NITROGEN MUSTARD PRODRUGS WITH NOVEL LIPOPHILIC PROTECTING GROUPS, AND PROCESSES FOR THEIR PRODUCTION

Abstract: The invention provides compounds of formula (I) and (II) wherein X and Y are independently chlorine, bromine, iodine, a mesyl group CH3SO3 or a tosyl group OSO2phenyl (wherein phenyl is optionally substituted by 1, 2, 3, 4 or 5 substituents independently selected from C1-4alkyl, halogen, cyano or nitro; R<1> and R<2> are independently 1 to 4 optional substituents; Z<1> and Z<2> are each independently -O- or -NH-; R<3> is hydrogen, <u>t</u>-butyl or allyl; Z<3> is a hydrocarbyl group such as carboxyethyl, optionally containing heteroatoms, and physiologically acceptable derivatives thereof. The compounds can be converted in situ into nitrogen mustard agents by the actions of enzymes such as carboxypeptidase or nitroreductase and are useful for the treatment of cancer.

Description: NITROGEN MUSTARD PRODRUGS WITH NOVEL LIPOPHILIC PROTECTING GROUPS. AND PROCESSES FOR THEIR PRODUCTION The present invention relates to prodrugs and their use in the treatment of disease, including cancers. The use of prodrugs represents a clinically very valuable concept in cancer therapy since, particularly where the prodrug is to be converted to an anti-tumour agent under the influence of an enzyme that is linkable to a monoclonal antibody that will bind to a tumour associated antigen, the combination of such a prodrug with such an enzyme monoclonal/antibody conjugate represents a very powerful clinical agent. This approach to cancer therapy, often referred to as "antibody directed enzyme/prodrug therapy" (ADEPT) is disclosed in WO88/07378. More recently, a similar approach ("GDEPT", Gene-Directed Enzyme Prodrug Therapj ) has been proposed where in place of an antibody/enzyme conjugate, tumour cells are targeted with a viral vector carrying a gene encoding an enzyme capable of activating a prodrug. The gene may be transcriptionally regulated by tumour specific promoter or enhancer sequences. The viral vector enters tumour cells and expresses the enzyme, in order that a prodrug is convened to an active drug only in the vicinity of the tumour cells (Huber et al. Proc. Natl. Acad. Sci. USA (1991) 88, 8039). The enzyme may be targeted to paπicular subcellular locations or expressed at the cell surface. T e present invention provides novel prodrugs which contain protecting groups which are more lipophilic than compounds of the prior art such as those disclosed in WO88/07378. Accordingly, the present invention provides compounds of the formula (I): wherein X and Y are independently chlorine, bromine, iodine, a mesyl group CH 3 SO 3 or tosyl group OSO 2 phenyl (wherein phenyl is optionally substituted by 1 , 2, 3, 4 or substituents independently selected from C alkyl, halogen, cyano or nitro: n is an integer from 0 to 4 and each group R\ which may be the same or different when is greater than 1. is fluorine, chlorine, bromine, iodine, hydroxy. mercapto. amino, nitro, C,_ alkyl optionally containing one double bond or one triple bond, C M alkoxy, -CONR 7 R (wh in R 7 and R 8 are as defined below), -NH(C -alkyl), -N(C M -alkyl), or C 5 altaαoyl; or when n is 2 or more and two groups R 1 are on adjacent positions on the phenylene ring, 2 adjacent R 1 groups together represent a) C4 alkylene optionally having one double bond; b) C3 alkylene: or c) -CH = CH-CH = CH-. -CH=CH-CH ; - or -CH,-CH=CH- each optionally substituted wit 1 , 2, 3 or 4 substituents said substituents each independently selected from the grou consisting of C M alkyl, C alkoxy, halogen, cyano, nitro, C 2 . 5 alkanoyl and -CONR7R ''wherein R7 and R8 are as defin d below): m is an integer from 0 to 4 and R 2 , which may be the same or different when m is greate than 1 , is fluorine, chlorine, bromine, iodine, hydroxy, mercapto, amino. nitro, C alkyl optionally containing one double bond or one triple bond, C,_ 4 alkoxy, -CONR 7 R 8 (wherei R 7 and R 8 are as defined below), -NH(C -alkyl), -N(C -alkyl) 2 and C 2 5 alkanoyl; or whe n is 2 or more and two groups R 1 are on adjacent positions on the phenylene ring, 2 adjacen R 1 groups together represent a) C4 alkylene optionally having one double bond; b) C3 alkylene; or c) -CH=CH-CH=CH-, -CH=CH-CH 2 - or -CH 2 -CH=CH- each optionally substimted with 1, 2. 3 or 4 substituents said substituents each independently selected from the group consisting of C alkyl, C alkoxy, halogen, cyano, nitro, C 2 . s alkanoyl and -CONR7R8 (wherein R7 and R8 are as defined below); 7} and Z 2 are each independently -O- or -NH-: R 3 is hydrogen, t-butyl or allyl; Z 1 ι< eroup -V-W where V is -CH-.-T- in which T is -CH,-, -O-, -S-, (SO)- or -(SO,> (provided that when V has sulphur or oxygen as its second atom, W is other than -COOH) and said group V optionally further carrying one or two substituents Ql and/or Q2 on carbon; wherein Q 1 and Q 2 each independently represents C M alkyl or halogen; or, when Ql and Q2 are bonded to adjacent carbon atoms, Q 1 and Q : together may additionally represent a C,-C 4 alkylene radical optionally substimted with 1. 2, 3 or 4 substituents independently selected from the group consisting of C,., alkyl and halogen and W represents (1) COOH, (2) -(C = O)-O-R6 wherein R6 represents a C alkyl, C 3 . 6 cycloalkyl or aryl (as defined in 3 below) group; (3) -(C=O)-NR7R8 wherein R7 and R8 each independently represent hydrogen or a Cl- 6 alkyl, C3-6 cycloalkyl, aryl. heteroaryl linked to N via carbon or C7-9 aralkyl group wherein aryl is phenyl; heteroaryl is a 5 or 6 membered ring containing 1 to 3 heteroatoms selected from the group consisting of nitrogen and sulphur; the aryl moiety pet se. the heteroaryl moiety and the aryl moiety of the aralkyl group may be substimted on carbon with 1-4 substituents selected from the group consisting of -COOH, -OH, -NH ; , -CH 2 -NH 2 , -(CH2) -COOH. tetrazol-5-yl and -SO 3 H and the alkyl moiety may optionally carry a methyl group; (4) -SO,NHR9 wherein R9 is as defined for R7 but may additionally represent -CF 3 , - CH 2 CF 3 or aryl as defined above; (5) SO : ,R10 in which RIO represents H. C w alkyl or C 3 . 6 cycloalkyl, (6) PO 3 R10R10 (wherein the RIO radicals, which may be the same or different, are as herein defined) ' "r , a tetraz l-5-yl group: (8) -CONH-SO 2 Rl l in which Rl l represents (a) C 3 . 7 cycloalkyl; (b) C.^-alkyl optionally substimted with substituents selected from the group consisting of aryl as defined below, C -alkyl, CF 3 or halogen; and (c) perfluoro-C,. alkyl; wherein aryl is phenyl or phenyl having 1-5 substituents wherein the substituents are selected from the group consisting of halogen, -NO 2 . -CF 3 , C,. alkyl. C M alkoxy. -NH 2 , -NHCOCH 3 , -CONH , -OCH 2 COOH, -NH(C -alkyl), -N(C,. 4 -alkyl) 2 , -NHCOOC alkyl, -OH, -COOH, -CN and -COOC M alkyl; and (9) -M-Het wherein M represents S, SO or SO 2 and Het represents a 5 or 6 membered heterocyclic aromatic ring linked to M via a carbon atom of the aromatic ring, said aromatic ring containing 1, 2, 3 or 4 heteroatoms selected from the group consisting of O, N and S said aromatic ring optionally being substituted on carbon atoms of the ring with 1. 2. 3 or 4 sub timents selected from the group consisting of -OH. -SH. - CN, -CF 3 , NH, and halogen; and physiologically acceptable derivatives of the compound of formula (I). These prodrugs can be activated at the site of a tumour by a carboxypeptidase enzyme, for example, CPG2 as disclosed in WO88/07378 or WO94/02450. The carboxypeptidase enzyme may be delivered to the site of a tumour by a GDEPT system as described in the prior art. A carboxypeptidase enzyme is capable of removing the glutamic acid or glutamic acid- derivative moiety (which are preferably in the L-configuration) of compounds of the formula (I). While not wishing to be bound by any one particular theory, it is believed the removal oi glutamic acid moiety from the compounds produces an unstable intermediate compound which undergoes self-immolation to remove the 4-amino or 4-hydroxyl-benzylic system (simated between the groups Z 1 and Z 2 ) thus leading to the release of the alkylating nitrogen mustard drug. CPG enzyme is available from Sigma Chemical Company, Fancy Road, Poole, Dorset, U.K. CPG enzyme was described in: Goldman, P. and Levy. C.C., PNAS USA. 58: 1299-1306 (1967) and in: Levy, C.C. and Goldman P., J. Biol. Chem., 242: 2933-2938 (1967). Carboxypeptidase G3 enzyme has been described in Yasuda, N. et al.. Biosci. Biotech. Biochem., 56: 1536-1540 (1992). Carboxypeptidase G2 enzyme has also been described in European Patent 121 352. Large scale purification of CPG2 from Pseudomonas RS-16 was described in Sherwood et :! " "SS), Eur. J. Biochem., 148, 44^-453. PG2 may also be obtained by recombinant techniques The nucleotide coding sequence for CPG2 has been published by Minton, N.P. et al.. Gene, 3_i (1984), 31-38. Expression of the coding sequence has been reported in E.coli (Chambers, S.P. et al., Appl. Microbiol, Biotechnol. (1988), 29, 572-578) and in Saccharomvces cerevisiae (Clarke, L. E. et al., J. Gen Microbiol, (1985) 131, 897-904). Total gene synthesis has been described by M. Edwards in Am. Biotech. Lab (1987), 5. 