WO1998035982A1 - Compounds for use in adept - Google Patents

Abstract

Compounds of Formula (I) wherein A is optionally substituted phenyl or naphthyl; X is a direct bond, CH2 or oxygen; V is NH or O; r is 0-2, provided that when V is O then r is zero; Y?1 and Y2¿ are independently selected from chloro, bromo, iodo, -O-SO¿2?-C1-4alkyl and -O-SO2-phenyl; W is COOH or 1H-1,2,3,4-tetrazol-5-yl; R?1¿ is hydrogen, fluoro, chloro, bromo, C¿1-4?alkyl or C1-4alkoxy; p is 0-4 wherein values of R?1¿ may be the same or different and when p is less than 4 remaining substituent values are hydrogen; R2 is C¿1-6?alkyl, hydroxyC1-6alkyl, phenylC1-6alkyl, C1-4alkoxyC1-4alkyl, phenylC1-4alkoxyC1-4alkyl, C1-4alkylthioC1-4alkyl, phenylC1-4alkylthioC1-4alkyl or carbamoylC1-4alkyl; or an enantiomer, diastereoisomer, pharmaceutically acceptable salt, in-vivo hydrolysable ester or solvate thereof. These compounds are useful for cancer treatment using antibody directed enzyme prodrug therapy (ADEPT), particularly as carboxypeptidase B activatable prodrugs.

Description

COMPOUNDS FOR USE IN ADEPT

The invention relates to prodrug and drug compounds for use in ADEPT (antibody directed enzyme prodrug therapy) systems comprising mutant CPB (carboxypeptidase B) enzymes. The invention also relates to methods of manufacturing the compounds, pharmaceutical compositions and methods of treating diseases, especially cancer.

Targeting of drugs selectively to kill cancer cells in a patient has long been a problem for medical research. ADEPT is one approach to overcome the problem. ADEPT uses a tumour selective antibody conjugated (e.g. by gene fusion) to an enzyme. The conjugate is administered to the patient (usually intravenously), allowed to localise at the tumour site(s) and clear from the general circulation. Subsequently a prodrug is administered to the patient which is converted by the enzyme (localised at the tumour sites) into a cytotoxic drug which kills tumour cells. Since one molecule of enzyme can catalyse generation of many cytotoxic drug molecules an amplification effect is produced. Furthermore tumour cells not displaying the antigen recognised by the antibody (tumours usually display microheterogeneity) are also killed by enzymically amplified generation of the cytotoxic drug.

In International Patent Application WO 96/20011, published 4-Jul-96, we proposed a "reversed polarity" ADEPT system based on mutant human enzymes having the advantage of low immunogenicity compared with for example bacterial enzymes. A particular host enzyme was human pancreatic CPB (see for example, Example 15 [D253K]human CPB & 16

[D253R]human CPB therein) and prodrugs therefor (see Examples 18 & 19 therein). The host enzyme is mutated to give a change in mode of interaction between enzyme and prodrug in terms of recognition of substrate compared with the native host enzyme. In our subsequent International Patent Application WO 97/07769, published 6-Mar-97, further work on mutant CPB enzyme/ prodrug combinations for ADEPT was described.

A suitable enzyme mutation is a polarity change in its active site such that it turns over a prodrug with a complementary polarity; the prodrug not being significantly turned over by the unmutated host enzyme. For example, the natural host enzyme recognises its natural substrate by an ion pair interaction and this interaction is reversed in the design of mutated enzyme and complementary prodrug. Point mutations will be referred to as follows: natural amino acid (using the 1 letter nomenclature) , position, new amino acid. For example "D253K" means that at position 253 of mature active HCPB an aspartic acid (D) has been changed to lysine (K). Multiple mutations in one enzyme will be shown between square brackets with individual mutations separated by commas.

The present invention relates to the discovery of a new class of prodrugs for use in ADEPT systems based on human CPB.

According to one aspect of the present invention there is provided a compound of Formula la

wherein A is

and A is optionally substituted with upto 4 substituents independently selected from fluoro, chloro, bromo, C_]-4alkyl or C_!-4alkoxy;

X is a direct bond, CH or oxygen; Y and Y are independently selected from chloro, bromo, iodo, -0-S0₂-C_1-4alkyl and -O-

SO₂-phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from

C_1- alkyl, halo, cyano or nitro;

R is hydrogen, fluoro, chloro, bromo, C_!-4alkyl or C_1-4alkoxy; p is 0-4 wherein values of R may be the same or different and when p is less than 4 remaining substituent values are hydrogen;

R is C_1-6alkyl, hydroxyC_]-6alkyl, phenylCι_-6alkyl, C_1- alkoxy C]_-4alkyl, phenylCι_-4alkoxyCj_-4alkyl, C_1-4alkylthioC_1- alkyl, phenylC].₄alkylthioCι_-4alkyl or carbamoylC j _-4alkyl; or an enantiomer, diastereoisomer, an in-vivo hydrolysable ester, a pharmaceutically acceptable salt or a carboxypeptidase activatable prodrug thereof with the proviso that the following compounds and salts thereof are excluded N-(4-{4-[N,N-bis-(2-chloroethyl)-amino]-3-methyl-phenoxy}-benzoyl)-L- alanine, N-(4- {4- [N,N-bis-(2-chloroethyl)-amino] -phenoxy } -benzoyl)-L-alanine, N-[N-(4-{4-[N,N- bis-(2-chloroethyl)-amino]-3-methyl-phenoxy}-benzoyl)-L-alanyl]-L-glutamic acid and, N- [N-(4- { 4- [N,N-bis-(2-chloroethyl)-amino] -phenoxy } -benzoyl)-L-alanyl]-L-glutamic acid.

The excluded compounds relate to one of our earlier patent filings, International Patent Application WO 97/07769, published 6-Mar-97.

Preferably A is optionally substituted phenyl, X is oxygen, Y¹ and Y2 are independently selected from chloro, bromo or iodo, p = 0 or p = 2 and Ri is methyl, and R is C_1-6alkyl. Preferably the carboxypeptidase activatable prodrug is of Formula I

Formula I wherein A is

and A is optionally substituted with upto 4 substituents independently selected from fluoro, chloro, bromo, C_1-4alkyl or C_1-4alkoxy; X is a direct bond, CH₂ or oxygen; V is NH or O; r is 0-2, provided that when V is O then r is zero;

1 2

Y and Y are independently selected from chloro, bromo, iodo, -0-S0₂-Ci_-4alkyl and -O-

S0₂-phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from C_!-4alkyl, halo, cyano or nitro;

W is COOH or lH-l,2,3,4-tetrazol-5-yl

R¹ is hydrogen, fluoro, chloro, bromo, C_1-4alkyl or C_)-4alkoxy; p is 0-4 wherein values of R may be the same or different and when p is less than 4 remaining substituent values are hydrogen;

R is C_1-6alkyl, hydroxyC_1-6alkyl, phenylC_1-6alkyl, Cι_-4alkoxyC_1-4alkyl, phenylC_I-4alkoxyC_1-4alkyl, C_{) -4}alkylthioC]_-4alkyl, phenylC_1-4alkylthioC_1-4alkyl or carbamoylC ] _-4alkyl; or an enantiomer, diastereoisomer, pharmaceutically acceptable salt, i -vivo hydrolysable ester or solvate thereof with the proviso that the following compounds and salts thereof are excluded, N-[N-(4-{4-[N,N- bis-(2-chloroethyl)-amino]-3-methyl-phenoxy}-benzoyl)-L-alanyl]-L-glutamic acid and

N-[N-(4-{4-[N,N-bis-(2-chloroethyl)-amino]-phenoxy}-benzoyl)-L-alanyl]-L-glutamic acid. Preferably W is COOH. Preferably X is oxygen. Preferably Y¹ and Y2 are independently selected from chloro, bromo or iodo. Preferably p = 0 or, p = 2 and R' is methyl. Preferably R2 is

Preferably V is oxygen. Preferably r is 1. Preferably

A is optionally substituted phenyl.

Preferred compounds which are prodrugs are any one of the following compounds or a pharmaceutically acceptable salt thereof: (N-[4-(4-[N,N-bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanyl)-L-glutamic acid;

(N- [4-(4- [N,N-bis(2-chloroethyl)amino] -3 -methoxyphenoxy)benzoy 1] -L-alany l)-L-glutamic acid;

(N-[4-(4-[N,N-bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanyl)-L-glutamic acid;

(N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)benzoyl]-L-alanyl)-L-glutamic acid; and

(N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)-3,5-dimethylbenzoyl]-L- alanyl)-L-glutamic acid.

The compound (N-[4-(4-[N,N-bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L- alanyl)-L-glutamic acid or a pharmaceutically acceptable salt thereof is especially preferred. Preferred compounds which are drugs are any one of the following compounds or a pharmaceutically acceptable salt thereof:

N-[4-(4-[N,N-bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanine ; N- [4-(4- [N,N-bis(2-chloroethy l)amino] -3 -methoxyphenoxy)benzoyl]-L-alanine; N-[4-(4-[N,N-bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanine ; N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)benzoyl]-L-alanine; and N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)-3,5-dimethylbenzoyl]-L- alanine.

In this specification the generic term "alkyl" includes both straight-chain and branched-chain alkyl groups. However references to individual alkyl groups such as "propyl" are specific for the straight-chain version only and references to individual branched-chain alkyl groups such as "isopropyl" are specific for the branched-chain version only. An analogous convention applies to other generic terms.

Any phenyl ring in R is optionally mono- or di-substituted with substituents independently selected from Cι _4alkyl, halogen, OH, Cι_4alkoxy, C \ _4alkanoyl,

C i _4alkanoyloxy, amino, C \ _4alkylamino, di(Cι_4alkyl)amino, C^alkanoylamino, nitro, cyano, carboxy, carbamoyl, Cι _4alkoxycarbonyl, thiol, Cj^alkylsulfanyl, Cι_4alkylsulfinyl, C]_4alkylsulfonyl, C]_4alkylsulfonamido, carbamoylC_1-4alkyl, N-(monoC_1-4alkyl)- carbamoylC_1-4alkyl, N-(diC_1-4alkyl)carbamoyl-Cι_₄alkyl, hydroxyC_1-4 alkyl and C_1-4alkoxyC_1-4 alkyl.

The term "halogen" refers to fluorine, chlorine, bromine and iodine. The term "BOC" refers to tert-butyl-O-C(O)-.

Examples of Cι_-6alkyl include methyl, ethyl, propyl, isopropyl, sec-butyl, tert-butyl and pentyl; examples of Cj.₄alkyl include methyl, ethyl, propyl, isopropyl, sec-butyl and tert- butyl; examples of C_1-4alkoxy include methoxy, ethoxy and propoxy; examples of

Cj_4alkanoyl include formyl, acetyl and propionyl; examples of Cj_4alkanoyloxy include acetyloxy and propionyloxy; examples of C_1-4alkylamino include methylamino, ethylamino, propylamino, isopropylamino, sec-butylamino and tert-butylamino; examples of di- (Cι_-4alkyl)amino include di-methylamino, di-ethylamino and N-ethyl-N-methylamino; examples of Cι_4alkanoylamino include acetamido and propionylamino; examples of Cι_-4alkoxycarbonyl include methoxycarbonyl, ethoxycarbonyl and propoxy carbonyl; examples of Cj_4alkylsulfanyl include methylsulfanyl, ethylsulfanyl, propylsulfanyl, isopropylsulfanyl, sec-butylsulfanyl and tert-butylsulfanyl; examples of Cι_4alkylsulfinyl include methylsulfinyl, ethylsulfinyl, propylsulfinyl, isopropylsulfinyl, sec-butylsulfinyl and tert-butylsulfinyl; examples of Cj^alkylsulfonyl include methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, sec-butylsulfonyl and tert-butylsulfonyl; examples of Cι_4alkylsulfonamido include methylsulfonamido, ethylsulfonamido, propylsulfonamido and isopropylsulfonamido; examples of carbamoylC]_-4aIkyI include carbamoylmethyl, carbamoylethyl and carbamoylpropyl; examples of N-(monoCι_-4aIkyl)carbamoylCι_-4alkyl include N-methyl-carbamoylmethyl and N-ethyl-carbamoylethyl; examples of N- (diCι_-4alkyl)carbamoyl-C_1-4aIkyl include N,N-dimethylcarbamoylethyl and N-methyl-N- ethylcarbamoylethyl; examples of hydroxyCι_-4alkyl and hydroxyCι_-6alkyl include hydroxymethyl, hydroxyethyl, hydroxypropyl, 2-hydroxypropyl, 2-(hydroxymethyl)propyl and hydroxybutyl; examples of C_1-4alkoxyCj_-4alkyl include methoxyethyl, ethoxyethyl and methoxybutyl; examples of phenylCι_₆alkyl include benzyl, 4-hydroxybenzyl and phenethyl; examples

include benzyloxymethyl and benzyloxyethyl; examples of Cι_-4alkylthioCι_-4alkyI include methylthioethyl and ethylthioethyl; and examples

include benzylthioethyl and phenethylthioethyl.

