and J.M.C. Gutteridge (1985) 'Free Radicals in Biology and Medicine,

Clarendon Press, Oxford p.162. [18] O.H. Lowry, N.H. Rosebrough, A.L. Farτ and R.J. Farr (1951) J Biol Chem 193;265 [19] P.H. Beswick, K. Cheeseman, G. Poli and T.F. Slater (1981) 'Comparison of methods ued for measuring lipid peroxidation in rat liver microsomes and isolated hepatocytes' from 'Recent advances in lipid peroxidation and tissue injury' London:

Brunei University Press p.156-77. [20] B. Kalyanaraman, V.M. Darley-Usmar, J. Wood, J. Joseph and S. Parthasarathy

(1992) J Biol Chem 267(10;6789-95.

Claims

1. Use of a thiol group free compound comprising one or more α-amino acid residues of general formula (I):

R¹ R²

\ / C (I)

/ \ - NR CO -

wherein R is selected from the group consisting of hydrogen, optionally substituted Ci -C20 alkyl, Ci -C20 alkenyl, aryl and aralkyl groups and a group R^-OCO- where R is selected from Cj-C2o- 1-C20 alkenyl, aryl and aralkyl groups;

R* and R² are independently selected from the group consisting of hydrogen and optionally substituted C_j-Cg alkyl, Cj-Cg alkenyl, C7-C12 arylalkyl and aryl groups;

or R* and R² together represent a group =CR"R', wherein R" and R' are independently selected from hydrogen, halogen, and optionally substituted C \ -Cg alkyl and aryl groups; or a pharmaceutically acceptable salt or physiologically functional derivative thereof in the preparation of a medicament suitable for the treatment of a disease state caused by production of free radical species or in which free radical species are produced.

2. Use as claimed in claim 1 wherein at least one of R'and R² is a non-thiol group - (CH2)_n-L, where n is an integer from 1 to 3 and L is a leaving group or a group -O-L*, -S- L * , -CO-L^ or -CS-L* where L' is a leaving group wherein L and L* are selected from those groups which leave under physiological conditions on interaction of the compound with a free radical.

3. Use as claimed in claim 1 or claim 2 wherein the compound does not contain methionine.

4. Use as claimed in any one of the preceding claims wherein in each case the optional substitution is independently with CI, Br, F, I, OH, -S-alkyl where alkyl is C_j-Cg alkyl, -S-alkenyl where alkenyl is C₂-C₆, -SC(O)C₆H₅; -SC(O)C₆H₅-CH₃, -O-SO₂C₆H₅, -O- SO₂C₆H₅-CH₃, -COOCH₂C₆H₅ or -COOCH₂CH₂C₆H₅;

5. Use as claimed in any one of the preceding claims wherein one or more of the o- amino acid residues are in their D-isomeric form.

6. Use of a thiol free compound as claimed in any oneof claims 1 to 5 wherein the compound is of general formula (Ia)

R¹ R² \ /

C (la)

wherein o and p are each 0 or an integer, and the sum of o + p + 1 is no more than 500;

A' is selected from the groups

R¹ R² R¹ R² R¹ R² R¹ R²

\ / \ / \ / \ /

/ \ / \ / \ / \

- NR CO- -NR CH₂- -CH₂ CO- -CH₂ CH₂-

R¹ R² R¹ R² Rl R² R¹ R²

\ / \ / \ / \ / C ; C ; C and C

/ \ / \ / \ / \ CO- CH₂- -CH₂ -NR is selected from the groups

R¹ R² R¹ R² Rl R² R¹ R²

\ / \ / \ / \ / C ; C ; C ; C

/ \ / \ / \ / \ NR CO- -NR CH₂- -CH₂ CO- -CH₂ CH₂- Rl R² Rl R² Rl R² Rl R²

\ / \ / \ / \ / C C C and C

/ \ / \ / \ / \ -NR -CH₂ CH - RN-

Y is hydroxy or optionally substituted, Ci -Cg alkyl, C_j-Cg alkenyl, C_j-Cg alkoxy, C_j-Cg alkenylyoxy, arylalkoxy, phenyl, phenyloxy group, amino or alkylamino group;