38-44. The present invention further provides nitrogen mustard prodrugs of the formula (II): wherein X, Y, R 1 , n, Z . R and m are as defined for compounds υf the formula (I), and physiologically acceptable derivatives thereof. These prodrugs can be activated at the site of a mmour by a nitroreductase enzyme, for example, as disclosed in WO93/08288. The nitroreductase enzyme may be delivered to the site of a mmour by a GDEPT system as described in the prior art. Physiologically acceptable derivatives of said prodrugs of formulae (I) and (II) include salts. esters and salts of esters. Esters include carboxy lie acid esters in which the non-carbonyl iioιeι> r the e->ter grouping is selected from straight or branciied chain C,. 6 alkyl. (methyl. n-propyl, n-butyl or t-butyl); or C-^cyclic alkyl (e.g. cyclohexyl). Salts inclu physiologically acceptable base salts, eg derived from an appropriate base, such as alk metal (e.g. sodium), alkaline earth metal (e.g. magnesium) salts, ammonium and N (wherein R is C M alkyl) salts. Other salts include acid addition salts, including t hydrochloride and acetate salts. Preferred values of X and Y are independently chloro, iodo and mesyl. Preferably, n either is zero or is 1 or 2 and the group(s) R l are independently fluorin chlorine, bromine, iodine or C M alkyl. Similarly, it is preferred that either m is zero or 1 or 2 and the group(s) R 2 are independently fluorine, chlorine, bromine, iodine or C ilk R 3 is preferably hydrogen. V is preferably a group -V-W where V is -CH 2 CH : - and W is a group -COOH, -(C=O)-O where R" i<* a? defined above, or -(C = O)-NR 7 R R where R 7 and R 8 are indepen ent hydrogen or C-. 6 alkyl. Preferred compounds of the formula (I) are those in which Z\ Z 2 , n and m are as defin above, and: X and Y are independently selected from chloro. iodo or mesyl; each phenylene group which may be optionally substimted by R 1 and R 2 is unsubstimted if either is substimted. the group(s) R 1 and/or R : are independently selected from fluorin ch'orine. bromine. ; ndinε or C . alkyl: R 3 is hydrogen; and Z 3 is a group -CH 2 CH 2 -COOH. Compounds of the formula (I) which are especially preferred are those in which X and independently represent iodo or chloro; n and m are both zero; R, represents hydrogen; Z, and Z, each independently represent -NH- or -O-; and represents CH,CH : COOH. Preferred compounds of the formula (II) are those in which Z 1 is -O- or -NH-, X and Y are The independently selected from iodo and mesyl, each phenylene group which may be optionally substimted by R 1 and R 2 is unsubstimted or if either is substimted, the group(s) R 1 and/ or R 2 are independently selected from fluorine, chlorine, bromine, iodine or C,^ alkyl, m ut preferably fluorine, and in which case n and/or m are 1 or 2 Compounds of the formula (II) which are especially preferred are those in which X represents iodo; n represents zero; Z 1 represents -O- or -NH-; R 2 represents hydrogen or fluorine: and m represents an integer from 1 or 2. The invention further provides pharmaceutical formulations. Such formulations comprise a compound of formula (I) or (II) together with one or more pharmaceutically acceptable carriers or diluents. Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral or parenteral (e.g. intramuscular or intravenous) administration. The formuk' ions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. For example, formulations suitable for parenteral administration include aqueous and non¬ aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostatis and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the polypeptide to blood components or one or more organs. Suitable liposomes include, for example, those comprising the positively charged lipid (N[l- (2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA), those comprising dioleoyl- phosphatidylethanolamine (DOPE), and those comprising 3/3[N-(n',N'-dimethylaminoethane) carbamoyl]cholesterol (DC-Choi) . In GDEPT the prodrug will usually be administered following the administration of a deliver "-ystem containing t e gene encoding the enzyme. This includes administration of a modifie virus encoding an enzyme. Typically, the virus will be administered to the patient and the the uptake of the virus by infected cells monitored, for example by recovery and analysis o a biopsy sample of targeted tissue. A wide range of viral delivery systems for gene therapy are known in the art. For exampl EP-A-415 731 describes molecular chimeras comprising a promoter which may be activate in a mmour cell is operably linked to a heterologous gene encoding an enzyme capable o converting a prodrug into a cytotoxic agent. Such molecular chimeras may be used to expres carboxypeptidase or nitroreductase in mmour cells, in order to activate prodrugs of th invention. EP-A-415 731 describes incorporation of such molecular chimeras into vira vectors, e.g. adenoviral υr retroviral vectors. Such viral vectors may also be adapted fo utilization in the present invention. Other recombinant viral vector delivery systems are described in WO91/02805. WO92/14829 WO93/10814, WO94/21792, WO95/07994 and WO95/14091 the disclosures of which ar incorporated herein by reference. Methods for producing vector delivery systems based o the above-mentioned disclosures may be used to deliver vectors encoding nitroreductase o carboxypeptidase to target cells. The exact dosage regime for GDEPT will, of course, need to be determined by individua clinicians for individual patients and this, in turn, will be controlled by the exact namre o the prodrug and the cytotoxic agent to be released from the prodrug but some genera guidance can be given. Chemotherapy of this type will normally involve parentera administration of both the prodrug and the enzyme or a vector encoding the enzyme an administration by the intravenous route is frequently found to be the most practical. In GDEPT systems, the amount of vector delivered will be such as to provide an effectiv . llular concentration of so i at ihe prodrug may be activated in sufficien concentrations at the site of a mmour to achieve a therapeutic effect, e.g. reduction in the mmour size. This may be determined by clinical trials which involve administering a range of trial doses to a patient and measuring the degree of infection or transfection of a target cell or mmour. The amount of prodrug required will be similar to or greater than that for ADEPT systems nf the type mentioned above. For example, suitable dose of prodrugs in such systems are described in Bagshawe et al, 1991 in Antibody, Immunoconjugates and Radio-Pharmaceuticals I. 915-922. Such doses eg. from 5 to 2000 mg/n (eg about 200 mg/m 2 per dose (eg every one or two days or weekly) of prodrugs of the invention may be used, although doses outside this range may be used at the discretion of the physician. The present invention also provides a system for use in the control of neoplasia in a human or animal subject comprising a vector or other delivery system capable of selectively infecting mmour cells in said subject, said vector carrying a DNA or RNA sequence encoding an enzyme, in association with a prodrug of formula (I) or (II) capable of being converted to a nitrogen mustard compound by the action of said enzyme. The present invention extends to a method of treating neoplasia in a human or animal subject requiring such treatment which comprises administering to the host an effective amount of a prodrug of the invention and a modified virus, said modified virus capable of selectively infecting mmour cells in said subject, said virus carrying a DNA or RNA sequence encoding an enzyme capable of converting said prodrug to an active nitrogen mustard compound. The present invention also extends to a method of treating neoplasia in a human or animal subject requiring such treatment which comprises administering to the host an effective amount of a prodrug of the invention and a non viral vector system, said non-viral vector system capable of being selectively introduced into mmour cells in said subject, said vector system carrying a DNA or RNA