According to another aspect of the present invention there is provided a prodrug of Formula I'

wherein A is

optionally substituted with upto 4 substituents independently selected from fluoro, chloro, bromo, C_1-4alkyl or C_1-4alkoxy; X is a direct bond, CH₂ or oxygen;

V is NH or O; r is 0-2, provided that when V is O then r is zero;

Y and Y are independently selected from chloro, bromo, iodo, -O-S0₂-C_1-4alkyl and -O- SO₂-phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from

C_1-4alkyl, halo, cyano or nitro;

R¹ is hydrogen, fluoro, chloro, bromo, Cι_-4alkyl or C_)-4alkoxy; p is 0-4 wherein values of R may be the same or different;

R is C_]-6alkyl, hydroxyC_1-6alkyl, phenylCι_-6alkyl, C_1- alkoxyC_I-4alkyl, phenylC_1- alkoxyC_]-4alkyl, C_1-4alkylthioC_1-4alkyl, phenylC_1-4alkylthioC_1-4alkyl or carbamoylC _{\ -4}alkyl ; or an enantiomer, diastereoisomer, pharmaceutically acceptable salt, in-vivo hydrolysable ester or solvate thereof with the proviso that

N-[N-(4-{4-[bis-(2-chloroethyl)-amino]-3-methyl-phenoxy}-benzoyl)-L-alanine]-L- glutamic acid and

N-[N-(4-{4-[bis-(2-chloroethyl)-amino]-phenoxy}-benzoyl)-L-alanine]-L-glutamic acid are excluded.

General, preferred and specific values for variable groups are as set out for the corresponding compounds of Formula I. The excluded compounds relate to one of our earlier patent filings, International Patent Application WO 97/07769, published 6-Mar-97.

In another aspect of the present invention there is provided a compound of Formula la

or an in-vivo hydrolysable ester or a pharmaceutically acceptable salt thereof with the proviso that N-(4- {4- [bis-(2-chloroethyl)-amino] -3 -methyl-phenoxy } -benzoyl)-L- alanine and N-(4-{4-[bis-(2-chloroethyl)-amino]-phenoxy}-benzoyl)-L-alanine are excluded. The excluded compounds relate to one of our earlier patent filings, International Patent Application WO 97/07769, published 6-Mar-97. General, preferred and specific values for variable groups are as set out for the corresponding compounds of Formula I.

The compounds of Formula la are mustard drugs obtainable by hydrolysis of the terminal amino acid from a corresponding carboxypeptidase activatable prodrug such as the terminal amino acid (or analogue thereof) from the right hand side of a compound of Formula I or I' by a reversed polarity CPB enzyme. Examples of reversed polarity CPB enzymes include any one of [D253KJHCPB, [G251T.D253K] HCPB and

[A248S,G251T,D253K]HCPB. The drug compounds may also be obtained by conventional chemical synthesis. General, preferred and specific values for variable groups are as set out for the corresponding compounds of Formula I.

A is preferably

A is most preferably unsubstituted but if A is substituted then A is preferably mono- or di- substituted, especially mono-substituted. Preferred substituents on A are fluoro, C_1-4alkyl or C]- alkoxy; more preferably fluoro or Cι_-4alkyl; more preferably fluoro or methyl. A preferred value for V is NH. When V is NH then r is preferably 1. Preferred values for X are a direct bond or -O- and X is especially -0-. Y and Y are preferably independently selected from chloro, bromo and iodo, more preferably bromo and iodo and especially bromo. Y and Y are preferably the same. Preferably R is hydrogen or C_1-4alkyl; more preferably hydrogen or methyl and especially hydrogen. If R¹ takes a value other than hydrogen then p is preferably 1 or 2, especially 1. R is preferably C_1-6alkyl or phenylC_1-6alkyl optionally mono or di substituted on phenyl with hydroxy; more preferably C_{] -6}alkyl or phenylC_1-6alkyl optionally mono substituted on phenyl with hydroxy; more preferably methyl, phenylmethyl or 4-hydroxyphenylmethyl; and especially R² is methyl.

The compounds of the present invention possess chiral centres and the present invention covers all stereoisomers and mixtures thereof. It is preferred that the chiral centres of the "dipeptide moiety" in Formula I have the configuration indicated in the Formula below which will generally give L-stereochemistry in the corresponding amino acid.

A corrresponding preferred stereochemistry applies to compounds of Formulas la and r.

Preferred individualised compounds of the invention are as follows: (N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanyl)-L-glutamic acid ;

N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanine;

(N-[4-(4- N,N-Bis-(2-bromoethyl)amino]phenyl)-benzoyl]-L-alanyl)-L-glutamic acid;

N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenyl)-benzoyl]-L-alanine;

(N-[4-(4-[N,N-Bis-(2-iodoethyl)amino]phenyl)-benzoyl]-L-alanyl)-L-glutamic acid; N-[4-(4-[N,N-Bis-(2-iodoethyl)amino]phenyl)-benzoyl]-L-alanine;

(N-[4-(4-[N,N-bis-(2-bromoethyl)amino]benzyl)-benzoyl]-L-alanyl)-L-glutamic acid;

N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]benzyl)-benzoyl]-L-alanine; and pharmaceutically acceptable salts thereof.

Compounds of the invention may form salts which are within the ambit of the invention. Pharmaceutically acceptable salts are preferred although other salts may be useful in, for example, isolating or purifying compounds.

When the compound contains a basic moiety it may form pharmaceutically acceptable salts with a variety of inorganic or organic acids, for example hydrochloric, hydrobromic, sulphuric, phosphoric, trifluoroacetic, citric or maleic acid. A suitable pharmaceutically- acceptable salt of the invention when the compound contains an acidic moiety is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a pharmaceutically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine. Solvates, for example hydrates, are also within the ambit of the invention and may be prepared by generally known methods. An in vivo hydrolysable ester of a compound of the Formula I containing carboxy group is, for example, a pharmaceutically-acceptable ester which is hydrolysed in the human or animal body to produce the parent acid. Suitable pharmaceutically-acceptable esters for carboxy include C]_-6alkoxymethyl esters for example methoxymethyl, C_1-6alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C_3-8cycloalkoxycarbonyloxyCι_-6alkyl esters for example 1-cyclohexylcarbonyloxyethyl; l,3-dioxolen-2-onylmethyl esters for example 5-methyl-l,3-dioxolen-2-onylmethyl; and C_1-6alkoxycarbonyloxy ethyl esters for example 1 -methoxycarbonyloxyethyl and may be formed at any carboxy group in the compounds of this invention. According to another aspect of the invention there is provided a pharmaceutical composition comprising a compound as defined in Formula I, I' or Formula la or an individual end product compound described herein together with a pharmaceutically acceptable diluent or carrier. A preferred pharmaceutical composition is one suitable for parenteral administration such as for example a solution, preferably sterile and suitable for injection or infusion. The compound may be supplied in dry form, such as freeze-dried, ready for solution in a suitable solvent such as for example an aqueous buffer or water.

The compositions of the invention may preferably be in a form suitable for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, or intramuscular dosing or as a suppository for rectal dosing). The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art.

The pharmaceutical compositions in the form of a sterile injectable aqueous or oily suspension, may be formulated according to known procedures using one or more appropriate dispersing or wetting agents and suspending agents. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent.

Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols. For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

The size of the dose for therapeutic or prophylactic purposes of a compound of the Formula I will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. As mentioned above, compounds of the Formula I are useful in treating tumours.

The anti-tumour activity can be demonstrated in the following model. LoVo tumour cells (10 ) (ECACC No: 87060101) are injected subcutaneously on the flank of athymic nude mice. When the tumour xenograf s are 4-5 mm in diameter antibody-mutant-CPB enzyme (for example as described in International Patent Application WO 96/20011) is administered i.v. at a dose of 2.5 mg/kg (500U CPG2 activity /kg). Following a time interval (about 72 hr) to allow the conjugate to localise to the tumour and clear from blood and normal tissues, prodrug (3 X 70 mg/kg) is administered i.p. as 3 bolus doses, hourly, over a two hour period. Tumour regression and tumour growth delays are measured. Typical conjugate doses of 0.25-10 mg/kg in combination with prodrug (3 X 50-90 mg/kg) may be tested in this model. Using 3 doses of prodrug over 2hr is preferred to a single dose.

Based on anti-tumour data with a known ADEPT system comprising F(ab')2A5B7-CPG2 a clinical dose of conjugate would generally be in range 0.025-1.0 mg/kg, and more preferably 0.5-1.0 mg/kg. Prodrug would be administered once the conjugate had localised to the tumour and cleared from blood and normal tissues (conjugate level in plasma < 1 μg/ml). Based on clinical data with the F(ab')2A5B7-CPG2 conjugate (Bagshawe, Clinical Pharmacokinetic Concepts 27, 368, 1994) this is likely to be after 48-96 hr following conjugate administration. However based on pharmokinetic data obtained with conjugates suitable for prodrugs of the present invention, prodrug would preferably be administered 24- 72 h after conjugate. The dose of prodrug would generally be administered intravenously as 3 bolus doses, hourly, over a two hour period. Based on the mouse xenograft data with F(ab')2A5B7-CPG2 conjugate/ N_r(4-[N₃N-bis(2-iodoethyl)amino]- phenoxycarbonyl)-L-glutamic acid, anti-tumour activity in patients would generally be expected with a total dose of 15-150 mg/kg. However, standard dose escalation studies of prodrug would be used to define the optimal therapeutic dose.

Compounds of this invention may be useful in combination with known anti-cancer and cytotoxic agents. If formulated as a fixed dose such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically active agent within its approved dosage range. Sequential use is contemplated when a combination formulation is inappropriate.

Drug compounds of Formula la are useful as cytotoxic agents in their own right for example by direct application (for example by injection) into tumours. Such compounds are also useful in the context of being produced in situ by a suitable ADEPT conjugate from a corresponding prodrug. Prodrugs of Formula I may also exhibit anti-tumour activity in their own right when administered in vivo; the anti-tumour activity thereof is expected to be enhanced when used in conjunction with an antibody-enzyme conjugate in ADEPT.

According to another aspect of the invention there is provided a compound of Formula I or a pharmaceutically-acceptable salt thereof, for use as a medicament. According to another aspect of the invention there is provided a compound of Formula

I or a pharmaceutically-acceptable salt thereof, for use in preparation of a medicament for treatment of tumours.