Z and R are independently selected from the group consisting of hydrogen, optionally substituted C1-C20 alkyl, C2-C20 alkenyl, aryl and arylalkyl groups, and a group R^-OCO- wherein R^ is selected from C1-C20 alkyl, C2-C20 alkenyl, aryl and arylalkyl; or is XΗ2⁺ where X is halogen; or Z and Y together represent a single covalent bond; and

Rl and R², for each repeat unit of the o or p units, are independently selected from the group consisting of hydrogen and optionally substituted Cj-Cg alkyl, C_j-Cg alkenyl, C7-C12 arylalkyl and aryl groups; or Rl and R² together represent a group =CR R⁷, wherein R" and R ' are independently selected from hydrogen, halogen, and optionally substituted C _j -Cg alkyl and aryl groups;

or a pharmaceutically acceptable salt thereof.

7. Use as claimed in claim 6 wherein Z and R are independently selected from hydrogen, C_j -Cg alkyl, C2-Cg alkenyl, Cg aryl and C7-C12 arylalkyl.

8. Use as claimed in any one of the preceding claims wherein the compound is of formula (lb):

wherein: m is an integer from 1 to 500;

and Y, Z and R, Rland R² are as defined above in connection with formula (Ia);

9. Use as claimed in any one of the preceding claims wherein the compound is a free or protected α-amino acid, peptide or peptide analogue.

10. Use as claimed in any one of claim 2 to 9 wherein the sum of (o + p + 1 ) or m is an integer of from 2 to 100.

1 1. Use as claimed in claim 9 wherein the sum of (o + p + 1 ) or m is from 2 to 10.

12. Use as claimed in claim 9 wherein the sum of (o + p + 1) or m is 2 or 3.

13. Use as claimed in any one of the preceding claims wherein the peptide is a cyclic peptide.

14. Use as claimed in any one of claims 2 to 13 wherein Z and Y form a covalent bond.

15. Use as claimed in any one of the preceding claims wherein the compound is a dipeptide of formula (II) Rl R²

\ / C NH CO (II)

/ \ / \ / \ ZNH CO C Y / \

R3 R⁴ wherein

R and R-⁵ are independently selected from H, CgH5, C_j_g alkoxy-substituted phenyl, e.g. p-CH3OCgH4 and optionally substituted Cj-Cg alkyl or C .12 arylalkyl; wherein optional substituents are independently selected from CI, Br, F, I, OH, -S-alkyl where alkyl is C_j-Cg alkyl; -S-alkenyl where alkenyl is C2-Cg alkenyl, -SC(O)CgH₅, -OSO₂-p- CgH₅CH₃, -COOCH₂CgH₅ , -COO(CH₂)gCgH₅ and -COOCH₂CH₂C₆H₅;

R² and R⁴ are independently selected from H and optionally substituted Ci -Cg alkyl wherein optional substituents are independently selected from CI, Br, F, I, OH, -SC(O)C₆H₅, -OSO₂p CgH₅-CH₃, -COOCH₂CgH₅. -COO(CH₂)gCgH₅ and -COOCH₂CH₂CgH5; or

Rl and R² together and/or R- and R⁴ respectively together represent =CH2;

Y represents an amino group or a group OR° where R° is independently selected from H or C_j-Cg alkyl;

and Z represents R^OCO wherein R is independently selected from CJ-C20 alkyl (preferably C1-C5 alkyl), phenylalkylcarbonyloxy, e.g. CgH₅CH₂OCO, C9H19OCO; or a hydro-halo salt; or Y and Z together represent a single covalent bond.