sequence encoding an enzyme capable of converting said prodrug to an active nitrogen mustard operably linked to a promoter effective in expressing said enzyme in said cells. Vectors capable of selectively infecting mmour cells are known in the art. By "selectively infecting" it is meant that the vector, for example a modified virus, will primarily infect u.u. . cells and that die proportion of non-tumour cells infected is such that the damage to non-mmour cells by administration of the prodrug will be acceptably low, given the namr of the disease being treated. Ultimately, this will be determined by the physician. It will also be understood that the DNA or RNA sequence encoding an enzyme carried by th virus will be linked to suitable expression control signals such that expression of the enzym will occur in the targeted mmour cells. Non- viral vector systems will be capable of being selectively introduced into tumour cell utilizing methods such as those mentioned above, e.g. calcium phosphate co-precipiu..tion microinjection, liposomes, direct DNA uptake, and receptor-mediated DNA transfer (Morga & French Anderson, Annu. Rev. Biochem., 1993,62; 191). Prodrugs of the present invention may also be used as reagents in in vitro systems to test th activity of candidate enzymes which may be incorporated into GDEPT systems. For example, a tumour cell mas be infected or transfected in vitro by a vector containing a enzyme. The enzyme will be one which is, or suspected to be, capable of converting prodrug of the invention into an active drug. The prodrug is then added to the culture an the amount of cell killing or inhibition of cell growth is measured (for example by using vital stain to record the number of viable cells or by replating a sample of the culture to cou the number of viable cells). This model system may also be used to test the toxicity, activi or selectivity of compounds of the present invention against a range of mmour cell type suitable mmour cells for use in these model systems include ovarian, colon, glioma, breast _.mu.' .ell -i d non-small cell lung cancer cells, and melanomas. The invention further provides processes for the production of compounds of formulae (I) an (II). Thus the invention provides a process for the production of a compound of formula (I as defined above which comprises reacting a compound of the formula (III) in which X, Y, R 1 and n are as defined for formula (I) and Z 4 is -O- or -NH- with a compound of formula (IV) in which R 2 . m. Z 2 . Z 3 and R 3 are as defined above, and Q is a leaving group. The reaction may be performed in an aprotic solvent and in the presence of a base. The invention also provides a process for the production of compounds of the formula (II) which comprises reacting a compound of formula (III) as defined above with a 4-nitrobenzyl chloroformate optionally substimted with from 1 to 4 group(s) R 2 as defined above. The invention further provides compounds of the formula (IV); in which R 2 , m, Z 2 , R 3 and Z 3 are as defined above for compounds of the formula (I), an Q is a leaving group, with the proviso that when R 2 is fluorine, chlorine, bromine, iodin hydroxy, mercapto, amino, nitro, C alkyl, C haloalkyl, C alkoxy, C haloalkoxy, C 2 lkenyl, C 2 _. alkynyl. and C _, hal-^lkenyl. R 3 is hydrogen or a C , . υ alkyl group, and Z 3 is group -CH 2 CH,COOX 10 where X 10 is hydrogen or a Cl-6alkyl group, then Q is not a N ---uccinimidyloxy group. These compounds are useful as linkers which may be attached t active cytotoxic compounds to provide prodrugs of said compounds with inci ease lipophilicity. The compounds of formula (IV) may be linked to such cytoxic compounds vi free -OH or -NH 2 groups on such compounds, using methods and reactions described below or modified using routine experimentation. Such protected cytotoxic compounds may be useful in GDEPT methods, or in vitro assay used to studv such methods Brief Description of the Drawings Figure 1 provides a reaction scheme used to make the compounds described in Example 9 Figure 2 provides a reaction scheme used to make the compounds described in Example 15 Figure 3 provides a reaction scheme used to make the compounds of the invention describe in Example-. 1. 2, 3 and 4. ' Compounds of the invention may be made using reactions and methods known per se in th art of chemistry. The following methods are of particular use. and are further illustrated b reference to the Examples where indicated. Such reference does not limit the general reactio schemes to those compounds alone. The schemes may be used for other compounds of th invention: A: Compounds of formula (I) where Z' is -O-: (i) Compounds of formula (I) in which Z 1 = -O- and Z 2 = -NH-, may be prepared by the reaction of a nitrogen mustard of formula (III) wherein X. Y, R 1 and n are as defined above and Z 4 is -O- with a linker of formula (IV) where R 2 , m. R 3 and Z 3 are as defined above, Z 2 = -NH- and Q is a leaving group. Preferred leaving groups Q include a succinimidyloxy group, a 4-nitrophenyloxy group, a pentafluorophenyloxy group and a tetrachoroethyloxy CH(C1)CC1 3 group. The reaction may be carried out in aprotic solvents in the presence of a base, for example THF and triethylamine or acetonitrile and basic resins (Example 1). (ii) Compounds of the formula (III) may be made starting from 4-nitrophenol ■ (' optionally substimted with the group(s) R' (n , (a., d fined above). The phenolic group is protected as an adamantanyloxycarbonyl-derivative (by reacting the starting materials wi adamantanyl-fluoroformate and triethylamine in THF at rt). The protected 4-nitrophen carbonate is reduced to the corresponding amine by hydrogen transfer in ethanol usi ammonium formate and Pd/C 10% as catalyst at rt. The amine is then hydroxyethylated wi ethylene oxide in AcOH at 20°C and then reefed to the desired nitrogen mustard. Referen may be made to EP-A-433 360 or EP-A-490970 for suitable conditions. The compounds m be purified by column chromatography Deprotection to remove the adamantyl group m be carried out in trifluoroacetic acid. (iii) Alternatively, the nitrogen mustard of formula (III) may be activated as chloroformate by treatment with phosgene or triphosgene in an aprotic solvent a triethylamine followed by coupling with a compound of formula (V): where R 2 . m. Z\ R 3 and Z 3 are as defined above. This may be carried out in THF or oth aprotic solvents in the present of a base for example triethylamine or pyridine). (iv) A further alternative route of synthesis of compounds of the formula (I) in which is -O- involves direct coupling of 4-nitrophenol optionally substimted with the group(s) R' (as defined above) with the compound of the formula (IV) or by reaction of the sa optionally substimted 4-nitrophenol compound chloroformate with the compound of formu (VI). in which Z 2 is -NH- (see below) followed in each case by the reaction described abo to convert the nitro group, via an amine. to a mustard group. (v). A way of obtaining compound (I) where Z 1 = -O- and Z 2 = -O- is from nitrogen mustard (III) where Z 4 = -O-, by transforming it to the trimethylsilyl derivative. The silylated phenol is then coupled with a compound of formula (V) where Z 2 = -O-. Compound (V) is first activated as the fluoroformate with KF, phosgene and crown ether at -78 °C, in an aprotic solvm '- * ι?e Example 2). prior to reaction with silyated phenol. B: Compounds of formula (I) where Z 1 is -NH-: (i) Compounds of the formula (I) in which Z 1 is -NH- may be made by reaction of a compound of formula (III) in which Z 4 is -NH- with a linker of the formula (IV) in aprotic solvents and in the presence of a base. Compounds of the formula (III) in which Z 4 is -NH- may be made from a l-halo-4-nitrophenyl compound, optionally substimted with the group(s) R' (n) (as defined above). This is converted to the corresponding l-bis-hydroxyethylamino-4- nitrophenyl compound by reaction with diethanolamine with heat and the resulting product purified by column chromatography. The corresponding 4-nitro nitrogen mustard may be made by for example mesylation using mesyl chloride in pyridine and subsequent reaction to other halo mustards, e.g. bromo or iodo mustards if required. The 4-nitro group may be reduced by hydrogen transfer in ethanol using ammonium formate and a Pd/C 10% catalyst at 20° C. (ii) Alternatively the l-bis-hydroxyethylamino-4-nitrophenyl compound mentioned above can be reduced using ammonium formate and Pd/C 10% as catalyst in ethanol at 20°C to provide the corresponding phenylene-diamino derivative. This derivative can be converted into the corresponding 1 -. * itiino nitrogen mustard