According to another aspect of the present invention there is provided a method of treating tumours, by administering an effective amount of a prodrug compound of Formula I or a pharmaceutically-acceptable salt thereof, to a mammal in need of such treatment, the mammal having a tumour localised ADEPT conjugate for prodrug activation at the tumour. According to another aspect of the present invention there is provided an ADEPT product containing a conjugate and a prodrug compound of Formula I or a pharmaceutically- acceptable salt thereof as a combined preparation for sequential use in treatment of tumours. According to a further aspect of the present invention we provide a method for the 5 delivery of a cytotoxic drug to a site which comprises administering to a host a first component, which first component comprises an antibody or fragment thereof capable of binding a given antigen, the antibody or fragment thereof being conjugated to a mutant CPB enzyme (preferably mutant human pancreatic CPB) capable of converting a compound of Formula I or pharmaceutically acceptable salt thereof into a cytotoxic drug; followed by

10 administration to the host of a second component, which second component comprises a compound of Formula I or a pharmaceutically acceptable salt thereof convertible under the influence of the enzyme to the cytotoxic drug. Preferably the components are delivered in effective amounts. The preferred host is human. Preferably the enzyme is [A248S,G251T,D253K]HCPB and the antibody is preferably humanised CDR grafted

15 806.077 antibody. The site to which the cytotoxic drug is to be delivered is preferably tumour cells which will generally be present in a tumour-bearing mammalian host such as a human.

When the said first component is administered to the tumour bearing host, the antibody or antibody fragment moiety of the conjugate directs the conjugate to the site of the tumour and binds the conjugate to the tumour cells.

20 Once unbound conjugate has been substantially eliminated from the host to be treated, for example by clearance from the host after the elapsing of an appropriate time or after accelerated clearance, for example as described in Br. J. Cancer (1990), 6J_, 659-662, the second component may be administered to the host. It is highly desirable to substantially eliminate unbound conjugate from the host before administration of the second component,

25 since otherwise cytotoxic drug may be generated other than at the site of the tumour thus resulting in general toxicity to the host rather than site specific toxicity.

Specific tumours of interest include carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, prostate, stomach, cervix, thyroid and skin. CEA positive tumours are particularly preferred such as for example CEA positive colorectal

30 tumours. Note that targetting of the ADEPT system to different tumours is accomplished by changing the targetting component of the conjugate. Antibody may be obtained from a hybridoma using standard techniques known in the art such as documented in Fenge C, Fraune E & Schuegerl K in "Production of Biologicals from Animal Cells in Culture" (Spier RE, Griffiths JR & Meignier B, eds) Butterworth-Heinemann, 1991, 262-265 and Anderson BL & Gruenberg ML in "Commercial Production of Monoclonal Antibodies" (Seaver S, ed), Marcel Dekker, 1987, 175-195. The cells may require re-cloning from time to time by limiting dilution in order to maintain good levels of antibody production.

Antibodies useful in ADEPT have been described as follows. Antibody BW 431/26 was described in Haisma, H.J. et al., Cancer Immunol. Immunother., 34: 343-348 (1992). Antibodies L6, 96.5, and 1F5 were described in European Patent 302 473. Antibody 16.88 was described in International Patent Application WO90/07929. Antibody B72.3 was described in European Patent No. 392 745. Antibody CEM231 was described in European Patent No. 382 411. Antibodies HMFG-1 and HMFG-11 (Unipath Ltd, Basingstoke, Hants, United Kingdom) react with a mucin-like glycoprotein molecule on milk fat globule membranes and may be used to target breast and ovarian cancers. Antibody SM3 (Chemicon International Ltd, London, United Kingdom) reacts with core protein of mucin and may be used to target breast and ovarian cancer. Antibodies 85A12 (Unipath Ltd, Basingstoke, Hants, United Kingdom) and ZCEA1 ( Pierce Chemical Company, Chester, United Kingdom) react with tumour antigen CEA. Antibody PR4D1 (Serotec, Oxford, United Kingdom) reacts with a colon tumour associated antigen. Antibody E29 (Dako Ltd, High Wycombe, United Kingdom) reacts with epithelial membrane antigen. Antibody C242 is available from CANAG Diagnostics, Gothenberg, Sweden.

Generally, antibodies useful in ADEPT are poorly internalised by the tumour cells they recognise. This allows the targeted prodrug-activating enzyme to be resident on the cell surface and thus generate active drug at the tumour site from circulating prodrug.

Internalisation of antibody may be assayed by known techniques, for example as set out in Jafrezou et al., Cancer Research 52: 1352 (1992) and in Press et ak, Cancer Research, 48: 2249 (1988).

According to another aspect of the present invention there is provided a matched two component system designed for use in a host in which the components comprise: (i) a substantially non-immunogenic first component that is a targeting moiety capable of binding with a tumour associated antigen, the targeting moiety being linked to a mutated form of CPB enzyme capable of converting a prodrug of Formula I into an antineoplastic drug and; (ii) a second component that is a prodrug of Formula I convertible under the influence of the enzyme to the antineoplastic drug, the prodrug not being significantly convertible into antineoplastic drug in the host by natural unmutated host enzyme.

The term "the prodrug is not significantly convertible into antineoplastic drug in the host by natural unmutated host enzyme" means that the prodrug does not give undue untargeted toxicity problems on administration to the host. The term "substantially non-immunogenic" means that the first component can be administered to the host on more than one occasion without causing significant host immune response as would be seen with for example the use of a mouse antibody linked to a bacterial enzyme in a human host.

Preferably the mutated enzyme is based on an enzyme from the same species as the host for which the system is intended for use but the mutated enzyme may be based on a host enzyme from a different species as long as the structure of the enzyme is sufficiently conserved between species so as not to create undue immunogenicity problems. More preferably the mutated enzyme is any one of [D253K]HCPB, [G251T,D253K] HCPB and [A248S,G251T,D253K]HCPB. [A248S,G251T,D253K]HCPB is especially preferred. Preferably the targeting moiety is an antibody, especially an antibody fragment such as for example F(ab')2- Linkage to enzyme for conjugate synthesis may be effected by any suitable method such as for example use of heterobifunctional reagents as cross-linkers or preferably by gene fusion. Antibody may be from the same host (eg use of mouse antibody in mice) or the antibody may be manipulated such that it is not significantly recognised as foreign in the chosen host (eg use of chimeric, CDR grafted or veneered mouse antibodies in humans). Preferably the first component is a fusion protein between an anti-CEA antibody and a reversed polarity human CPB enzyme. A preferred anti-CEA antibody is the antibody obtainable from hybridoma 806.077 deposited as ECACC deposit no. 96022936. Hybridoma 806.077 antibody was deposited at the European Collection of Animal Cell Cultures (ECACC), PHLS Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom on 29th February 1996 under accession no. 96022936 in accordance with the Budapest Treaty. Humanisation of antibody 806.077 and production of fusion proteins thereof has been described in International Patent Application WO 97/42329, Zeneca Limited, published 13-Nov-97.

Transplantation of the variable domains of rodent antibodies into the constant domains of human antibodies (chimeric antibodies) or building the antigen binding loops (CDRs) of rodent antibodies into a human antibody (CDR grafting) have both been shown to greatly decrease the immunogenicity of the rodent antibody in preclinical studies in monkeys and in patients. Even CDR grafted antibodies incorporate a large number (>50) of amino acids from the rodent antibody sequence into the human framework. Despite this in monkeys and patients greatly reduced immunogenicity has been reported. This provides evidence that mutating a very limited number of amino acids in the catalytic site of a host enzyme is likely to result in an enzyme with minimal immunogenicity and certainly lower immunogenicity than a non-host enzyme. The reader is directed to the following references: A. Mountain and J. R. Adair, Biotechnology and Genetic Engineering Reviews 10, 1-142, 1992; G. Winter and W. J. Harris, Trends in Pharmacological Sciences, 14, 139-143, 1993; I.I. Singer et al, J. Immunol, 150. 2844-57, 1993; J. Hakimi et al, J. Immunol, 147, 11352-59, 1991 and; J. D. Isacs et al, The Lancet, 340, 748-752, 1992. The constant region domains may be for example human IgA, IgE, IgG or IgM domains. Human IgG2 and 3 (especially IgG2) are preferred but IgG 1 and 4 isotypes may also be used. Human antibodies per se may also be used such as those generated in mice engineered to produce human antibodies. (Fishwald et al. in Nature Biotechnology (1996), \4, 845-851).

According to another aspect of the invention there is provided a matched two component system designed for use in a host in which the components comprise: (i) a substantially non-immunogenic first component that is a targeting moiety capable of binding with a tumour associated antigen, the targeting moiety being linked to a mutated form of CPB enzyme capable of converting a prodrug of the invention into an antineoplastic drug and;

(ii) a second component that is a prodrug of the invention convertible under the influence of the enzyme to the antineoplastic drug, the prodrug not being significantly convertible into antineoplastic drug in the host by natural unmutated host enzyme. Preferably the enzyme is [A248S,G251T,D253K]HCPB and the targeting moiety is humanised CDR grafted 806.077 antibody.

Although the compounds of the Formula I and la are primarily of value as therapeutic agents for use in warm-blooded animals (including man), they are also useful as pharmacological standards for use in the development of new biological tests and in the search for new pharmacological agents.

In another aspect the present invention provides a process for preparing a compound of the Formula I or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof which process comprises a) deprotecting a compound of the Formula II:

Formula II

, or b) deprotecting a compound of the Formula Ha,

Formula Ha

1 1 wherein Pr and Pr independently represent hydrogen or carboxy protecting groups which may be the same or different, other variable groups are as hereinbefore defined, and wherein any other functional group is optionally protected with the proviso there is at least one protecting group and optionally, if desired, forming a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof. In one embodiment, deprotection step a) is preferred.

Protecting groups may in general be chosen from any of the groups described in the literature or known to the skilled chemist as appropriate for the protection of the group in question, and may be introduced by conventional methods. Protecting groups may be removed by any convenient method as described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with minimum disturbance of groups elsewhere in the molecule. Specific examples of protecting groups are given below for the sake of convenience, in which "lower" signifies that the group to which it is applied preferably has 1-4 carbon atoms. It will be understood that these examples are not exhaustive. Where specific examples of methods for the removal of protecting groups are given below these are similarly not exhaustive. The use of protecting groups and methods of deprotection not specifically mentioned is of course within the scope of the invention.

A carboxyl protecting group may be the residue of an ester-forming aliphatic or arylalkyl alcohol or of an ester-forming silanol (the said alcohol or silanol preferably containing 1-20 carbon atoms).

Examples of carboxy protecting groups include straight or branched chain (l-12C)alkyl groups (eg isopropyl, t^butyl); lower alkoxy lower alkyl groups (eg methoxymethyl, ethoxymethyl, isobutoxymethyl); lower aliphatic acyloxy lower alkyl groups, (eg acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl); lower alkoxycarbonyloxy lower alkyl groups (eg 1-methoxycarbonyloxyethyl, 1 -ethoxycarbonyloxy ethyl); aryl lower alkyl groups (eg benzyl, p-methoxybenzyl, o^nitrobenzyl, r nitrobenzyl, benzhydryl and phthalidyl); tri(lower alkyl)silyl groups (eg trimethylsilyl and t^butyldimethylsilyl); tri(lower alkyl)silyl lower alkyl groups (eg trimethylsilylethyl); and (2-6C)alkenyl groups (eg allyl and vinylethyl).

Methods particularly appropriate for the removal of carboxyl protecting groups include for example metal-catalysed hydrogenolysis or acid-, base-, or enzymically-catalysed hydrolysis. It should be noted however that base catalysed hydrolysis is not suitable for mustard compounds due to potential damage thereof in the presence of base. Examples of hydroxyl protecting groups include lower alkyl groups (eg t-butyl), lower alkenyl groups (eg allyl); lower alkanoyl groups (eg acetyl); lower alkoxycarbonyl groups (eg t-butoxycarbonyl); lower alkenyloxycarbonyl groups (eg allyloxycarbonyl); aryl lower alkoxycarbonyl groups (eg benzoyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl); tri lower alkylsilyl (eg trimethylsilyl, tibutyldimethylsilyl) and aryl lower alkyl (eg benzyl) groups.