16. A thiol group free compound comprising one or more α-amino acid residues of general formula (I):

Rl R² \ /

C (I)

/ \

- NR CO -

wherein R is selected from the group consisting of hydrogen, optionally substituted C1-C20 alkyl, C _j -C20 alkenyl, aryl and aralkyl groups and a group R^-OCO- where R^ is selected from Cι-C20_> 1-C20 alkenyl, aryl and aralkyl groups;

Rl and R² are independently selected from the group consisting of hydrogen and optionally substituted C_j-Cg alkyl, C_j-Cg alkenyl, C7-C12 arylalkyl and aryl groups; wherein at least one of Rl and R² has a substituent selected from CI, Br, F, I, -SC(O)CgH5, -SO₂-p-C₆H₅-CH3, -COOCH₂CgH₅, -COOCH₂CH₂CgH5, -COO(CH₂)gCgH₅ or Rl and R² together represent a group =CR"R⁷, wherein R^ and R' are independently selected from hydrogen, halogen, and optionally substituted Ci -Cg alkyl and aryl groups;

or a pharmaceutically acceptable salt thereof for use in therapy.

17. A compound as claimed in claim 16 wherein the compound is a cyclic peptide.

18. A compound as claimed in claim 16 wherein the compound is a diprotected peptide.

19. A compound as claimed in claim 16 wherein the compound is an N-protected peptide.

20. A compound as claimed in claim 16 wherein the compound is a C-protected peptide.

21. A compound as claimed in claim 16 wherein the compound is a 2.5-diketopeperazine.

22. Novel compounds of general formula (lb).

Rl R²

\ /

C Y (lb)

/ / \\ // Z - -NR CO- m

wherein:

m is an integer from 1 to 500;

and Y, Z and R, Rland R² are as defined in claim 6.

23. A compound as claimed in claim 22 wherein Rl and R² together represent =CHR R⁷, wherein R" and R ' are independently selected from hydrogen, halogen, and optionally substituted Ci-Cg alkyl and aryl groups;

24. A compound as claimed in claim 22 wherein m is an integer from 2 to 10.

25. A compound as claimed in claim 22 wherein: R is independently selected from H, CH₃ and C H5;

R² is independently selected from H, CH₃, and CH2OH; R³ is independently selected from H, -CH OH, and -CH OSO2-p-CgH₅-CH₃; or R³ and R⁴ together represent =CH2; and

R⁴ is H.

25. A compound as claimed in claim 22 being thiol group free wherein at least one of Rl and R². or R³ and R⁴, is a group -(CH2)_n- , where n is an integer from 1 to 3 and L is a leaving group or a group -0-L . -S-Ll, -CO-LI or -CS-L1 where Li is a leaving group wherein L and L' are selected from those groups which leave the compound under physiological conditions on interaction of the compound with a free radical species.

26. A compound as claimed in claim 25 wherein L is a halogen atom or L 1 is hydrogen, an arylsulphonylalkyl group, e.g. tosyl or -COC H5, an arylalkyl, eg.-OCH2CgH5 or - OCH₂CH₂CgH5. or. -SCOC₆H₅.

27. N-carbobenzyloxy-DL-alanine-Δ-alanine methyl ester [1ME].

28. N-carbobenzyloxyglycyl-O-tosyl-L-serine methyl ester [1HT].

29. N-carbobenzyloxyglycyl-Δ-alanine [2HE].

30. N-carbobenzyloxy-DL-2-phenylglycyl-DL-serine [2PO].

31. 3,6-Bis-methylene 2,5-piperazinedione [5EE].

32. N-carbobenzyloxyglycyl-Δ-alanine methyl ester [1HE].

33. N-carbobenzyloxy-DL-2-phenylglycyl-Δ -alanine methyl ester [ 1 PE] .

34. N-carbobenzyloxy-Δ -alanine methyl ester [1EE].

35. A method for the treatment of free radical molecule generating and/or free radical molecule caused disease comprising the administration of a clinically useful amount of compound of formula (I), (Ia) or (lb) or a pharmaceutically acceptable salt or physiologically functional derivative thereof in a pharmaceutically useful form.

36. A pharmaceutical formulation comprising a compound as described in any one of the claims 1 to 16 or a pharmaceutically acceptable salt or physiologically functional derivative thereof together with a pharmaceutically acceptable carrier therefor.

37. A method for the preparation of a pharmaceutical formulation comprising bringing into association a compound as described in any one of claims 1 to 16 or a pharmaceutically acceptable salt or physiologically functional derivative thereof, and a pharmaceutically acceptable carrier therefor.