as described in the above paragraph, e.g. initially by reaction with mesyl chloride. (iii) In an alternative route, the nitrogen mustard (III) Z 4 = -NH- may be activated as an isocyanate by treatment with phosgene or triphosgene in an aprotic solvent followed by coupling with a compound of formula V where R 2 , m, Z 2 , R 3 and Z 3 are as defined above. The coupling may be carried out in aprotic solvents and in the presence of a catalyst, for example dibutyltin dilaurate (see Examples 3 and 4). C: Compounds of formula (II): (i) Compounds of the formula (II) may be obtained by coupling nitrogen mustard phenol compounds described in section A(ii) above with 4-nitrobenzyl choloroformate "■rnionally substimted with hp gmup(s) R 2 (n , (as defined above) in the presence or absence of triethylamine at 20°C. This is used to provide compounds of formula (II) in which Z 1 is -O- (ii) Alternatively aniline nitrogen mustards as described in section B(ii) above may be used with the chloroformate as described in section C(i) above. This is used to provide compounds of the formula (II) in which Z 1 is -NH-. D: Compounds of the formula (IV) in which Z 2 is -NH-: (i) Compounds of the formula (IV) in which Z 2 is -NH- may be made from a 4- itro benzylic alcohol optionally substimted with the group(s) R : (m) (as defined above). The hydroxyl function is protected as a pyanyl- or t-butyl-dimethylsilyl (TBDMSi)-ethcr by treatment at 20°C with 3,4-2H-dihydropyran and pyridinium-p-toluensulfonate (PPTS) in an aprotic solvent or with TBDMSi chloride and imidazole in dimethylformamide (DMAT), respectively (Example 5). The intermediate thus obtained is reduced to the corresponding amine by hydrogen transfer in ethanol using ammonium formate and Pd/C 10 as catalyst at 20°C. This amine is converted to a glutamyl ester intermediate of formula (VI) (Example 6): where R 2 , m, R 3 and Z 3 are as defined above, Z 2 is -NH- and Pr is the pyanyi- or - * -butyl-dimethylsilyl (TBDMSi)-ether protecting group. This may be done by treating the amine with triphosgene and triethylamine in toluene at 60°C to provide the corresponding ■-.ocyante. which is coupled with a glutamate derivative of formula R 3 O-C(O)-CH(NH 2 )-Z 3 •■■• here R 3 and Z are as defined above Alternatively the corresponding glutamyl-isocyanate obtained from the corresponding glutamate by treatment with triphosgene and triethylamine in toluene at -78°C may be reacted with the amine in a one pot procedure (Example 7). Glutamate derivatives of formula R 3 O-C(O)-CH(NH 2 )-Z 3 may be obtained by reference to WO94/02450. Where necessary or desired, the group Z 3 may be converted to another group Z 3 using routine chemistry known er se following production of the compound of formula VI). or after reaction of that to the compound of formula (I). (ii) The compound of formula (VI) is deprotected to remove the TBDMSi or pyranyl • roups by treatment with mild acidic media (AcOH. THF and H 2 O or PPTS. EtOH. 55°C) (Example 8). This yields a compound of formula (VI) in which Pr is hydrogen. Compounds of the formula (IV) in w hich Q is a leav ing group may be prepared using standard leac .ions known in the art (Example 8). (iii) Where Q is a succinimidyl group the compound of formula (VI) where Pr is hydrogen may be treated with disuccinimidyl-carbonate and triethylamine in acetonitrile. Where a 4-nitrophenyl carbonate group is desired treatment with 4-nitrophenyl chloroformate and triethylamine in THF may be used (Example 9). A pentafluorophenyl carbonate may be added by in si phosgenation of pentafluorophenol followed by coupling to the linker of formula (VI) in which Pr is' hydrogen. (iv) The compound of formula (VI) in which Pr is hydrogen may be obtained directly by coupling of the 4-aminobenzylic alcohol with glutamyl-isocyanate in aprotic solvents (Example 10). E: Compounds of the formula (IV) in which Tr is -O-: (i) The starting materials for the linkers possessing a carbamic bond are -inbub titu ed or substimted (with the grυup(s) R-,,, v (as defined above)) 4-hydrθ4\y-benzylic alcohols. These type of linkers may require an extra electron withdrawing group on th aromatic nucleus in order to undergo 1 ,4-elimination. The 4-hydroxy group is specificall protected as an acetate by treating the starting material with acetyl-v-triazolo-[4,5-b]pyridin and IN NaOH in THF at 20°C. The alcohol function of the acetate is further protected a c pyranyl- or TBDMSi-ether by the procedures described in section D above. The acetat function is then deprotected to restore the 4-hydroxy group in NaHCO 3 aq. MeOH at 20° The resulting phenol compounds are reacted in a one pot procedure with a protecte glutamyl-isocyanate as described in section D(i) above. This yields a compound of th formula of (VI) as shown above in which Z 2 is -O- and Pr is the pyanyl- o t-butyl-dimethylsilyl (TBDMSi)-ether protecting group. (ii) Deprotection of this compound yields a compound of the formula (VI) in which is hydrogen. This may be converted to compounds of the formula (IV) by methods analogou to t'.o e described in section D (iii) above. . (iii) An alternative procedure starts from 4-hydroxy-benzaldehyde which is protectec wit 1 ,3-propane-dithiol in the presence of BF ? .Et 2 O, at room temperature, to give the 4[2'-(r,3 S,S-dithianyl)]phenol. The coupling of the protected phenol with glutamyl isocyanate i toluene in the presence of Et-N provides the di-terr-butyl.O-{4-[2'(r,3'-S, dithianyl)]phenyl}oxycarbonyl-L-glutamate. The deprotection of this intermediate to th corresponding aldehyde, is carried out with Hg(ClO 4 ) 2 in THF at room temperature. Th reduction of the aldehyde with a reducing agent like sodium cyano-borohydryde yields th desired compound of formula (VI) in which Pr is hydrogen and Z 2 is -O- (Example 11). (iv) Another procedure to obtain the deprotected aldehyde is by direct coupling of hydroxy-benzaldehyde with the glutamyl-isocyanate (Example 12). (v) An alternative route to the compound of formula (VI) in which Z 2 is -O- and Pr 5 hydrogen is by direct coupling of the 4-hydroxy-benzylic alcohol, as potassium phenolat i obtained from the starting material and potassium tert-butoxyde in the presence of crow ether) with the corresponding glutamyl-isocyanate in aprotic solvents (Example 13). (vi) The compound of formula (IV) in which Z 2 is -O- and Q is a leaving group may be prepared using standard reactions known in the art. For example where Q is a 4-nitrophenyl carbonate group the compound of formula (VI) where Pr is hydrogen may be treated with 4- nitrophenyl chloroformate and triethylamine in THF (Example 14). F: Alternative synthesis of compounds of formula (IV): Compounds of the formula (TV) in which Q is hydrogen, fluoro, chloro, bromo or -O-(N- succinimide) may also be obtained by reference to WO95/03830 published on 9 February 1995 or WO95/02420 published 26 January 1995, the contents of which are hereby incorporated by reference. The following Examples illustrate the invention. The numbering of compounds given in the examples is according to that shown in schemes 1-3. Example 1. DiaIlyl.f(4 , -(N.N-bis(2-chloroethvI)amino)phenyloxycarbonyl-oxy-benzyl-4-carbamoyl-L- glutamate! (22) Activated linker 15b (0.20 g, 0.37 mmol) and 4-N,N-bis(2-chloroethyl)aminophenol 21 (0.14 g, 0.60 mmol) was dissolved in 10 mL acetonitrile. Then 0.13 g of a strongly basic resin- Amberlyst 27 (OH form) was added and the reaction mixture stirred with the resin for 20 h. The resin was filtered off, and an additional 0.08 g of fresh resin was added. After 30 min of stirring, the second portion of resin was filtered off, and the solvent evaporated. The product was purified by column chr< ~ -ι..atograph;. (eluent: cyclohexane :. < \cOEt 2: 1), to afford 22 (82 mg, 32%). 'H-NMR δ„: 1 -80-2.15 (2m, 2H, CH,CH(NH)-), 2.30-2.45 (m, 2H, CH,CO 2 -allyl), 3.72 (s, 8H, N(CH 2 CH 2 C1) 2 ), 4.30-4.40(m, 1Η, CΗ-G ), 4.55 (d, 2Η, J=5.41, CH,O allyl), 4.61 (d, 2H, J=5.31, CH 2 O allyl), 5.13 (s, 2H, PhCH 2 ), 5.10-5.40 (m, 4Η, CH 2 = allyl), 5.80-6.00 (m. 2H, CH= allyl), 6.64 (d, IH. J=8.13 Hz, NH-G), 6.75 (d, 2H, J=9.10 H^.^-), 7.04 (d, 2H, H^.^), 7.31 (d, 2H, J = 8.55Hz, H-^ , 7.41 (d. 2H. H a . 0! 3 +5 ), 8.69 (s, lH.PhNH). MS (FAB), m/z: 636 (M + l , 60), 658 (M +23, 7). anal (C, 0 Η 35 N 3 O g Cl 2 ): calcd C 56.61, H 5.54. N 6.60; found C 56.49, H 5.69, N 6.27. 4 , -fN.N-bis(2-chloroethvπamino1phenyloxycarbonyl-oxy-benzyl-4-carbamoyl-L-glutami acid (23) To a solution of 22 (100 mg, 0.16 mmol), PPh 3 (92 mg, 0.35 mmol) and Pd(PPh 3 ) 4 (16 mg 0.014 mmol) in CH 2 C1 2 (8 mL), acetic acid (1 mL) were added and the mixture stirred fo 5 h under argon. The solvent was evaporated, and the residue kept under pump vacuum fo 2 h, then dissolved in acetone: AcOEt 1 : 1. The acetone was evaporated affording 23. PPh -.