Examples of amino protecting groups include formyl, aralkyl groups (eg benzyl and substituted benzyl, p-methoxybenzyl, nitrobenzyl and 2,4-dimethoxybenzyl, and triphenylmethyl); di-p-anisylmethyl and furylmethyl groups; lower alkoxycarbonyl (eg t- butoxycarbonyl); lower alkenyloxycarbonyl (eg allyloxycarbonyl); aryl lower alkoxycarbonyl groups (eg benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl; trialkylsilyl (eg trimethylsilyl and t-butyldimethylsilyl); alkylidene (eg methylidene); benzylidene and substituted benzylidene groups. Methods appropriate for removal of hydroxy and amino protecting groups include, for example, acid-, base-, metal- or enzymically-catalysed hydrolysis, for groups such as p-nitrobenzyloxycarbonyl, hydrogenation and for groups such as o-nitrobenzyloxycarbonyl, photolytically.

The reader is referred to Advanced Organic Chemistry, 4th Edition, by Jerry March, published by John Wiley & Sons 1992, for general guidance on reaction conditions and reagents. The reader is referred to Protective Groups in Organic Synthesis, 2nd Edition, by Green et al, published by John Wiley & Sons for general guidance on protecting groups.

The compound of Formula II may be prepared by reacting a compound of Formula III

Formula III

with a compound of Formula IV, Y^a-O- Y , or with a compound of Formula IV^a, Y^a- L, wherein L is a leaving group (such as for example Cl or Br), Y^a and Y rb° i-ndependently represent -SO₂-C_!-4alkyl and -S0 -phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from C_]-4alkyl, halo, cyano or nitro and other variable groups are as hereinbefore defined, under suitable conditions (for example using a polar aprotic organic solvent and a base at a non-extreme temperature) to give a compound of Formula II where Y and Y represent -0-S0₂-C_]-4alkyl and -0-SO₂-phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from C_1-4alkyl, halo, cyano or nitro; and optionally, if desired, treating the compound thus obtained with an alkali metal chloro, bromo or iodo salt under suitable conditions (for example using a polar aprotic solvent) to give a

1 2 compound of Formula II where Y and Y represent chloro, bromo or iodo. Compounds of ι 2

Formula II where Y and Y represent chloro, bromo or iodo may also be prepared directly from compounds of Formula III by reaction with a phosphine in the presence of a chloro, bromo or iodo reagent. Suitable examples include use of triphenylphosphine in the presence of I₂, CBr₄ or CCl₄.

The compound of Formula III may be prepared by reacting a compound of Formula V

COOPr²

Formula V with ethylene oxide under suitable conditions. Suitable conditions include the presence of an aqueous protic solvent at a non-extreme temperature.

The compound of Formula V may be prepared by hydrogenating a compound of Formula VI

COOPr¹

Formula VI under suitable conditions. Suitable conditions include hydrogen in the presence of a catalyst such as palladium on carbon, and an organic solvent such as for example ethyl acetate at a non-extreme temperature.

The compound of Formula VI may be prepared by reacting a compound of Formula VII

Formula VII with a compound of Formula VIII

COOPr¹

Formula VIII under suitable amide bond forming conditions. Typically a carbodiimide coupling reagent is used in the presence of an organic solvent (preferably an anhydrous polar aprotic organic solvent) at a non-extreme temperature, for example in the region -10 to 40°, typically ambient temperature of about 20°. Compounds of Formulas VII and VIII are either commercially available or readily synthesised from known starting materials using standard techniques.

The compound of Formula Ila may be prepared by reacting a compound of Formula Ilia

Formula Ilia

with a compound of Formula IV, Y^a-O-Y , or with a compound of Formula IV^a, Y^a- L, wherein L is a leaving group (such as for example Cl or Br), Y^a and Y^b independently represent -SO₂-C_1-4alkyl and -S0₂-phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from C_1-4alkyl, halo, cyano or nitro and other variable groups are as hereinbefore defined, under suitable conditions (for example using a polar aprotic organic solvent and a base at a non-extreme temperature) to give a compound of Formula Ila where Y and Y represent -O-SO₂-C_1-4alkyl and -O-S0₂-phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from Cι_-4alkyl, halo, cyano or nitro; and optionally, if desired, treating the compound thus obtained with an alkali metal chloro, bromo or iodo salt under suitable conditions (for example using a polar aprotic solvent) to give a

1 2 compound of Formula Ila where Y and Y represent chloro, bromo or iodo. Compounds of

1 2

Formula Ila where Y and Y represent chloro, bromo or iodo may also be prepared directly from compounds of Formula Ilia by reaction with a phosphine in the presence of a chloro, bromo or iodo reagent. Suitable examples include use of triphenylphosphine in the presence of I₂, CBr₄ or CCl₄. The compound of Formula Ilia may be prepared by reacting a compound of Formula

Va

Formula Va with ethylene oxide under suitable conditions. Suitable conditions include the presence of an aqueous protic solvent at a non-extreme temperature. The compound of Formula Va may be prepared by hydrogenating a compound of

Formula Via

Formula Via under suitable conditions. Suitable conditions include hydrogen in the presence of a catalyst such as palladium on carbon, and an organic solvent such as for example ethyl acetate at a non-extreme temperature. The compound of Formula Via may be prepared by reacting a compound of Formula

VII as defined above with a compound of Formula Villa

COOPr²

Formula Villa under suitable amide bond forming conditions. Typically a carbodiimide coupling reagent is used in the presence of an organic solvent (preferably an anhydrous polar aprotic organic solvent) at a non-extreme temperature, for example in the region -10 to 40°, typically ambient temperature of about 20°.

Compounds of Formulas VII and Villa are either commercially available or readily synthesised from known starting materials using standard techniques. Compounds of Formula II or Ila can also be prepared by reacting a compound of

Formula IX

with a compound of Formula XIV

Formula XIV wherein Wa represents a carboxyl group in protected form or a tetrazol-5-yl group, under suitable amide bond forming conditions. Typically a carbodiimide coupling reagent is used in the presence of an organic solvent (preferably an anhydrous polar aprotic organic solvent) at a non-extreme temperature, for example in the region -10 to 40°, typically ambient temperature of about 20°.

Compounds of Formula XIV can be prepared by reacting a compound of Formula XV

Formula XV wherein Pr³ represents an amino group in protected form, with a compound of Formula XVI

Formula XVI wherein Wa represents amino or hydroxyl, under suitable amide bond forming conditions. Typically a carbodiimide coupling reagent is used in the presence of an organic solvent (preferably an anhydrous polar aprotic organic solvent) at a non-extreme temperature, for example in the region -10 to 40°, typically ambient temperature of about 20°. Compounds of Formula IX can be prepared by deprotecting a compound of Formula X

Formula X wherein PH represents a carboxyl group in protected form. Suitable reaction conditions include use of hydrogen in the presence of a catalyst such as palladium on carbon at ambient temperature.

Compounds of Formula X can be prepared by reacting compounds of Formula XI

Formula XI with a compound of Formula IV, Y^a-O-Y , or with a compound of Formula IV^a, Y^a- L, wherein L is a leaving group (such as for example Cl or Br), Y^a and Y independently represent -S0₂-C_l-4alkyl and -S0₂-phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from C_]-4alkyl, halo, cyano or nitro and other variable groups are as hereinbefore defined, under suitable conditions (for example using a polar aprotic organic solvent and a base at a non-extreme temperature) to give a compound of Formula X where Y¹ and Y represent -0-S0₂-C)_-4alkyl and -O-S0 -phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from C_{1 -4}alkyl, halo, cyano or nitro; and optionally, if desired, treating the compound thus obtained with an alkali metal chloro, bromo or iodo salt under suitable conditions (for example using a polar aprotic solvent) to give a

1 2 compound of Formula X where Y and Y represent chloro, bromo or iodo. Compounds of

1 2

Formula X where Y and Y represent chloro, bromo or iodo may also be prepared directly from compounds of Formula XI by reaction with a phosphine in the presence of a chloro, bromo or iodo reagent. Suitable examples include use of triphenylphosphine in the presence ofI₂, CBr₄ or CCl₄.

Compounds of Formula XI can be prepared by reacting compounds of Formula XII

Formula XII with ethylene oxide under suitable conditions. Suitable conditions include the presence of an aqueous protic solvent at a non-extreme temperature. Compounds of Formula XII can be prepared by hydrogenating compounds of Formula XIII

Formula XIII under suitable conditions. Suitable conditions include hydrogen in the presence of a catalyst such as palladium on carbon, and a polar aprotic organic solvent at a non-extreme temperature.

Compounds of Formula XIII are either commercially available or readily synthesised from known starting materials using standard techniques.

Biological activity was tested in various ways as described herein and pharmacological properties of the compounds of the Formula I vary with structural change as expected. No physiologically unacceptable toxicity was observed at the effective dose for compounds tested of the present invention.

The invention will now be illustrated in the following non-limiting Examples (and supporting Reference Examples) in which, unless otherwise stated :- (i) evaporations were carried out by rotary evaporation in vacuo and work-up procedures were carried out after removal of residual solids by filtration;

(ii) operations were carried out at room temperature, that is in the range 18-25°C and under an atmosphere of an inert gas such as argon;

(iii) column chromatography (by the flash procedure) was performed on Merck KIESELGEL™ silica (Art. 9385) obtained from E. Merck, Darmstadt, Germany;

(iv) yields are given for illustration only and are not necessarily the maximum attainable;

(v) the end-products of the Formula I and la have satisfactory microanalyses and their structures were confirmed by nuclear magnetic resonance (NMR) and mass spectral techniques.

(vi) intermediates were not generally fully characterised and purity was assessed by thin layer chromatography, HPLC, infra-red (IR) or NMR analysis; (vii) melting points are uncorrected and were determined using a Mettler SP62 automatic melting point apparatus or an oil-bath apparatus;

(viii) the following abbreviations have been used:- ADEPT antibody directed enzyme prodrug therapy

BOC tert-butoxycarbonyl

CPB carboxypeptidase B

DCCI 1 ,3 -dicyclohexylcarbodiimide

DMA N,N-dimethylacetamide

DMAP 4-dimethyl-aminopyridine

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

EDCI 1 -(3 -dimethylaminopropy l)-3 -ethy 1-carbodiimide hydrochloride

EEDQ 2-ethoxy- 1 -ethoxycarbonyl- 1 ,2-dihydroquinoline

HCPB human carboxypeptidase B, preferably pancreatic

HOBT 1 -hydroxybenzotriazole hydrate

RT room temperature

TFA trifluoroacetic acid

THF tetrahydrofuran

TMSI trimethylsilyliodide

Z benzyloxycarbonyl

(ix) Chemical shifts are in δ (ppm) and peak multiplicities are designated as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; dt, doublet of triplets; q, quartet; m, multiplet; br, broad.

(x) All temperatures are in degrees centigrade.

(xi) CELITE™ was used as a commercial source of diatomaceous earth. Reference Example 1 (see Scheme 6)

Preparation of a) (N-[4-(phenoxy)-benzoyI]-L-alanyl)-L-glutamic acid

and b) N-[4-(phenoxy)-benzoyl]-L-alanine

To a solution of starting material dibenzyl (N-[4-(phenoxy)-benzoyl]-L-alanyl)-L-glutamate

(1.7 g) in ethyl acetate (50 ml) was added 10% palladium on carbon (50% moist; 0.3g). The mixture was stirred under an atmosphere of hydrogen for lh. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate evaporated to give the desired end product a) as an oil 1.2 g (91%).

NMR (DMSO-d₆): 8.5 (t, IH), 8.2 (t, IH), 8.0 (d, 2H), 7.5-7.0 (m, 7H), 4.6 m (s, IH), 4.3 (m,

IH), 2.4-1.9 (m, 4H), 1.4 (d, 2H).

The starting material was made from 4-phenoxybenzoic acid and L-alanyl-L-glutamic acid di- tert-butyl ester as described in Example 3.

NMR (DMSO-d₆): 8.5 (t, IH), 8.2 (t, IH), 8.0 (d, 2H), 7.5-7.0 (m, 7H), 4.6 m (s, IH), 4.3 (m,

IH), 2.4-1.9 (m, 4H ), 1.4 (d, 2H).

End product b) was made using analogous methodology.

Example 1.

Preparation of (N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanyl)-L- glutamic acid

(Compound 6, Scheme 2, X = O, R = Me )

A) Starting material (N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L- alanyl)-L-glutamic acid dibenzyl ester (1.5 g) was dissolved in ethyl acetate (100 ml) and 10% palladium on carbon (50% moist with water, 1 g) added. The mixture was stirred under an atmosphere of hydrogen for 2 h. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate evaporated to give the desired end product as an oil 0.8 g (68%). NMR (DMSO-d₆) δ: 8.3 (d,lH) ; 8.1 (d,lH); 7.8(d,lH); 7.0-6.9 (m,6H); 4.5 (m,lH); 4.1 (m, IH); 3.8 (t, 4H); 3.6 (t, 4H); 2.3-1.8 (m,4H); 1.3 (d,3H). The starting material was prepared as follows.

B) 4-(4-Nitrophenoxy)benzoic acid (Rarick, Brewster and Daines J.A.C.S. 1993, 55, 1289-90) (3.8g) was dissolved in DMF (65 ml) and L-alanyl-L-glutamic acid dibenzyl ester hydrochloride (Beilharz G.R. et al Aust J. Chem. 1983, 36, 751-8 ) ( 6.5 g ) added, followed by HOBT (6.5 g), then EDCI (8.4g) and then triethylamine (6 ml). The mixture was stirred at RT for 4 h, poured into sodium hydrogen carbonate solution (750 ml) and extracted twice with ethyl acetate. The combined organic extracts were washed with water, 0.5M citric acid solution, dried and evaporated to dryness. The residue was chromatographed on silica gel eluting with 1.1 hexane/ethyl acetate to give N-(4-(4-nitrophenoxy)-benzoyl)-L-alanyl-L- glutamic acid dibenzyl ester as an oil, 4.5 g (47%).

NMR (CDC1₃) δ: 8.3 (d, 2H), 7.9 (d, 2H), 7.3 (m, 10H), 7.1-6.9 (m, 6H), 5.2 (s, 2H), 5.1 (s, 2H), 4.7 (m, 4H), 2.5-2.0 (m, 4H), 1.5 (d, 3H).

C) Product from step B (4.5 g) was dissolved in ethyl acetate (100 ml) and 10% Platinum on carbon (50% moist with water, 0.9 g) added. The mixture was stirred under an atmosphere of hydrogen for 3 h. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate evaporated to dryness. The residue was chromatographed on silica gel, eluting with 1.1 hexane/ethyl acetate to give (N-[4-(4-aminophenoxy)-benzoyl]-L-alanyl)-L- glutamic acid dibenzyl ester as an oil 3.0 g (71%). NMR (CDCI₃) δ: 7.7 (d, 2H), 7.3 (m, 10H), 7.1-6.8 (m, 10H), 5.2 (s, 2H), 5.0 (s, 2H), 4.7 (m, 2H), 2.5-2.0 (m, 4H), 1.4 (d, 3H).

D) Product from step C (3.0 g) was dissolved in 1 : 1 acetic acid/water (60 ml) and ethylene oxide (3 g) passed in during 1 h. The mixture was allowed to stand at RT for 18 h, evaporated to dryness, the residue treated with sodium hydrogen carbonate solution (150 ml) and extracted twice with ethyl acetate. The combined organic extracts were washed with water and evaporated to dryness. The residue was chromatographed on silica gel eluting with ethyl acetate to give (N-[4-[4-[N,N-bis-(2-hydroxyethyl)amino]phenoxy]-benzoyl]-L-alanyl)-L- glutamic acid dibenzyl ester as an oil 2.3 g (67%). NMR (CDCI₃) δ: 7.7 (d, 2H), 7.3 (m, 10H), 7.1-6.6 (m, 10H), 5.2 (s, 2H), 5.0 (s, 2H), 4.7 (m, 2H), 3.9 (t, 4H), 3.6 (t, 4H), 2.5-2.0 (m, 4H). 1.5 (d, 3H).

E) The product from step D (2 g) in dichloromethane (40 ml) was cooled to -10° under an inert atmosphere and triethylamine (1.6 ml) added. To this mixture was added a solution of methanesulphonic anhydride (2.06 g) in dichloromethane (5 ml) keeping the temperature at -10° to -5°. The reaction was held at this temperature for a further lh, washed twice with ice- cold water, dried, and evaporated to an oil. This oil was dissolved in DMF (25 ml) and lithium bromide (2.4 g) was added. The mixture as heated at 80° for 15 min, cooled, poured into water (300 ml) and extracted twice with diethyl ether . The combined organic extracts were washed with water, dried and evaporated to dryness. The residue was chromatographed on silica gel eluting with 1 :1 hexane/ethyl acetate to give the desired starting material as an oil (1.5 g). NMR (CDCI₃) δ: 7.8 (d, 2H), 7.4 (m, 10H), 7.0 (m, 5H), 6.8 (d, 3H), 5.2 (s, 2H), 5.1 (s, 2H), 4.7 (m, 2H), 3.8 (t, 4H), 3.5 (t, 4H), 2.4-2.0 (m, 4H), 1.5 (d, 3H).

Example 2 Preparation of N-[4-(4-[N,N-bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanine

Compound 6 (Scheme 3, X = O, R = Me)

A) To a solution of starting material N-[4-[4-[N,N-Bis-(2-bromoethyl)amino]phenoxy]- benzoyl]-L-alanine tert-butyl ester (313 mg) in dichloromethane (2 ml) was added TFA

(5 ml). The mixture was allowed to stand at ambient temperature for 1 hour and then evaporated to dryness. The residue was azeotroped twice with ethyl acetate and evaporated to give the desired end product as an oil (320 mg). NMR (CDC1₃) δ: 7.8 (d, 2H), 7.2-6.8 (m, 8H), 4.8 (m,lH), 3. 8 (t, 4H), 3.6 (t, 4H), 1.6 (d, 3H).

B) The starting material was made using an analogous procedure to that described in Example 1 but using L-alanine tert-butyl ester hydrochloride in place of L-alanyl-L-glutamic acid dibenzyl ester hydrochloride at step B.

NMR data for compounds in Scheme 3

Compound 2 (X = O, R = Me) (CDC1₃) δ: 8.2-7.1 (d, 8H), 6.8 (m, IH), 4.6 (m, IH), 1.4 (m,

12H).

Compound 3 (X = O,R = Me) (CDC1₃) δ: 7.7-6.6 (m, 9H), 4.6 (m, IH), 1.4 (m, 1 IH). Compound 4 (X = 0,R = Me) (CDC1₃) δ: 7.7-6.7 (m, 9H); 4.6 (m,lH); 3.9 (t, 4H); 3.6(t, 4H),

1.5 (m, 12H)

Compound 5 (X = 0,R = Me) (CDC1₃) δ: 7 .7-6.6 (m, 9H), 4.6 (m, IH), 3.7 (t, 4H), 3.4 (t,

4H), 1.5 (m, 12H).

Example 3 Preparation of prodrug a) (N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenyl)-benzoyl]-L-alanyl)-L-gIutamic acid

(Compound 6, Scheme 1, X = bond) and corresponding drug; b) N- [4-(4- [N,N-Bis-(2-bromoethyl)amino] phenyl)-benzoyl] -L-alanine

(Compound 6, Scheme 3, X = direct bond, R = Me).

A) Starting material, di-tert-butyl N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenyl)- benzoyl]-L-alanyl-L-glutamate (305 mg) in dichloromethane (10 ml) and TFA (5 ml) was allowed to stand at RT for 2 h and then evaporated to dryness. The residue was azeotroped twice with ethyl acetate and evaporated to give the desired end product a) as an oil (354 mg). The starting material was prepared as follows.

B) 4-(4-Nitrophenyl)benzoic acid (Byron D.J. et al J. Chem. Soc. 1966, 840-5) (625 mg) was dissolved in DMF (25 ml) and L-alanyl-L-glutamic acid di-tert-butyl ester (518 mg, see Japanese patent application JP 54009224, Ciba-Geigy) was added, followed by HOBT (212 mg) then EDCI (361 mg). The mixture was stirred at RT for 18 h, poured into sodium hydrogen carbonate solution and extracted twice with ethyl acetate. The combined organic extracts were washed with water, 0.5M citric acid solution, dried and evaporated to dryness. The residue was chromatographed eluting with 7:3 hexane/ ethyl acetate to give di-tert-butyl N-(4-(4-nitrophenyl)-benzoyl)-L-alanyl-L-glutamate (622 mg).

C) The product from step B (645 mg) was dissolved in ethyl acetate (35 ml) and 10% Palladium on carbon (50% moist with water, 65 mg) was added. The mixture was stirred under an atmosphere of hydrogen until uptake ceased. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate was evaporated to dryness. The residue was chromatographed eluting with 1.1 hexane/ethyl acetate to give di-tert-butyl N-[4-(4- aminophenyl)-benzoyl]-L-alanyl-L-glutamate (506 mg).

D) Product from step C (470mg) was dissolved in 1 : 1 acetic acid/water (50 ml) and ethylene oxide (2.8 g) passed in during lh. The mixture was allowed to stand at RT for 18 h. After evaporation to a small volume the residue was treated with sodium hydrogen carbonate solution (150 ml) and extracted twice with ethyl acetate. The combined organic extracts were washed with water and evaporated to dryness. The residue was chromatographed, eluting with ethyl acetate to give di-tert-butyl N-[4-(4-[N,N-bis-(2-hydroxyethyl)amino]phenyl)-benzoyl]- L-alanyl-L-glutamate (436 mg). E) The product from step D (307 mg), triphenylphosphine (525 mg), imidazole (136 mg) and carbon tetrabromide (664 mg) were dissolved in dry dichloromethane(20 ml). The mixture was stirred at RT for 3 h under nitrogen, filtered and the filtrate was evaporated to dryness. The residue was purified by chromatography using 1 : 1 hexane/ethyl acetate as eluent, to give the desired starting material (333 mg).

Note: The corresponding drug (b) was made using the procedure described above but using L-alanine tert-butyl ester in place of L-alanyl-L-glutamic acid di-tert-butyl ester (see Scheme 3).

NMR data for compounds in Scheme 1; Synthesis of prodrug Compound 6 (X = Direct bond)

Compound 2

NMR (DMSO-d₆) δ: 1.34 (d, 3H), 1.36 (s, 18H), 1.77 (m, IH), 1.91 (m, IH), 2.29 (m, 2H),

4.13 (m, IH), 4.53 (m,lH), 7.89 (d, 2H), 8.05 (m, 4H), 8.18 (d, IH), 8.32 (d, 2H), 8.59 (d, IH); MS 554 [M-H]^". Compound 3 NMR (DMSO-d₆) δ: 1.34 (d, 3H), 1.38 (s, 18H), 1.76 (m, IH), 1.92 (m, IH), 2.28 (m, 2H),

4.14 (m, IH), 4.32 (m, IH), 5.31 (s, 2H), 6.63 (d, 2H), 7.43 (d, 2H), 7.62 (d, 2H), 7.88 (d, 2H), 8.14 (d, IH), 8.38 (d,lH); MS 526 [MH]⁺.

Compound 4

NMR (DMSO-d₆) δ: 1.33 (d, 3H), 1.35 (s, 18H), 1.75 (m, IH), 1.89 (m, IH), 2.28 (m, 2H),

3.45 (m, 4H), 3.52 (m, 4H), 4.11 (m, IH), 4.50 (m, IH), 4.75 (t, 2H), 6.74 (d, 2H), 7.54 (d,

2H), 7.65 (d, 2H), 7.89 (d, 2H), 8.18 (d, IH), 8.22 (d, IH); MS 614 [MH]⁺.