*• a precipitate in AcOEt. The solid was filtered, washed with AcOEt and then dissolved i 50 mL CHC1, and washed with aqueous citric acid 10% (2x50 mL). The organic layer wa collected, H 2 O was added and the CHC1 3 evaporated yielding 23 (28 mg, 32%) as precipitate which was filtered from the aqueous solution. 'H-NMR d H : 1.80-2.00 (2m, 2H CH 2 CH(NH)-), 2.15-2.25 (t, 2H, CH CO 2 H), 3.72 (s, 8H, N(CH,CH,C1) 2 ), 1.10 4.25(rn.PhCH. ). 6.49 (d. 1Η. J=7.34 Ηz. NΗ-G), 6.75 (d, 2Η. J=9.10 Hz. H-^.^*), 7.0 (d, 2H. H^.^.), 7.29 (d, 2H, J=8.53 Hz, H,,^), 7.41 (d, 2H, H * ^), 8.75 (s I H.PhNH). MS (FAB), m/z: 556 (M + l . 55). 578 (M +23, 100). Accurate mas (C 24 Η 2 -,Ν 3 O g Cl 2 Νa): calcd. 578.1073; found 578.1070; . Example 2. 4 , -πy.N-b-s(2-chloroethyl)aminolphenol-trimethylsilyl ether (25) Phenol nitrogen mustard 21 (60 mg, 0.26 mmol) and Me 3 SiCN (40 μL. 0.3 mmol) wer stirred at room temperature without solvent for 30 min; then 10 mL boiling hexane was adde to the reaction mixture and the remaming solid filtered off. After evaporation of the hexane 25 (70 mg, 88%) resulted as an oil. Η-NMR δ H : 0.20 (s, 9H, Si(CH 3 ) 3 ), 3.55-3.70 (m. 8H N(CH 2 CH 2 C1) 2 ), 6.65 (d, 2Η, J=9.30 Hz, H-,^^), 6.73 (d, 2H, H ^ ^^) MS (FAB), m/z 304 (M + , 100). Accurate ."..-.:>. (C i3 H 21 Cl ; NOSi) calcd. 305.0769, found 305.0765. diallyl.f(4'-(N.N-bis(2-chloroethyl)amino)phenyloxy-carbonyl-oxy-benzyl-4-oxycarbonyl-L glutamate! (26) To a stirred solution of 14b (100 mg, 0.26 mmol) in CH 2 C1 2 (5 mL) at -78°C, was adde phosgene (20% solution in toluene, 150 μL, 0.3 mmol), KF (200 mg, 3.45 mmol) and 18 crown-6 ether (25 μL 0.12 mmol). After 1 h at -78°C. fluoroformate 24 was formed a indicated by IR spectra (ι> OCOF = 1827 cm "1 ). Silylated phenol 25 (70 mg, 0.23 mmol) i CH-CU (3 mL) was added and the solution stirred 1 h more at -78°C. then filtered and th iα -is. evaporated. After purification by preparative HPLC (eluent: cyclohexane:AcOEt 4: 1) 26 (60 mg, 41 %) resulted. Η-NMR δ H : 1.90-2.20 (2m, 2H. CH,CH(NH)-), 2.45-2.60 (m, 2H. CH,CO 2 -allyl), 3.72 (s, 8H, N(CH 2 CH 2 C1) 2 ), 4.15-4.25(m, 1Η, CΗ-G), 4.57 (d 2Η. J = 5.40. CH 2 O allyl), 4.63 (d, 2H, J=5.28 Hz, CH 2 O allyl), 5.23 (s, 2H, PhCH 2 ), 5.15- 5.35 (m, 4Η, CH 2 = allyl), 5.85-6.00 (m, 2H, CH= allyl), 6.75 (d, 2H, J=9.11Hz, H * V 7.06 (d. 2H. H^ ..■>. 7 14 (d. 2H. J=8.51 Hz. H^^), 7.41 (d, 2H, U m « , .„). 8.31 (d, IH, J=7.77, NH-G); MS (FAB) m/z: 637 (M, 100) 4 , -[N.N-bis(2-chloroethyl)amino1phenyl-oxy-carbonyl-oxy-benzyl-4-oxycarbonyl-L- glutamic acid (27) To a solution of 26 (40 mg, 0.063 mmol), PPh3 (63 mg, 0.24 mmol) and Pd(PPh 3 ) 4 (12 mg, 0.01 mmol) in CH 2 C1 2 (3 mL), acetic acid (80 μL) was added and the mixmre stirred for 2 h under argon. The solvent was evaporated and the residue dissolved in AcOEt (25 mL), washed with aqueous citric acid 5% (25 mL) and with H 2 O (25 mL). The organic layer was dried over MgSO^ and the solvent evaporated After purification by preparative HPLC (eluent CH 2 Cl,:EtOH:AcOH 90:5:5) 27 (8 mg, 25%) resulted. Η-NMR (CD 3 COOD), OH.:.05-2.20 (CH.CH(NH)-). 2.61 (t, 2H, CH 2 CO 2 H), 3.60-3.75 (m, 8H, N(CH.C// 2 .;i) 2 ), 4.45-4.55 (m. 1Η, CΗ-G), 5.23 (s, 2Η PhCH 2 ), 6.73 (d, 2H, J=9.20 Hz, H^.^-), 7.06 (d. 2H. H-^., 5 *), 7.19 (d, 2H, J = 8.59 Hz, H-,^^)), 7.45 (δ, 2H, H^^); MS, (ESI) m/z: 557 (M + l , 3). Example 3. di-t-butyl.r(4 , -(N.N-bis(2-chloroethvπamino)phenylcarbamoyl-oxy-benzyl-4-oxycarbonyl- L-glutamatel (17) T«_ ._, solution f 14a (60 mg 0.1:. mmol) in toluene (5 mL) was added isocyanate 16 (0.60 mmol) in toluene (5 mL) and dibutyltin dilaurate (10 μL). The reaction mixmre was stirred for 12 h at room temperature. The solid which formed was filtered off and the filtrate evaporated. Purification was achieved by column chromatography (eluent: cyclohexane: AcOEt 3: 1). when product 17 (90 mg, 90%) was obtained as an oil. 'H-NMR δ H 1.41 (s, 9H, t-Bu), 1.42 (s, 9H, t-Bu), 1.65-2.05 (2m, 2H, CH 2 CH(NH)- ), 2.30-2.40 (m, 2H, CH 2 CO 2 -t-Bu ), 3.68 (s, 8H, N(CH CH,C1) ; ). 3 95-4.15 (m, 1Η. CΗ-G ), 5.10 (s, 2Η. PhCH, ), 6.70 (d. 2Η. J=8.75 Hz, H-,^ 5 *), 7.10 (d, 2H, J=8.31 Hz, , H arorn3 _- 5 ), 7.28 (d, 2H, H^.^.), 7.42 (d, 2H. H ^ ,^), 8.92 (d. IH. J=7.80 Hz. NH-G), 9.36 (s. IH. Ph'-NH). MS. m/z (FAB): όfcT M + l . 5 ). 689 ( *r23. 100) 4 , -rN.N-bis ( 2-chloroethyl ) aminoIphenylcarbamoyl-oxy-benzyl-4-oxycarbonyl-L-gluι_ami * acid (18) Di-t-butyl ester 17 (90 mg, 0.135 mmol) was dissolved in formic acid (10 mL) and stirred under argon at 12 °C for 24 h. The reaction mixmre was lyophilised, the freeze-dried material τ trirur-u d with CH 2 C1 : . 'he solvent removed an the solid kept in a dessicator 24 h under vacuum to afford 18 (65 mg, 87%). Η-NMR δ H : 1.75-2.15 (2m, 2H. CH.CH(NH)- ), 2.30- 2.50 (m. 2H, CH_,CO 2 H ). 3.68 (s, 8H. N(CH 2 CH,C1) : ). 3 95-4.10 fm. IΗ, CΗ-G), 5.10 (s. 2Η. PhCH 2 „ 6.70 (d. 2Η, J=8.66Hz, H^.^), 7.11 (d, 2H. J = 8.29, H voπύ+5 ), 7.28 (d, 2H, H^.^.), 7.42 (d, 2H, H,^^, 8.07 (d, IH, J=7.80 Hz, NH-G), 9.36 (s, IH, Ph'NH). MS, m z (FAB): 555 (M, 90), 578 (M+23, 100). Accurate mass: calcd: (C 24 Η 2 ,Ν 3 O g Cl,Νa), 578.1073, found: 578.1070. Example 4. diallvU(4 , -(N.N-bis(2-chloroethyl)amino)phenylcarbamoyl-ox>-benzyl-4-carbamoyl-L- glutamate! (19) A similar procedure as for 17 was used, starting from isocyanate 16 (0.54 mmol) in to,uene (5 mL). 8b (0.10 g, 0.27 mmol) dissolved in CH 2 C1 2 (5 mL). and dibutyltin dilaurate (10 μL), to yield 19 as a solid. After further recrystallisation from hexane:AcOEt 9:2 19 (0.11 mg. 64%) resulted. Η-NMR δ H : 1.75-2.15 (2m. 2H, CH,CH(NH)-), 2.35-2.50 (m. 2H, CH 2 CO : -Allyl ), 3.68 (s, 8H, N(CH,CH,C1) 2 ), 4.25-4.40(m, 1Η, CΗ-G ), 4.55 (d, 2Η, J = 5.39 Hz, CH 2 O allyl), 4.61 (d, 2H, J = 5.37 Hz, CH 2 O allyl), 5.01 (s, 2H, PhCH, ), 5.15- 5.35 (m. 4Η. CH 2 = allyl), 5.80-6.05 (m, 2H, CH= allyl). 6.61 (d, IH, J=7.97 Hz. NH-G), 6.69 (d, 2H, J = 8.99Hz, H-^ ^*), 7.28 (d, 4H, J=7.79 Hz, , H^^+H^.^.), 7.39 (d, --I ri ^ϋ ^ / . 8*64 (s, lH,PhΛH. ), 9.30 (s, 1Η, PhWH). MS, m/z (FAB): 635 (M+ l . 5), 657 (M +23, 5). 4 , -rN.N-bis(2-chloroethyl)aniinolphenylcarbamoyl-oxy-benzyl-4-carbamoyl-L-glutamic acid (20) Diallyl ester 19 (40 mg, 0.06 mmol) dissolved in CΗ 2 C1 2 (2 mL) was stirred with Pd(PPh,) 4 (4 mg) and pyrrolidine (15 μL, 0.15 mmol) The reaction mixmre was kept under argon for 45 min then diluted with 2 mL toluene. The pyrrolidine salt of the acid 20 precipitated and 0 was recovered by filtration. The free acid was obtained by extracting the precipitate with _ji 'J 'aqueous citric auu 10% . i ύe organic layer was washed with H,O and evaporated to - 15 - give the free acid (20 mg, 60% ) after filtration of the remaining water. Η-NMR δ H : 1.70- 2.05 (2m, 2H, CH.CH(NH)-), 2.20-2.35 (m, 2H, CH 2 CO 2 -allyl), 3.68 (s, 8H, N(CH,CH,C1) 2 ), 4.15-4.30(m. 1Η, CΗ-G ), 5.01 (s, 2Η, PhCH 2 ), 6.45 (d, IH, J = 7.98Hz, NH-G),6.69 (d, 2H, J = 9.05Hz, H^*^ . , 7.27 (d, 4H. J=8.60Hz, H-^^+H^ +6 ), 7.39 5 (d. 2Hj.« , 2 . +5 *), 8.65 (s, IH, Ph/VH), 9.30 (s, 1Η, Ph'NΗ). MS (FAB), m z: 554 (M. 50), 577 (M+23, 100). Accurate mass: calcd: (C 24 Η 28 N 4 O 7 Cl,Na) 577.1233, found: 577.1230. Example 5. (4-nitro-benzyl) t-butyl-di-phenyl-silyl ether (2a). To a stirred solution of 4-nitrobenzyl alcohol (1.00 g, 6.50 mmol) and imidazole (0.97 g, IC 14.10 mmol) in DMF (10.0 mL), t-butyl-diphenyl-chlorosilane (1.98 g, 1.87 mL, 7.20 mmol) was added over 10 min under nitrogen at room temperature. The reaction mixmre was stirred toi an additional 5 h, diluted with 75 mL Et 2 O, washed with H 2 O (5 times, 15 mL), dried f'MgSOj) and evaporated under vacuum. An oil which crystallised on standing, resulted; yield: 2.54 g. After recrystallisation from EtOH 70%, 2.36 g (93 %) of a white soiid is 15 obtained mp 80-81 °C. v n c l (film)- 2931 . 2857 (CH 2 , asym.. sym.). 1521. 1345 (NO,): Η-NMR, δ H : 1.06(9H, s, t-Bu), 4.92 (2H, s, CH 2 ), 7.42-7.46 (5H, m, Ph), 7.63-7.65 (7H, m, Ph+H-,^^), 8.23. (2H, d, J = 8.23 Hz, H-, om3+5 ); MS, (El), (391.54); m/z: 334 (M - t-Bu, 100), 288 (M - t-Bu - NO 2 , 10), 256 (M - t-Bu - Ph, 20), 199 (P^SiOH * , 100); C, 3 H 25 NO 3 Sι. 0 4-nitro-benzyl -2'-tetrahvdropyranyl ether (2b). To a stirred solution of 4-mtrobenzyl alcohol (1.53 g, 10.0 mmol) in CH 2 C1 2 (50 mL), 3,4- dihydropyran ( 1 ^6 ml.. 1 .26 g. 15 0 mmol ) and pyridinium-p- t oluene sulphonate (0 25 g) were added. After 1.5 h the reaction mixmre was evaporated to 4-5 mL, AcOEt (25 mL) was added and the solution washed with water (3x50mL), then brine, dried (MgSO 4 ) and 5 evaporated under vacuum. The product, 2b, (2.46 g, 99%) resulted as an oil. 'H-NMR, δ H : 1.51-1.63 (m. 4H, 2H 4 +2H 5 -), 1.66-1.74 (m, IH, 2H 3 ) 3.50 (s, IH, H 6 .), 3.78 (m, IH, H 6 ), 4.62 (d, IH, J=3.8 Hz, CH 2 -Ph), 4.72 (m, IH, H 2 ), 4.81 (d, IH, CH,-Ph), 7.62 (d, 2H. J = 8.5 Hz, H arorn ), 8.22 (d. 2H. H ^ ; MS, (FAB), (237. 52); m/z: 260 (M + +23. 15), 236 (M - 1 , 45), 136 (4-NO 2 C 6 H 5 CH 2 + ,100). Example 6. (4-amino-benzyl) t-butyl-di-phenyl-silyl ether (3a) To a stirred solution of 2a (5.00 g, 12.80 mmol) in ethanol (100 mL) was added Pd/ 10% (1.50 g) and ammonium formate (4.60 g) in one portion at 20 °C. After 1.5 h the cataly was remo ed by filtration, the filtrate concentrated under vacuum and the residue π-rtition between EtOAc :H 2 O. The organic layer was dried (MgSO 4 ) and concentrated to give 3a n oil; yield: 4.24 g (91 %) ■ -• _ /cm 1 TilmV 3433. 3378 (NH,), 2931 2857 (CH : . asym sym.): Η-NMR, δ H : 1.00 (s, 9H. t-Bu), 4.57 (s, 2H. CH 2 ), 4.98 (s broad, 2H. NH 2 ), 6. (d, 2H, J = 8.25 Hz. H,^^), 6.96 (d, 2H, H^^), 7.42-7.46 (m, 5H, Ph). 7.62-7.65 ( 5H, Ph): MS, (El), (361.56); m z: 361 (M + , 8), 304 (M - t-Bu. 100). 199 (Ph 2 SiOH + , 100 anal (C 23 H 27 NOSi), C. H, N. (4-amino-benzyl)-O-2'-tetrahvdropyranyl ether (3b) A similar method as for 3;» •'•■ -is employed using 2b (8.53 g. 35.9 mmol) in ethanol (150 mL Pd/C 10% (3.50 g) and ammonium formate (12.0 g, 180.0 mmol). An oil (6.40g, 86 resulted after work up, which was purified by column chromatography (cyclohexane: AcO 1 : 1 ): The desired compound eluted first as an oil (2.59 g. 38%). (film): 3447, 33 (NH,, sym, asym), 2943, 2870 (CH,, asym. , sym.); 'H-NMR, δ H : 1.42-1.53 (m, 4 2H, +2H< ). 1 .55-1.72 (m. IH. 2H. ) 3.45 (s, IH. H Λ ), 3.79 (m, IH, H„ ), 4.22 (d. I J = 11.1 Hz, CH,-Ph), 4.37 (d. IH, CH,-Ph), 4.60 (d, IH. H, ), 5.00 (s, 2H, NH,), 6.52 ( 2H. J = 8.23 Hz. H ^ , 6.97 (d, 2H, H„J; MS, (El), (207, 27); m/z: 207 (M ~ , 100), 1 (H,NCJL,CH,\ 100): anal (C,,H r NO,, C. H. N. Example 7. di-r-butyl . (t-butyl-di-Dhenyl-silyl-O-benzyl)-4-carbamoyl-L-g!utamate (7a) Method A: A suspension was formed by adding 0.31 g (0,43 mL, 3.1 mmol) triethylami to a solution of di-t-butyl-glutamate hydrochloride (0.46 g, 1.55 mmol) in 7 mL THF. T isocyanate, 4a. (O.60 g, 1.55 mmol) dissolved in 3 mL THF (dry) was added to this at roo temperature. After 2 h the precipitate was filtered and the solvent removed under vacuu giving 0.87 g (87%) of an oil. The product was purified by column chromatography (AcOE cyclohexane 2: 1) yielding 0.53 g (53 %) of pure 7a, as a white solid, mp-= 89-90 °C. i /cπ (film): 3359 (NH). 2932. 2857 (CH,, asym. , sym.). 1729 (C = O, ester), 1670 (C = O, urea 1 154 tC- . sir. ): Η-NMR. δ H : 1.03 (s. 9H. t-Bu). 1 . -KWs °TI. t-Bu-βlu). 1.43 (s. 9H. t-B glu). 1.68-2.00 (2m, 2H. CH(NH)CH 2 ), 2.18-2.32 (m, 2Η, CH,CO,-t-Bu), 4.08-4.12 ( 1H. CH(NH)CH,). 4.68 (s, 2H, PhCH 2 ), 6.38 (d, 1Η, J = 8.12 Ηz, NΗ-glu). 7.19 (d. 2Η, J = 8.41 Hz, H-,^ 5 ), 7.32-7.47 (m, 7H. Ph+H^^), 7.62-7.70 (m, 5H. Ph), 8.54 (s, IH. NH-Ph); MS, (El), (646.90); m/z: 590 (M - r-Bu + 1, 2), 534 (M - 2r-Bu + 2, 5), 478 (M - 3t-Bu + 3, 100), 199 (PfcSiOH , 100); anal (C 37 H 50 N 2 O 6 Si), C, H, N. Method B_: One pot synthesis of compound 7a : To a solution of di-t-butyl-glutamate hydrochloride (4.14 g, 14.0 mmol) and triphosgene (1.39 g, 4.67 mmol) in toluene, cooled at -78 . 3 90 L (2.83 g 28 mmol) triethylamine in 10 mL toluene were added dropwise over 30 min. The reaction was allowed to reach room temperature and was completed by 1 h. To this mixmre, a solution containing (4-amino-benzyl)t-butyl-diphenyl-silyl ether 3a (5.00 g, 13.8 mmol) and triethylamine (1.95 mL, 14.0 mmol) was added over 5-10 min. The reaction was completed by 20 h. The reaction mixmre was filtered, washed with H,O (200 mL). HCl solution 1 % (200 mL). Na,CO, solution 1 % (200 mL), H 2 O (twice. 200 mL). dried (MgSO 4 ) and evaporated under vacuum, yielding a thick oil. Yield: 9.90 g. (TLC, AcOEt : cyclohexane 3: 1). The product was used for deprotection without further purification. diallvI.(2 etrahvdropyranyl-O-benzyl)-4-carbamoyl-L-glutamate (7b) A similar one pot procedure was used, starting from diallyl glutamate- -toluene-sulphonate (1.08 g, 3.1 mmol), triphosgene (0.30 g, 1.03 mmol) and triethylamine (0.86 mL, 0.63 g, 6.2 mmol) in toluene (20 mL) at -78 °C to prepare the diallyl -L-glutamyl isocyanate 6b. To the reaction mixmre 3b (0.52 g, 2.5 mmol) and triethylamine (0.41 mL) in 5mL toluene was added. An oil resulted (1.17 g) which was separated by preparative HPLC using cyclohexane: AcOEt 1.5:1 as eluent. The desired compound separated as an oil (0.75 g, 65%). υ^/cm- 1 (film): 3362 (NH-j), 2944, 2871 (CH 2 , asym., sym.); 'H-NMR, δ H : 1.47-1.50 (m, 4H. 211. +2H S .), 1.60-l .oS . 3H, 2H 3 + CH,CH(NH)-), 1.88-2.05 (m, IH, - H,CIUNH)- ), 2.37-2.45 (m, 2H, CH 2 CO 2 A), 3.45 (s, IH, H 6 ), 3.80 (m, IH, H 6 ), 4.20-4.31 (m, IH, - CH(NH)-), 4.35 (d. IH, J = 11.55 Hz, CH,-Ph), 4.53-4.63 (m, 5H, CH 2 -Ph+CH,O allyl), 5.18-5.33 (m. 4H, CH,= allyl), 5.80-6.00 (m, 2H. CH= allyl), 6.60 (d, IH, J = 8.03 Hz, NH-G), 7.20 (d, 2H. J = 8.47 Hz, H^), 7.35 (d, 2H, H^ , 8.47 (s, IH. NH-Ph): MS, (El), (460, 53); anal (C 24 H 32 N 2 O 7 ), C, H, N. Example 8. di-t-butyl.(4-hvdroxybenzyl)carbamoyl-L-glutamate (8a) Tetra-butylammonium fluoride solution in THF (1M) 20.0 mL (2.5 eq) was added t compound 7a (5.15 g, 8.0 mmol) in 100 mL THF at room temperature. The reaction wa ini He*-! 'a 3 !ι (TLC \cOEt ■ cyclohexane 3: 1) The reaction mixmre was evaporated unde vacuum. The oil thus obtained was dissolved in AcOEt (50mL), washed with water (3x10 mTΛ dried (MgSO 4 ) and evaporated again. An oil (5.08 g) was obtained The deprotecte compound. 8a. was purified by column chromatography (AcOEt : cyclohexane 3: 1) yieldin 1.88g (58.5 %) of an oil which crystallised on standing. Recrystallisation from 60% aqueou MeOH afforded a white solid. Mp : 103-104 °C; ijc -' (film): 3370 (broad, NH + OH), 2967 (CH 3 ), 2930, 2857 (CH,, asym., sym.), 1716 (C=O, ester), 1678 (C=O, amide), 115 ( C-O, str.); 'H-NMR. δ H : 1.40 (s. 9H. t-Bu), 1.42 (s, 9H. t-Bu). 1.72-2.00 (2m. 2H. CH(NH)CH,), 2.20-2.31 (m, 2Η, CH,CO,-t-Bu), 4.10-4.18 (m, 1Η. CH(NΗ)CΗ 2 ), 4.39 (d, 2H, J = 5 36 Hz, CH,). 4.99 (t. III. OLOH). 6.38 (d. 1Η. J = 8.11 Ηz. NΗ- u). 7.16 (d, 2Η, J = 8.35 Hz, H- ^), 7.31 (d, 2H, H^,,), 8.50 (s, IH, NH-Ph); MS, (El), (408.49); m/z: 408 (M \ 10), 352 (M - i-B + 1 , 4), 296 (M - 2t-Bu + 2, 14): ana (C,,H 3 ,N 2 O 6 . C.H,N. diallyl, (4-hvdroxybenzyl)carbamoyl-L-g!utaπιate (8b) The intermediate 7b (0.61 g, 1.3 mmol) was hydrolysed at 45 °C in 24.5 mL of a mixmre AcOH.THF.H,O 4:2.1 lor 3.5 h. (monitored by TLC, eluent, AcOEt: cyclohexane 1 : 1). The reaction mixmre was diluted with 50 mL H 2 O, extracted with ether (2x25 mL), then wit AcOEt (2x30 mL). The pooled organic layers were washed with water (2x30 mL), drie Λlfc C . ,, and evaporated to drynej>s (with addition oi toluene, 2x3ϋιnL). An oil resulted (0.5 g) which was purified by preparative HPLC (cyclohexane: AcOEt 1:2), yielding compoun 8b (0.221 g, 45%). .Jem-' (film): 3354 (NH,, OH broad), 1737 (C=O, ester), 165 (C=O, amide); 'H-NMR, δ H : 1.85-1.93 (m, IH, CH,CH(NH)-), 2.00-2.05 (m, IH, - CH,CH(NH)-), 2.46 (t, 2H, J = 5.42 Hz, CH,CO,A), 4.26-4.35 (m, IH, -CH(NH)-), 4.4 (d, 2H, J=5.64 Hz. CH 2 -Ph), 4.55 (d, 2H, J=5.32 Hz, CH,O allyl), 4.61 (d, 2H, CH,O ally!). 4.98 (t, III, OH), 5.17-5.37 (m, 4H, CH,= allyl), 5.85-5.94 (m, 2H. CH= allyl), 6.56 <d, IH. J = 8.03 Hz. NH-G). 7.17 (d, 2H, J = 8.48 Hz. H^ , 7.32 (d, 2H, H^ , 8.52 (s. IH. NH-Ph): MS. (El). (376. 41): m/z: 399 (M + +23, 35). 377 (M + + 1. 100). 359 (M + t- * - 4,, anal <C lβ H 24 N,O β ). C, H, N. Example 9. di-t-butyl.f4(4 , nitrophenylox\carbonyl)oxybenzyll-carbamoyl-L-glutaιnate (9a) To a stirred solution of 8a (0.20 g, 0.49 mmol) in dry THF (10 mL) were added 0.11 g (0 .5 mmol) 4-nitrophenyl chloroformate and triethylamine (0.1 mL, 0.6 mmol) at room tempeiature. The reaction was complete by lh. The precipitate formed was filtered and the solution concentrated under vacuum. AcOEt (10 mL) was added, the solution washed with brine ( 2x10 mL). dried and evaporated again, giving an oil (0.28 g). The product was purified by column chromatography (0.16 g, 57%) and repurified by preparative HPLC (0.14 g). jcm- 1 (film): 3349 (NH 2 ), 2979, 2932 (CH 2 , asym., sym.), 1767 (C=O, carbonate), 1716 (C=O. ester), 1652(C=O, amide), 1527, 1349 (NO,); 'H-NMR, δ H : 1,39 (9H, s, t-Bu), 1.42 (9H, s, t-Bu), 1.67-2.05 (2H, 2m, CH 2 CH(NH)-), 2.20-2.38 (2H, 2m, CH 2 CO,-t-Bu), 4.08-4.21 (IH. m. CH(NH)-). 