Compound 5

NMR (DMSO-d₆) δ: 1.34 (d, 3H), 1.35 (s, 18H), 1.76 (m, IH), 1.92 (m, IH), 2.29 (m, 2H),

3.61 (m, 4H), 3.82 (m, 4H), 4.13 (m, IH), 4.52 (m, IH), 6.83 (d, 2H), 7.60 (d, 2H), 7.69 (d,

2H), 7.92 (d, 2H), 8.14 (d, IH), 8.42 (d, IH); MS 740 [MH]⁺ (2xBr). Compound 6

NMR (DMSO-d₆) δ: 1.34 (d, 3H), 1.79 (m, 18H), 1.98 (m, IH), 2.32 (m, 2H), 3.60 (m, 4H), 3.82 (m, 4H), 4.24 (m, IH), 4.52 (m, IH), 6.83 (d, 2H), 7.62 (d, 2H), 7.68 (d, 2H), 7.91 (d, 2H), 8.1 1 (d, lH),,8.42 (d, IH); MS 626 [M-H]^~ (2xBr).

NMR data for compounds in Scheme 3; synthesis of drug Compound 6 (X = Direct bond, R = CH₃)

Compound 2 NMR (DMSO-d₆) δ: 1.37 (d. 3H), 1.39 (m, 9H), 4.33 (m, IH), 7.88 (m, 2H), 8.02 (m, 4H),

8.32 (m, 2H), 8.75 (d, IH);

MS 371 [MH]⁺.

Compound 3

NMR (DMSO-d₆) δ: 1.35 (d, 3H), 1.38 (s, 9H), 4.33 (m, IH), 5.30 (s, 2H), 6.63 (d, 2H), 7.43 (d, 2H), 7.63 (d, 2H), 7.87 (d, 2H), 8.55 (d, IH);

MS 341 [MH]⁺.

Compound 4

NMR (DMSO-d₆) δ: 1.36 (d, 3H), 1.41 (s, 9H), 3.45 (m, 4H), 3.53 (m, 4H), 4.32 (m, IH),

4.72 (m, 2H), 6.75 (d, 2H), 7.53 (d, 2H), 7.67 (d, 2H), 7.89 (d, 2H), 8.55 (d, IH); MS 429 [MH]⁺.

Compound 5

NMR (DMSO-d₆) δ: 1.37 (d, 3H), 1.39 (s, 9H), 3.62 (m, 4H), 3.83 (m, 4H), 4.52 (m,lH), 6.84

(d, 2H), 7.61 (d, 2H), 7.69 (d, 2H), 7.91 (d, 2H), 8.58 (d, 2H);

MS 555 [MH]⁺ (2xBr). Compound 6

NMR (DMSO-d₆) δ: 1.39 (d, 3H), 3.61 (t, 4H), 3.84 (t, 4H), 4.43 (m, IH), 6.85 (d, 2H), 7.63

(d, 2H), 7.70 (d, 2H), 7.91 (d, 2H), 8.60 (d, IH);

MS 499 [MH]⁺ (2xBr). Example 4

Preparation of (N-[4-(4-[N,N-Bis-(2-iodoethyl)amino]phenyl)-benzoyl]-L-alanyl)-L- glutamic acid

(Compound 8, Scheme 1, X = Direct Bond)

A) The starting material N-[4-(4-[N,N-Bis-(2-iodoethyl)amino]phenyl)-benzoyl]-L- alanyl-L-glutamic acid di-tert-butyl ester (0.465 g) was dissolved in dry dichloromethane (15 ml) under nitrogen at ambient temperature. To this solution was added TMSI (0.379 ml) and the reaction stirred for 2 h at RT. The reaction mixture was then poured onto a stirred solution of diethyl ether (120 ml) and acetic acid (5 ml). The yellow solid produced was filtered off, washed with diethyl ether and dried under vacuum to give the desired product as a hydroiodide salt (0.304 g).

NMR (DMSO-d₆) δ: 1.34 (d, 3H),1.76 (m,lH),1.94 (m,lH), 2.28 (t, 2H), 3.36 (t, 4H), 3.78 (t, 4H), 4.02 (m,lH), 4.49(m,lH), 6.77 (d, 2H), 7.61 (d, 2H), 7.66 (d, 2H), 7.89 (d, 2H), 8.12

(d, lH), 8.4 (d, IH).

MS 720 [M-H]^"

The starting material was prepared as follows.

B) Triphenylphosphine (0.876 g), imidazole (0.231 g) and iodine (0.851 g) were dissolved in dry dichloromethane (20 ml). A solution of N-[4-[4-[N,N-Bis-(2- hydroxyethyl)amino]phenyl]-benzoyl]-L-alanyl-L-glutamic acid di-tert-butyl ester (0.5 g) in dichloromethane (20 ml) was added. The reaction mixture was stirred at RT for 3 h under nitrogen, filtered to remove imidazole hydroiodide and the filtrate was evaporated. The foamlike residue was purified by flash chromatography on silica gel using (6.5: 1) dichloromethane/ethyl acetate as eluent, to give the desired starting material (0.469 g) as a gum.

NMR (DMSO-d6) δ: 1.35 (s+d, 21H), 1.75 (m, IH), 1.9 (m, IH), 2.27 (t, 2H), 3.3 (t, 4H), 3.77 (t, 4H), 4.12 (m, IH), 4.5 (m,lH), 6.75 (d, 2H), 7.6 (d, 2H), 7.65 (d,2H), 7.9 (d,2H), 8.15 (d,lH), 8.42 (d,2H). MS 832 [M-H]-

The N-[4-[4-[N,N-Bis-(2-hydroxyethyl)amino]phenyl]-benzoyl]-L-alanyl-L-glutamic acid di- tert-butyl ester was prepared using analogous methodology with that set out in Example 3, steps B-D.

Example 5

Preparation of N-[4-(4-[N,N-Bis-(2-iodoethyI)amino]phenyl)-benzoyI]-L-alanine Compound 8, Scheme 3, X = Direct Bond, R = Me

A) The starting material N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenyl)-benzoyl]-L- alanine tert-butyl ester (0.578 g) was dissolved in dry dichloromethane (15 ml) under nitrogen at RT. To this solution was added TMSI (0.303 ml) and the reaction mixture was stirred for 1.5 h at RT. The reaction mixture was then poured onto a stirred solution of diethyl ether (120 ml) and acetic acid (5 ml). The yellow solid produced was filtered off, washed with diethyl ether and dried under vacuum to give the desired end product as a hydroiodide salt

(0.524 g).

NMR (DMSO-d₆/acetic acid-d₄) δ: 1.39 (d, 3H), 3.3 (t, 4H), 3.76 (t, 4H), 4.42 (q, IH),

6.75 (d, 2H), 7.6 (d, 2H), 7.66 (d, 2H), 7.9 (d,2H). MS 591 [M-H]-.

The starting material was prepared as follows.

B) Triphenylphosphine (1.22 g), imidazole (0.32 g) and iodine (1.22 g) were dissolved in dry dichloromethane (20 ml). A solution of N-[4-(4-[N,N-Bis-(2- hydroxyethyl)amino]phenyl)-benzoyl]-L-alanine tert-butyl ester (0.5g) in dichloromethane (20 ml) was added to this solution. The reaction mixture was stirred at RT for 18 h under nitrogen, filtered to remove imidazole hydroiodide and the filtrate was evaporated. The foamlike residue was purified by flash chromatography on silica gel using 20:1 dichloromethane/ethyl acetate as eluent, to give the desired starting material (0.599 g) as a gum. NMR (DMSO-d₆) δ: 1.35 (d, 3H), 1.4(s, 9H), 3.32 (t, 4H), 3.77 (t, 4H), 4.33 (m, IH), 6.77 (d, 2H), 7.6 (d, 2H), 7.67 (d, 2H), 7.9 (d, 2H), 8.58 (d, IH). MS 649 [MH]⁺

The_N-[4-(4-[N,N-Bis-(2-hydroxyethyl)amino]phenyl)-benzoyl]-L-alanine tert-butyl ester was prepared using analogous methodology with that set out in Example 3, steps B-D.

Example 6

Preparation of (N-[4-(4-[N,N-bis-(2-bromoethyl)amino]benzyI)-benzoyl]-L-alanyI)-L- glutamic acid Compound 7 (Scheme 4)

A) Starting material di-tert-butyl N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]benzyl)- benzoyl]-L-alanyl-L-glutamate was deprotected using TFA by the procedure described in Example 3.

NMR compound 7: (d₆-DMSO) δ: 1.31 (d, 3 H), 1.78 (m,l H), 1.95 (m,l H), 2.28 (t, 2 H), 3.54 (t, 4 H), 3.71 (t, 4 H), 3.85 (s, 2 H), 4.10 (m,l H), 4.46 (m, 1 H), 6.65 (d, 2 H), 7.06 (d, 2 H), 7.26 (d, 2 H), 7.78 (d, 2 H), 8.09 (d, 1 H), 8.33 (d,l H); MS 640 [M-H]^"; Found: C, 47.7; H, 4.9; N, 5.8. C₂₆H₃₁Br₂N₃O₆.).0.7 Et₂O.0.5 CF₃CO₂H requires C, 47.7; H,5.2; N, 5.6. The starting material for this compound was prepared as follows.

B) Triethylamine (435 μl) and diphenylphosphoryl azide (791 μl) were added to a stirred solution of 4-carboxy-4'-ethoxycarbonyldiphenylmethane (A Wallon et al, Chem. Ber. 1990, 123, 375) (852 mg) in anhydrous dioxan (10 ml). After 1 h, thin layer chromatography indicated that a small amount of starting material remained. A second portion of diphenylphosphoryl azide (160 μl) was added and stirring was continued for a further 90 min. Anhydrous benzyl alcohol (930 μl) was added and the reaction mixture was warmed over 30 min to a temperature of 110°and stirred for 2 h at this temperature under reflux. The reaction mixture was cooled overnight and evaporated to dryness. The crude product was purified by flash column chromatography eluting with 0-4% v/v ethanol in CH₂C1₂ to yield ethyl 4- [4- (benzyloxycarbonylamino)benzyl]benzoate as an oil (1.768 g) containing aromatic impurities. NMR δ (d₆-DMSO): 1.26 (t, 3 H), 3.92 (s, 2 H), 4.25 (q, 2 H), 5.10 (s, 2 H), 7.10 (d, 2 H), 7.10-7.50 (m, 22 H), 7.84 (d, 2 H), 9.70 (s, 1 H); MS 390 [MH]⁺.

C) A solution of product from step B (1.76 g) in ethanol (24 ml) was stirred 18 h with 2N aqueous sodium hydroxide. At the end of the reaction the mixture was warmed gently to dissolve a fine white precipitate, acidified (2N HCl) to pH 1.0 and extracted with ethyl acetate (2 x 50 ml). The ethyl acetate solution was washed with brine, dried and evaporated to give an off-white solid (1.7 g). The solid, 4-[4-(benzyloxycarbonylamino)benzyl]benzoic acid, was triturated with diethyl ether, isolated in a sinter and vacuum dried (875 mg). NMR δ (d₆-DMSO): 3.90 (s, 2 H), 5.10 (s, 2 H), 7.12 (d. 2 H), 7.28 (d, 2 H), 7.38 (d, 2 H), 7.83 (d, 2 H), 9.68 (s, 1 H).

D) EDCI.HC1 (506 mg) and N-methylmorpholine (290 μL) were added to a stirred solution of the product from step C (865 mg) and 1 -hydroxybenzotriazole hydrate (356 mg) in redistilled DMF at 0°. Stirring was continued 2 h at 0° and a solution of L-alanyl-L-glutamic acid di-tert-butyl ester (see Japanese patent application JP 54009224, Ciba-Geigy) (prepared by the catalytic hydrogenation of 1.63 g Z-L-alanyl-L-glutamic acid di-tert-butyl ester over 10% Pd/C in ethyl acetate) in redistilled DMF (8 ml) was added. The mixture was stirred 2 h whilst allowing to warm to RT and the clear solution was kept overnight. The DMF was removed by rotary evaporation at high vacuum and the residue was partitioned between ethyl acetate (2 x 30 ml) and water (20 ml). The ethyl acetate solution was washed with aqueous NaHC0₃, brine, dried and evaporated to dryness. The crude product was purified by flash column chromatography, eluting with 0-5% EtOH in CH₂C1₂ to yield di-tert-butyl N-[4-(4-[N-(benzyloxycarbonyl)amino]benzyl)-benzoyl]-L-alanyl-L- glutamate (compound 4) as a hard white foam, 1.23 g.

NMR δ (d₆-DMSO): 1.31 (d, 3 H), 1.37 (s,18 H), 1.76 (m,l H), 1.90 (m,l H), 2.25 (t, 2 H), 3.90 (s, 2 H), 4.12 (m,l H), 4.48 (m, 1 H), 5.10 (s, 2 H), 7.10 (d, 2 H), 7.20 (d, 2 H), 7.35 (m, 7 H), 7.78 (d, 2 H), 8.11 (d, 1 H), 8.34 (d,l H), 9.65 (s, 1 H) MS 674 [MH]⁺. E) A solution of product from step D (1.22 g) in ethyl acetate (40 ml) was stirred 2 h over 10% Pd/C (300 mg) in an atmosphere of H₂. The solution was filtered through diatomaceous earth and the filtrate was evaporated to give di-tert-butyl N-[4-(4- aminobenzyl)-benzoyl]-L- alanyl-L-glutamate as a hard white foam (compound 5; 932 mg). NMR δ (d₆-DMSO): 1.30 (d, 3 H), 1.35 (s, 18 H), 1.75 (m, 1 H), 1.90 (m, 1 H), 2.25 (t, 2 H), 3.76 (s, 2 H), 4.12 (m, 1 H), 4.48 (m, 1 H), 4.84 (br s, 2 H), 6.47 (d, 2 H), 6.84 (d, 2 H), 7.23 (d,2 H), 7.78 (d, 2 H), 8.11 (d, 1 H), 8.32 (d,l H) MS 540 [MH]⁺.

F) The product of step E was reacted with ethylene oxide (as described in the corresponding step in Example 3) to give di-tert-butyl N-[4-(4-[N,N-Bis-(2- hydroxyethyl)amino]benzyl)benzoyl]-L-alanyl-L-glutamate.

NMR (DMSO-d₆): 8.3 (d, IH), 8.1 (d, IH), 7.8 (d, 2H), 7.2 (d, 2H), 6.9 (d, 2H), 6.6 (d, 2H), 4.7 (t, 2H), 4.8 (m, IH), 4.1 (m, IH), 3.8 (s, 2H), 3.4 (m, 4H), 3.3 (m, 4H), 2.3 (t, 2H), 1.8 (m, 2H), 1.4 (s, 18H), 1.3 (s, 3H). G) The product from step F (615 mg) in dichloromethane (20 ml) was cooled to 0° under an inert atmosphere and triethylamine (413 μl) was added. To this mixture was added a solution of methanesulphonic anhydride (426 mg) in dichloromethane (5 ml) keeping the temperature at 0°. The reaction was held at this temperature for 1 h, washed twice with ice- cold water, dried, and evaporated to dryness. The residue was dissolved in DMF (8 ml) and lithium bromide (853 mg) was added. The mixture was heated at 90° for 10 min and cooled to RT. The DMF was removed by rotary evaporation at 0.1 mmHg and the residue was partitioned between ethyl acetate (2 x 10 ml) and water (10 ml). The combined organic extracts were washed with water, dried and evaporated to dryness. The crude product was purified by chromatography, eluting with 0-5% EtOH/CH₂Cl₂ to yield the desired starting material.

NMR δ (d₆-DMSO): 1.3 (d, 3 H), 1.35 (s, 18 H), 3.5 (m, 3 H), 3.7 (m, 1 H), 3.85 (s, 2 H), 6.64 (d, 2 H), 7.05 (d, 2 H), 7.25 (d, 2 H), 7.77 (d, 2 H), 8.1 (d, 1 H), 8.3 (d, 1 H).

Example 7 Preparation of N- [4-(4- [N,N-Bis-(2-bromoethy l)amino] benzy l)-benzoy 1] -L-alanine

Compound 7 (Scheme 5)

A) Starting material N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]benzyl)-benzoyl]-L-alanine tert-butyl ester was deprotected by treatment with TFA as described in Example 3 to give the desired end product. NMR: δ (d₆-DMSO): 1.35 (d, 3 H), 3.52 (t, 4 H), 3.68 (t, 4 H), 3.85 (s, 2 H), 4.38 (m,l H), 6.64 (d, 2 H), 7.05 (d, 2 H), 7.28 (d, 2 H), 7.78 (d, 2 H), 8.50 (d, 2 H) MS 509/511/513 [M-H]^"; Found: C, 46.2; H, 4.4; N, 4.6. C₂₁H₂₄Br₂N₂O₃.0.55CF₃CO₂H requires C, 46.2; H, 4.3; N, 4.9. The starting material was prepared as follows.

B) Triethylamine (870 μl) was added to a stirred solution of 4-carboxy-4'- ethoxycarbonyldiphenylmethane (A Wallon et al, Chem. Ber. 1990, 123, 375) (1.70 g) in t- butanol (20 ml). Diphenylphosphoryl azide (1.77 ml) was added and the reaction mixture was stirred for 4 h under reflux. The cooled reaction mixture was partitioned between ethyl acetate and water. The ethyl acetate solution was filtered through diatomaceous earth to remove a fine white precipitate and evaporated to dryness. The crude product was purified by flash column chromatography eluting with 0-4% EtOH in CH₂C1₂ to yield ethyl 4- [4- (BOCamino)benzyl]benzoate as an oil (compound 2; 902 mg). NMR δ (d₆-DMSO): 1.28 (t, 3 H), 1.45 (s, 9 H), 3.90 (s, 2 H), 4.26 (q, 2 H), 7.09 (d, 2 H), 7.31 (d, 2 H), 7.36 (d, 2 H), 7.85 (d, 2 H), 9.22 (s, 1 H); MS 356 [MH]⁺.

C) The product from step B (1.15 g) was dissolved in HCl in ethyl acetate(3M,12 ml). A white precipitate slowly formed. After 3 h the suspension was evaporated to give ethyl 4-(4- aminobenzyl)benzoate as a white solid.

D) The white solid from step C was dissolved in acetic acid (25 ml) and water (5 ml) and cooled to 0°. Ethylene oxide (ca. 10 g) was condensed into the solution which was kept overnight at RT under a dry ice/isopropanol reflux condenser. The reaction mixture was evaporated to dryness and the residue was partitioned between ethyl acetate (2 x 30 ml) and water (20 ml) with the addition of solid NaHCO₃ until the aqueous phase had pH >7.0. The ethyl acetate solution was washed with brine, dried and evaporated. The crude oily product was purified by flash column chromatography, eluting with 0-6% EtOH in CH₂C1₂ to yield ethyl 4-(4-[N,N-bis-(2-hydroxyethyl)amino]benzyl)benzoate as a colourless oil (Compound 4, 2.06 g). NMR δ (d₆-DMSO): 1.28 (t, 3 H), 3.4-3.6 (m, 8 H), 3.98 (s, 2 H), 4.26 (q, 2 H), 6.58 (d, 2 H), 6.97 (d, 2 H), 7.30 (d, 2 H), 7.85 (d, 2 H); MS 344 [MH]⁺.

E) The product from step D (2.05 g) was stirred in a mixture of EtOH (16 ml) and 2N aqueous NaOH (16.5 ml) to give a clear solution which was kept overnight. The EtOH was removed by rotary evaporation and the aqueous residue was acidified (cone. HCl) to pH 3.0. The white solid precipitate was filtered off, washed with water and vacuum dried to give 4-(4-[N,N-bis-(2-hydroxyethyl)amino]benzyl)benzoic acid (Compound 5): 228 mg. NMR δ (d₆-DMSO): 3.33 (m, 4 H), 3.48 (m, 4 H), 3.83 (s, 2 H), 6.58 (d, 2 H), 6.97 (d, 2 H), 7.28 (d, 2 H), 7.82 (d, 2 H); MS 314 [M-H]^".

F) A solution of the product from step E (210 mg), H-Ala-O-tert-Bu.HCl (135 mg), and HOBT (100 mg) in redistilled DMF (5 ml) was stirred at 0°. EDCI.HC1 (142 mg) and N- methylmorpholine (81 μL) was added. Stirring was continued 2 h at 0° and at RT overnight. The solution was evaporated to dryness and the residue was partitioned between ethyl acetate (2 x 10 ml) and water (10 ml). The ethyl acetate solution was washed with aqueous NaHC0₃, brine, dried and evaporated. The crude product was purified by flash chromatography, eluting with 0-5% EtOH v/v in CH₂C1₂ to yield the desired starting material as a white foam (Compound 6), 253 mg. NMR δ (d₆-DMSO): 1.35 (d, 3 H), 1.39 (s, 9 H), 3.35 (m, 4 H), 3.48 (m, 4 H), 3.83 (s, 2 H), 4.30 (m,l H), 4.68 (t, 2 H), 6.58 (d, 2 H), 6.97 (d, 2 H), 7.28 (d, 2 H), 7.75 (d, 2 H), 8.50 (d, 2 H); MS 443 [MH]⁺. Example 8

Assay of activity of mutant HCPB and native HCPB against des-mustard analogues of glutamic acid prodrugs

For convenience, assessment of K_m and k_cat was performed using prodrug analogues (des mustard compounds) because these enzyme parameters are believed to be affected only by the "dipeptide moiety" of the prodrugs.

Purified mutant [A248S,G251T,D253K]HCPB enzyme and native human CPB were assayed for their ability to cleave glutamic acid from a glutamic acid prodrug analogue (compound without a mustard moiety). Cleavage liberates a mono-carboxylic acid compound from the di-carboxylic acid (glutamic acid containing) prodrug analogue. Conversion of "glutamic acid prodrug analogues" to the "drug analogues" was measured using a HPLC based assay.

Prodrug analogue was diluted in the range 1-0.003 mM in 0.025 M Tris-HCL buffer, pH 7.5. Where necessary prodrug samples were adjusted to pH 7.5 with 0.1M NaOH. [A248S,G251T,D253K]HCPB or native HCPB, both at a final concentration of 2- 0.005μg/ml, were added to the prodrug analogues (500μl reaction volume prewarmed to 37° for 2 min) to start the reaction. Samples were incubated for 15-30 minutes at 37°. The reaction was terminated by the addition of 500 μl 98.8 % MeCN, 0.2% TFA and the samples placed on ice. The amount of product (drug analogue) produced was then quantified by HPLC.

For example the compound of Reference Example 1 showed substrate recognition and turnover, K_m of 0.18 mM and a k_cat of 56 sec^"1, by [A248S,G251T,D253K]HCPB enzyme together with desired lack of turnover by native HCPB.

Example 9

Cytotoxicity of glutamic acid prodrug and the des-glutamate mustard drug in LoVo human colorectal tumour cells.

The differential cytotoxicity to tumour cells of the glutamic acid prodrug of Example 1 and corresponding des glutamate drug of Example 2 has been demonstrated by the following means. LoVo colorectal tumour cells were incubated with prodrug or drug over a final concentration range of 5 X 10^"4 to 5 X 10^"8 M in 96- well (2,500 cells/well) microtitre plates for 1 h at 37°. The cells were then washed and incubated for a further three days at 37°C. After washing to remove dead cells, TCA was added and the amount of cellular protein adhering to the plates was assessed by addition of SRB dye as described by P. Skehan et al, J. Natl. Cancer Inst. 82, 1107 (1990). Potency of the compounds was assessed by the concentration required to inhibit cell growth by 50% (IC50).