4.63 (2H. s. CH,Ph), 6.46 (IH, d. J = 7.99 Hz, NH- lu), 7.33 (2H. d, J = 8.61 Hz. H-,^), 7.42 (2H, d, H^^), 7.56 (2H, d, J = 9.17, H a™ ,,, . - . , ■ 8.30 (2H, d. H„ om2 + . 8.68 (IH. . NH-Ph). MS. (ED. (573. 60); anal (C 28 H 35 N 3 O,o), C. H, N. diallyl,f4(4 , nitrophem oxycarbonyl)oxybenzyll-carbamoyl-L-glutamate (9b) Starting from 8b (0.19 g, 0.50 mmol), 9b was obtained by a similar procedure and purified by preparative HPLC. Compound 9b resulted as a white solid CO 132 g. 48.6%). 'H-NMR. δ H : 1.86-1.95 (m, IH, CH,CH(NH)-), 2.03-2.12 (m, IH, -CH,CH(NH)-), 2.46 (t, 2H, J =7.40 Hz. CH,CO,A), 4.33-4.48 (m, IH, -CH(NH)-), 4.55 (d, 2H. J=5.33 Hz, CH,O allyl). 4 61 (d. 2H. CH,O allyl). 5.17-5.38 m. 4H, CH,= allyl). 5.22 (s. 2H. CH,Ph). 5.87- 5.96 (m, 2H, CH= allyl), 6.65 (d, IH. J=8.26 Hz, NH-G), 7.34 (d, 2H, J=8.67 Hz, H ^ , 7.43 (d, 2H, H^ , 7.56(d, 2H, J = .14 Hz, H^), 8.3.1 (d, 2H, vo , 8.71 (s. IH, NH-Ph); MS, (El), (541, ); m/z: 564 (M + +23, 60), 542 (M ÷ + l , 6); anal (C 26 H 27 N 3 O 10 ): C, H, N; accurate mass: calcd. (C 26 H 27 N 3 O I0 Na): 564.1594; found 564.1590. Example 10. diaϊlyl . (4-hvdroxybenzyl)carbamoyl-L-glutamate (8b) To a solution of diallyl, glutamyl isocyanate 6b, [prepared from diallyl glutamate- -toluene sulphonate (10.0 g, 25.0 mmol), triphosgene (2.46 g, 8.3 mmol) and triethylamine (10.5 mL, 75.0 mmol. 3 eq) in 100 mL THF. at -78 °C] was added dropwise 4-aminobenzyl alcohol, 2S. (J.U g, 24.3 mmol) and triethylamine (3.41 mL. 24.3 mmol) in 20 mL toluene, over 10 min at room temperature. The reaction was complete by 15 min. The reaction mixtur. wa filtered, evaporated to dryness and the residue dissolved in 20 mL AcOEt, washed with wate (2x20 mL), dried (MgSO 4 ) and re-evaporated. An oil resulted (10,25 g). 2.7 g of the obtaine product was submitted to purification by preparative HPLC when 1.52 g (63%) of pure 8 Example 11. di- -butyl.l4r2'fl , 3 , -S.S-dithienyl)Dhenvnoxycarbonyl-L-glutamate Ϊ12). To a solution of di-t-butyl-glutamyl isocyanate 6a (prepared from di-t-butyl-glutamat hydrochloride (1.50 g, 5.0 mmol), triphosgene (0.50 g, 1.66 mmol) and triethylamine (1.4 mL, 28.0 mmol) in 50 mL toluene, at -78 °C), was added 4[2'(1 '3 '-S,S-dithienyl} phenol 11 (1 lOg, 5.0 mmol) and triethylamine (0.70 mL). After 12 h. the solid formed was filtere and the solution washed with: NaOH 1 % (75 mL), water (2x75 mL), dried (MgSO 4 ) an evaporated. A white solid (2.30 g, 95.8%) was obtained Recrystallisation from aqueou EtOH 50% afforded the pure 12 (1.81 g, 72.7%). (film): 3347 (NH 2 ), 2977, 294 (CH,, asym. , sym.). 1728 (C = O, ester); 'H-NMR, δ H : 1,41 (s, 9H, t-Bu), 1.42 (s, 9H, t Bu). 1.62-1.90 (m. 2H. CH,-5'), 1.90-2.32 (2m. 2H, CH,CH(NH)-). 2.30-2.40 (m, 2H CH,CO,-t-Bu), 2.86-2.93 (m, 2H, CH,-4' or 6 ), 3.09 (t, 2H, J = 12.3 Hz, CH,-6' or 4') 5.41 (s. IH. H-2'). 7.08 (d. 2H. J = 8.50 Hz. H ^ -.,^)- 7.43 (d. 2H, H-_ orn2+6 ), 8.12 (d, IH J=7.81 Hz, NH-Ph); MS, (El), (497, 67 ); m/z, : 497 (M + , 2), 451 (M - t-Bu , 2), 359 (M 2t-Bu, 20); Anal. : (C 24 H 25 NO 6 S 2 ), C, H, N. di-t-butyl.(4-benzaldehyl)oxycarbonyl-L-glutamate (13a). To the solution of dithiane 12 (U.50 g, 1.0 mmol) in CHC1 3 (10 mL) and THF (5 mL; wa added dropwise a solution of Hg(ClO 4 ) 2 .3H,O (0.91 g, 2.0 mmol) in 5mL THF. After 5 mi the precipitate was filtered and the solution is washed with H,O (2x25 mL), sodium carbonat (25 mL), H,O (2x25 mL), dried (MgSO 4 ) and evaporated. The compound 13a (0.33g, 81 % was obtained as a clear oil. u ^ /cm '1 (film): 3347 (NH,), 2979, 2934 (CH,, asym.. sym.) 1731 (C=O, ester), 1712 (C=O, aldehyde); 'H-NMR, δ H : 1,41 (s, 9H, t-Bu), 1.43 (s, 9H t-Bu). 1.80-1.87 (m, IH, H,CH(NH)-), 1.96-2.02 (m, IH, CH,CH(NH)-), 2.32-2.39 (m 2H, CH,CO,-t-Bu). 3.99-4.04 (m, IH, CH-G), 7.34 (d, 2H. J = 8.39 Hz. H-, orn3+5 ), 7.9 (d. 2H. H -rnm _. b ). 8.29 (d. IH, J=7.72 Hz. NH-Ph), 9.98 (s. IH. CHO): di-t-butyl. (4-hvdroxybenzyl)oxycarbonyl-L-glutamate (14a) Compound 13a (0.150 g, 0.36 mmol) dissolved in 5.2 mL of a mixture of H,O:AcOH:CH 3 OH (1: 1 : 1) was reduced with sodium-cyanoborohydride (0.027 g, 0.43 mmol) at room temperature. After lh, AcOEt (20 mL) was added, the reaction mixmre -.vap "-rated, the residue dissolved in AcOEt (20 mL), the solution washed with H,O (20 mL), brine (2x20 mL), dried and evaporated to dryness. Compound 14a (0.127g, 86%) was obtained as an oil. The final purification was achieved by preparative HPLC (cyclohexane:AcOEt, 2: 1). u-^/cm ' (film): 3352 (NH,, OH, broad), 2979, 2945 (CH,, asym. , sym.), 1728 (C=O, ester), 1712 (C=O, aldehyde); 'H-NMR, δ H : 1 ,41 (s, 9H, t-Bu), 1.42 (s, 9H, t-Bu), 1.70-1.82 (m, IH, CH,CH(NH)-), 1.94-1.99 (m, IH, CH,CH(NH)-), 2.31-2.38 (m, 2H, CH 2 CO,-t-Bu), 3.97-4.02 (m, IH, CH-G), 4.48 (d, 2H, J = 5.70 Hz, PhCH,OH). 5.15 (t. IH. OH), 7.03 (d, 2H. J = 8.39 Hz. H-^ . . 7.31 (d. 2H. H aron 2+6 ), 8.04 (d. IH, J=7.88 Hz, NH-Ph), 9.98 (s, IH, CHO); anal: (C 21 H 31 NO 7 ), C. H, N. Example 12. To a solution of di-t-butyl-glutamyl isocyanate 6a [prepared from di-t-butyl-glutamate hydrochloride (0.200 g, 0.67 mmol), triphosgene (0.067 g, 0.22 mmol) and triethylamine (0.190 mL. 1.35 mmol) as above] in 15 mL CHC1 3 , were added 4-hydroxybenzaldehyde (0.082g, 0.67 mmol) and triethylamine (0.190 mL, 1.35 mmol) in 10 mL CHC1 3 over 10 min. The reaction was heated under reflux for 2 h, then the solvent was removed under vacuum. The compound was purified by preparative HPLC (eluent: cyclohexane: AcOEt 6: 1). Finally a clear oil was obtained (0.197 g, 72%). Example 13. diallyl.(4-hvdroxybenzyl)oxycarbonyl-L-glutamate (14b) A clear solution of diallyl-glutamyl isocyanate 6b [prepared from diallyl-glutamate p-toluene sulphonate (3.28 g. 8.20 mmol), triphosgene (0.81 g, 2.73 mmol) and triethylamine (3.44 mL. 24.6 mmol) in 50 mL THF, at -78 °C, according to the procedure described above] was obtained by filtering the reaction mixmre at room temperature. 4-hydroxybenzyl alcohol 29 (1.00 g. 8.10 mmol) was dissolved in THF (dry) (10 mL) and 18-crown-6 ether (0.20 mL) and potassium tert-butoxide (0.16 g. 1.4 mmol) were added. To this mixmre. the solution of isocyauate 6b was added dropwise at room temperature with vigoious stirring. The reaction was terminated by addition of AcOH (0.5 mL). The reaction mixmre was filtered and th solvent removed under vacuum. The residue was dissolved in AcOEt (20 mL), the solutio washed with aqueous NaOH 4% (20 mL), with H 2 O (2x20 mL), dried and evaporated t dryness. Compound 14b (1.62g, 53 %) resulted and was purified by preparative HPL 5 (eluent cyclohexane: AcOEt 1 : 1). Finally a clear oil was obtained ( 0 80 g. 26.5 %). 'H-NMR δ H : 1.90-2.01 (m, IH, CH,CH(NH)-), 2.05-2.09 (m, IH, -CH,CH(NH)-), 2.50-2.56 (m, 2H CH,CO,A), 3.96-4.05 (m, IH. -CH(NH)-), 4.48 (d. 2H, J = 5.67 Hz. PhCH,OH), 4.58 (d 2H. J=5.26 Hz. CH,O allyl), 4.63 (d, 2H, CH 2 O allyl), 5.16 (t. IH, OH), 5.19-5.38 (m 4H, CH, = allyl), 5.95-5.99 (m, 2H, CH= allyl), 7.04 (d, 2H, J = 8.47 Hz, H^-^), 7.3 10 (d, 2H, H ιrom2 +6 ), 8.24 (d, IH, j =7.82 Hz, NH-Ph); MS, (FAB), (377, ); m/z: 400 (M + +23 82), 378 (M * + l , 3); accurate mass (C 19 H 23 NO 7 Na): calc. 400.1372; found 400.1376 Example 14. di-t-hutyl 4(4'nitrophenylo\vcarbonvl)oxybenzvIl-oxycarbonyl-L-glutamate(15a) Activation of linker 14a (1.0 g, 2.7 mmol) was achieved by the same procedure as i !5 Example 9. After purification, an oil resulted (0.64 g, 41 %). u-^/cm 1 (film): 3367 (JJH,) 2980. 2933 (CH,. asym., sym.), 1767 (C=O, carbonate), 1728 (C=O, ester); 'H-NMR, δ H 1 ,41 (s, 9H, t-Bu), 1.43 (s, 9H, t-Bu), 1.89-1.94 (m, IH, CH,CH(NH)-), 1.96-2.03 (m, IH CH,CH(NH)-), 2.31-2.37 (m, 2H. CH,CO,-t-Bu), 3.96-4.05 (m, IH. CH(NH)-), 5.30 (s, 2H CH 2 Ph), 7.15 (d, 2H, J = 8.46 Hz, H^^), 7.49 (d, 2H, H^ , 7.83 (d, 2H, J = 9.1 20 Hz. H„ otll3 s .). 8. 12 (d, IH, J=7.83 Hz, NH-Ph); 8.31 (d. 2H. H wm2 +6 ); MS, (FAB), (574 50); m/z : 597 (M + +23, 12), 575 (M + + l, 4), 519 (M - t-Bu - 1, 10), 463 (M - 2t-Bu - 2 4); diallyl. f 4(4' nitrophenyloxycarbonyl)oxybenzyll-oxycarbonyl-L -glutamate (15b) Starting from 14b (0.4 g, 1.06 mmol), 15b was obtained by a similar procedure. Purificatio 25 yielded an oil (0.448g, 75.5 %). 'H-NMR, δ H : 1.80-2.01 (m, IH, CH(NH)CH 2 -), 2.03-2.0 (m, IH CH(NH)CH,), 2.50-2.55 (m, 2Η, CH,CO,A), 4.16-4.24 (m, IH, CH(NH)-), 4.5 (d, 2H, J=5.29 Hz, CH 2 O allyl), 4.63 (d, 2H, CH,O allyl), 5.19-5.38 (m, 4H, CH,= allyl) 5.31 (s, 2H. PhCH,), 5.90-5.98 ( . 2H. CH= allyl), 7.16 (d, 2H, J = 8.43 Hz. H aron , 3+5 ) 7.50 (d, 2H, H.,^^), 7.58 (d, 2H, H^-^), 8.11 (d, 2H, H^ ^.); MS, (FAB), (542 30 50); m/z: 563(M + +23, 10), accurate mass: calcd. (C 26 H 2 6 N,O--Na), 565.1434; foun . ■• .65. 1430.

Claims: CLAIMS A compound of the formula (I): wherein X and Y are independently chlorine, bromine, iodine, a mesyl group CH 3 SO 3 or a tosyl group OSO ; phenyl (wherein phenyl is optionally substimted by 1 , 2. 3. 4 or 5 substituents independently selected from C alkyl, halogen, cyano or nitro; n is an integer from 0 to 4 and each group R'. w hich may be the same or different when n is greater than 1, is fluorine, chlorine, bromine, iodine, hydroxy, mercapto, amino, nitro, C,^ alkyl optionally containing one double bond or one triple bond, C M alkoxy, -CONR 7 R 8 (wherein R 7 and R 8 are as defined below). -NH(C -alkyl), -N(C -alkyl) 2 or C,. 5 alkanoyl; or when n is 2 or more and two groups R 1 are on adjacent positions on the phenylene ring, 2 adjacent R 1 groups together represent a) C4 alkylene optionally having one double bond; b) C3 alkylene; or :) -CH =CH-CH =CH-» -CH = CII-CT r -CH,-CH=CH each optionally substimted with 1 , 2. 3 or 4 substiments said substituents each independently selected from the group consisting of C M alkyl, C alkoxy, halogen, cyano, nitro, C,. 5 alkanoyl and -CONR7R8 (wherein R7 and R8 are as defined below); m is an integer from 0 to 4 and R 2 , which may be the same or different when m is greater than 1 , is fluorine, chlorine, bromine, iodine, hydroxy, mercapto, amino, nitro, C alkyl optionally containing one double bond or one triple bond, C,__, alkoxy, -CONR 7 R 8 (wherein R 7 and R 8 are as defined below), -NH(C -alkyl), -N(C M -alkyl) 2 and C, 5 alkanoyl; or when n is 2 r more and two groups R' are on adjacent positions on the phenylene ring. 2 adjacent •\' groups together represent a) C4 alkylene optionally having one double bond; b) C3 alkylene; or c) -CH = CH-CH=CH-, -CH=CH-CH,- or -CH 2 -CH =CH- each optionally substimted wi 1 , 2, 3 or 4 substiments said substiments each independently selected from the grou of C M alkyl, C,_, alkoxy, halogen, cyano, nitro, C . alkanoyl and -CONR7R (wherein R7 and R8 are as defined below); ' ' aiid 7. -ire each independently O- or -NH-; R 3 hydrogen, t-butyl or allyl; Z 3 is a group -V-W where V is -CH 2 -T- in which T is -CH,-, -O-, -S-, -(SO)- or -(SO, (provided that when V has sulphur or oxygen as its second atom, W is other than -COO and said group V optionally further carrying one or two substiments Ql and/or Q2 on carbo wher in Q' and Q 2 each independently represents C, _, alkyl or halogen; or, when Ql and Q2 are bonded to adjacent carbon atoms, Q' and Q 2 together ma ...Jduionally represent a C 3 C 4 alkylene radical optionally substituted with 1 , 2. 3 or substiments independently selected from the group consisting of C M alkyl and halogen an W represents (1) COOH. (2) -(C = O)-O-R6 wherein R6 represents a C 1 6 alkyl, C 3 . 6 cycloalkyl or aryl (as define in 3 below) group: (3) -(C = O)-NR7R8 wherein R7 and R8 each independently represent hydrogen or a Cl 6 alkyl, C3-6 cycloalkyl, aryl, heteroaryl linked to N via carbon or C7-9 aralk group wherein aryl is phenyl; heteroaryl is a 5 or 6 membered ring containing 1 to 3. heteroatoms selected from th group consisting of nitrogen and sulphur; the aryl moiety per se, the heteroaryl moiety and the aryl moiety of the aralkyl grou may be substimted on carbon with 1 -4 substiments selected from the group consistin of -COOH, -OH, -NH,, -CH,-NH,. -(CH2) -COOH, tetrazol-5-yl and -SO 3 H an the alkyl moiety may optionally carry a methyl group; (4) -SO,NHR9 wherein R9 is as defined for R7 but may additionally represent -CF 3 , CH,CF 3 or aryl as defined above: (5) SOΛ110 in which RIO represents H, C,. 6 alkyl or C . 6 cycloalkyl, (b) PO 3 R10R10 (wherein the RIO radicals, which may be the same or different, are as herein defined) (7) a tetrazol-5-yl group; (8) -CONH-SO,Rll in which Rll represents ( Ά) C-, , cycloalkyl : (b) C.- 6 -alkyl optionally substimted with substiments selected from the group consisting of aryl as defined below, C -alkyl, CF 3 or halogen: and (c) perfluoro-C 1 . 6 alkyl; wherein aryl is phenyl or phenyl having 1-5 substiments wherein the substituems are selected from the group consisting of halogen, -NO,, -CF 3 , C M alkyl, C alkoxy, -NH 2 , -NHCOCH 3 , -CONH 2 , -OCH,COOH, -NH(C M -alkyl), -N(C,. 4 -alkyl) 2 , -NHCOOC 1-4 alkyl. -OH, -COOH. -CN and -COOC alkyl; and (9) -M-Het wherein M represents S, SO or SO, and Het represents a 5 or 6 membered heterocvclic aro atic ring linked to M via a carbon atom of the aromatic ring, said aromatic ring containing 1, 2, 3 or 4 heteroatoms selected from the group consisting of O. N and S said aromatic ring optionally being substimted on carbon atoms ι f the ring with 1, 2, 3 or 4 substiments selected from the group consisting of -OH, -SH, - CN, -CF 3 , NH, and halogen; and physiologically acceptable derivatives of the compound of formula (I). 2. A compound according to claim 1 wherein R, and R, are each independently selected from hydrogen and fluorine, R 3 is hydrogen and Z 3 is a group CH,CH,COOH. - A compound , - f 'r-'."* formula (II): wherein X, Y. R', n, Z', R 2 and m are as defined in claim 1, and physiologically acceptabl derivatives thereof. 4. A compound according to claim 3 wherein R' and R 2 are each independently selecte from hvdrogen and fluorine. 5 A pharmaceutical composition comprising a compound according to any one o claims 1 to 4 together with one or more pharmaceutically acceptable carriers or diluents. 6. 4'-[N,N-bis(2-chloroethyl)amino]phenyloxycarbonyl-oxy-benzyl-4-carbamoyl-L glutamic acid. 7. 4'-[N,N-bis(2-chloroethyl)amino]phenyloxycarbonyl-oxy-benzyl-4-oxycarbonyl-L glutamatic. 8 4 ' -[N .N-bis(2-chloroethyl)amino]phenylcarbamoylo.\y-benzyl-4-oxycarbor yl-L glutamic acid. 9. 4'[N,N-bis(2-chloroethyl)amino]phenylcarbamoyloxy-benzyl-4-carbamoyl-L-glutami acid. 10. Use of a compound according to any one of claims 1 to 9 in a method of treatmen or therapy of the human or animal body. 11. A two component system for use in the control of neoplasia in a human or anima subject which comprises a compound according to any one of claims 1 to 9 and a vector o other delivery system capable of selectively infecting mmour cells in said subject, said vecto carrying a DNA or RNA sequence encoding an enzyme capable of converting said compoun to a nitrogen mustard active against said tumour. 12. A compound of the formula (IN): where R 2 , m, Z 2 , R 3 and Z 3 are as defined in claim 1, and Q is a leaving group, with the proviso that when R 2 is fluorine, chlorine, bromine, iodine, hydroxy, mercapto, amino, nitro, C, ^ alkyl. C haloalkyl. C alkoxy. C M haloalkoxy, C 2-4 alkenyl, C^ al ynyl, and C 2-4 haloalkenyl, R 3 is hydrogen or a C.. 6 alkyl group, and Z 3 is a group -CH 2 CH 2 COOX'° where X'° is hydrogen or a Cl-6alkyl group, then Q is not a Ν-succinimidyloxy group. 13. A process for the production of a compound of formula (I) as defined in clr im 1 which comprises reacting a compound of the formula (III): in which X, Y, R' and n are as defined for formula (I) and Z 4 is -O- or -ΝH- with a compound of formula (IN) in which R 2 , m, Z 2 , Z 3 and R 3 are as defined above, and Q is a leaving group or with compound of formula (V): in which R 2 , m, Z 2 , Z 3 and R 3 are as defined above, and recovering me compound of form (I) produced. 14. A process for the production of a compound of formula (II) as defined in claim which comprises reacting a compound of formula (III) as defined in claim 13 with nitrobenzyl chloroformate optionally substimted with from 1 to 4 group(s) R 2 as defined claim 3, and recovering the compound of formula (II) produced. 15. A compound of formula (V) wherein R 2 . m. Z 2 . R 3 and Z 3 are as defined in claim 1.

Inventors: NICULESCU-DUVAZ ION, SPRINGER CAROLINE JOY, NICULESCU-DUVAZ, ION, SPRINGER, CAROLINE, JOY
Applicants: CANCER RES CAMPAIGN TECH, NICULESCU DUVAZ ION, SPRINGER CAROLINE JOY, CANCER RESEARCH CAMPAIGN TECHNOLOGY LIMITED, NICULESCU-DUVAZ, ION, SPRINGER, CAROLINE, JOY
Application year: 1996
Main classification: C07C275
IPC codes: A61K31, A61K48, A61P35, C07C271, C07C275, C07F7