Initial experiments with LoVo tumour cells treated with the prodrug of Example 1 showed an IC₅₀ of 300 μM while the cells treated with the drug of Example 2 showed an IC₅₀ of 10 μM (mean data from 3 separate studies). Thus the prodrug was over 25 fold less cytotoxic to LoVo colorectal tumour cells than the drug demonstrating usefulness in ADEPT with suitable mutants of HCPB. In later experiments, cells treated with the drug of Example 2 showed an IC₅₀ of about 30 μM. Prodrug compounds tested of the present invention were generally at least three fold less cytotoxic to LoVo colorectal tumour cells than the corresponding drug. The compound of Example 33 could be used as a direct cytotoxic agent because it did not exhibit differential cytotoxicity compared with its corresponding drug in this assay.

Example 10 Anti-tumour activity of prodrugs and humanised antibody-mutant HCPB fusion protein in xenografted mice.

The anti-tumour efficacy of suitable prodrugs and humanised anti-CEA antibody-mutant HCPB fusion protein can be demonstrated in the following model.

LoVo colorectal tumour cells (ECACC no. 87060101) (1 X 10⁷) are injected s.c. into athymic nude mice. When the tumours are 4-5mm in diameter the conjugate is administered i.v. at doses between 10-100 mg/kg. Following localisation of the fusion protein to the tumours and allowing a suitable time interval for residual conjugate to clear from the bloodstream and normal tissues (1-4 days, or alternatively 12-48 h, preferably 12-24 h) the prodrug is administered either i.v or i.p. to the mice in doses ranging between 10-1000 mg/kg either as a single or multiple doses. The combination of antibody-enzyme fusion protein and prodrug cause the tumours to grow significantly slower than untreated control tumours or tumours treated with either the same dose of conjugate or prodrug alone. These studies demonstrate that the combination of the humanised antibody-mutant CPB fusion protein and the mutant CPB prodrugs result in significant anti-tumour activity.

The time interval between conjugate administration and prodrug administration can be optimised as required. For example, residual conjugate levels could be monitored to allow for clearance of conjugate from the bloodstream and normal tissues whilst allowing tumour localised conjugate to be optimised relative thereto for maximal therapeutic effect.

Another example would be to set up experiments using a series of intervals and simply select the interval giving the best therapeutic effect.

Example 11

Pharmaceutical compositions

The following illustrates a representative pharmaceutical dosage form of the present invention which may be used for therapeutic purpose (ADEPT) in humans. Injectable solutions i) A sterile aqueous solution, for injection, containing per ml of solution:

Antibody-enzyme 1.0 mg

Sodium acetate trihydrate 6.8 mg

Sodium chloride 7.2 mg

Tween 20 0.05 mg

A typical dose of antibody-enzyme is 30 mg followed 3 days later by prodrug. ii) Assemble the following for final prodrug dosage form preparation: glass vials (3 x

20 ml) each containing 600 mg of prodrug (of Formula I, Example 1) ; 3 ampoules containing 11 ml of 2.15% (w/v) sodium hydrogen carbonate; needles (3 x 18G); hydrophobic filters for venting the vials; and 3 x single use sterile 0.22μ filters for aqueous solutions. All materials must be stored at 2-8°C. (Note a drug of Formula la such as prepared in Example 2 could replace the prodrug for non- ADEPT related direct use of active drug without antibody-enzyme.). These operations are preferably to be performed under sterile conditions. No more than 1 hour prior to dosing, one vial of prodrug is vented with a needle and hydrophobic filter.

Sterile 2.15% w/v sodium hydrogen carbonate (10 ml) is then added directly through the bung via a syringe and needle. With the vent still in place the vial is swirled gently to obtain a clear solution (this will be 50 mg/ml as free base). The required dose volume is withdrawn into a sterile syringe through a sterile filter. The filter is then replaced by a sheathed sterile needle and the syringe unit kept cool prior to administration. Each remaining vial is prepared in an identical manner at intervals of one hour to allow for example three separate doses to be given 1 hour apart.

Example 12

Preparation of (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyl]-L- tert-leucyl)-L-glutamic acid

A) Starting material dibenzyl (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3- methylphenoxy)benzoyl]-L-tert-leucyl)-L-glutamate (0.44 g) was dissolved in ethyl acetate (20 ml) and 10% palladium on carbon (50% moist with water, 0.4 g) added. The mixture was stirred under an atmosphere of hydrogen for 5 h. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate evaporated to give the desired end product as a yellow foam 0.3 g (88%).

NMR (DMSO-d₆) δ 8.2 (d, IH); 7.9 (d, 2H); 7.7 (d, IH); 7.3 (d, IH); 7.0 (d, 2H); 6.9 (s, IH);

8.85 (d, IH); 4.5 (d, IH); 4.2 (m, IH); 3.6 (t, 4H); 3.3 (t, 4H); 2.3 (m, 5H); 2.0 (m, IH); 1.8 (m, IH); 1.0 (s, 9H).

The starting material was prepared as follows.

B) Benzyl 4-hydroxybenzoate (2 g) was dissolved in DMF (20 ml) and ground potassium carbonate (1.5 g) added. The solution was stirred for 5 min under an inert atmosphere and then 5-fluoro-2-nitrotoluene (1.5 g) added. The reaction was heated to 120 ° for 4 h and then evaporated to dryness. The resulting residue was partitioned between brine and ethyl acetate and the aqueous phase extracted twice with ethyl acetate. The combined organic extracts were dried and evaporated to dryness to yield benzyl 4-(3-methyl-4-nitrophenoxy)benzoate as a brown oil, 3.1 g (97%).

NMR (DMSO-d₆) δ 8.1 (m, 3H); 7.4 (m, 5H); 7.2 (m, 3H); 7.1 (m, IH); 5.3 (s, 2H); 2.5 (s, 3H). C) Product from step B (33 g) was dissolved in ethyl acetate (300 ml) and 10% Platinum on carbon (4 g) added. The mixture was stirred under an atmosphere of hydrogen for 15 h. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate evaporated to give benzyl 4-(4-amino-3-methylphenoxy)benzoate as an oil, 29 g (96%). NMR (DMSO-d₆) δ 7.9 (d, 2H); 7.4 (m, 5H); 6.9 (d, 2H); 6.7 (m, 3H); 5.3 (s, 2H); 2.1 (s, 3H). D) Product from step C (15 g) was dissolved in 1 : 1 acetic acid/water (200 ml), cooled to -10 o and ethylene oxide (27 g) passed in. The reaction was allowed to stand at RT for 15 h, evaporated to dryness and the residue taken up in ethyl acetate and washed with water. The organics were dried and evaporated to dryness. The resulting oil was chromatographed on silica gel eluting with ethyl acetate to give benzyl 4-(4-[N,N-bis(2-hydroxyethyl)amino]-3- methylphenoxy)benzoate 12.8g (67%).

NMR (DMSO-d₆) δ 7.9 (d, 2H); 7.4 (m, 5H); 7.2 (d, IH); 7.0 (d, 2H); 6.9 (m, 2H); 5.3 (s, 2H); 3.4 (q, 4H); 3.0 (t, 4H); 2.2 (s, 3H).

E) Product from step D (12.8 g) was dissolved in dichloromethane (100 ml) and triethylamine (12.7 ml) added. The reaction was cooled to -10 ° and then methane sulphonyl chloride (5.9 ml) in dichloromethane (50 ml) added under an inert atmosphere over 6 min. The reaction was then allowed to warm to RT over 4.5 h with stirring. The solution was then diluted with dichloromethane (100 ml) and washed with water and brine. The organics were dried and evaporated to dryness to yield benzyl 4-(4-[N,N-bis(2-mesyloxyethyl)amino]-3- methylphenoxy)benzoate as an orange oil 17.4g (95%). F) Product from step E (17.4 g) was dissolved in DMF (100 ml) and lithium chloride (12.9 g) added. The reaction was heated to 100 ° for 2 h under an inert atmosphere and then evaporated to dryness. The resulting residue was partitioned between water and ethyl acetate and extracted into ethyl acetate. The combined organic extracts were dried and then evaporated to dryness to give benzyl 4-(4-[N,N-bis(2-chloroethyl)amino]-3- methylphenoxy)benzoate as a brown oil 12.8 g (93%). NMR (DMSO-d₆) δ 7.9 (d, 2H); 7.4 (m, 5H); 7.3 (d, IH); 7.05 (d, 2H); 6.95 (s, IH); 6.9 (d,

IH); 5.3 (s, 2H); 3.75 (t, 4H); 3.35 (t, 4H); 2.3 (s, 3H).

G) Product from step F (12.8 g) was dissolved in ethyl acetate (150 ml) and 10% palladium on carbon (50 % moist with water, 10 g) added. The mixture was stirred under an atmosphere of hydrogen for 15 h. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate evaporated. The resulting solid was triturated with 20 % diethyl ether/hexane to give 4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoic acid as a white powder, 8.2g (80%).

NMR (DMSO-d₆) δ 12.7 (bs, IH); 7.9 (d, 2H); 7.3 (d, IH); 7.0 (d, 2H); 6.95 (s, IH); 6.9 (d, IH); 3.5 (t, 4H); 3.38 (t, 4H); 2.25 (s, 3H).

H) N-Boc-L-tert-leucine (1 g) was dissolved in DMF (20 ml) and dibenzyl L-glutamate

(2.4 g) added, followed by HOBT (0.67 g), EDCI (0.95 g) and triethylamine (0.7 ml). The mixture was stirred at RT under an inert atmosphere for 15 h. The mixture was evaporated to dryness and the residue partitioned between 1 M citric acid and ethyl acetate. The organics were washed with saturated sodium hydrogen carbonate, dried and evaporated to dryness to give dibenzyl N-(N-Boc-L-tert-leucine)-L-glutamate 2.3 g (96%).

NMR (DMSO-d₆) δ 8.25 (d, IH); 7.3 (bs, 10H); 6.4 (d, IH); 5.1 (s, 2H); 5.05 (s, 2H); 4.35

(m, IH); 3.8 (d, IH); 2.4 (t, 2H); 2.1 (m, IH); 1.9 (m, IH); 1.3 (s, 9H); 0.9 (s, 9H).

I) Product from step H (2.3 g) was dissolved in HCl in ethyl acetate (3 M, 20 ml) and stirred for 4 h. The solution was evaporated to dryness and then azeotroped with toluene to give dibenzyl N-(L-tert-leucyl)-L-glutamate hydrochloride, 2 g.

NMR (DMSO-d₆) δ 8.9 (d, IH); 8.2 (bs, 2H); 7.3 (bs, 10H); 5.1 (s, 2H); 5.05 (s, 2H); 4.4 (m,

IH); 3.55 (bs, IH); 2.5 (t, 2H); 2.1 (m, IH); 1.9 (m, IH); 0.9 (s, 9H).

J) Product from step G (0.3 g) was dissolved in DMF (20 ml) and product from step I (0.429 g) added, followed by HOBT (0.12 g), EDCI (0.17 g) and N-methylmorpholine (0.19 ml). The mixture was stirred at RT under an inert atmosphere for 15 h. The mixture was evaporated to dryness and the resulting residue was chromatographed on silica gel eluting with 1.1 hexane/ethyl acetate to give the desired starting material as an oil, 0.44 g (62%).

NMR (DMSO-d₆) δ 8.4 (d, IH); 7.9 (d, 2H);7.7 (d, IH); 7.3 (bs, 1 IH); 7.0 (d, 2H); 6.9 (s, IH); 8.85 (d, IH); 5.1 (s, 2H); 5.05 (s, 2H); 4.5 (d, IH); 4.4 (m, IH); 3.6 (t, 4H); 3.3 (t, 4H);

2.5 (m, 2H); 2.3 (s, 3H); 2.1 (m, IH); 1.8 (m, IH); 1.0 (s, 9H). Example 13

Preparation of (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyl]-L- leucyl)-L-glutamic acid

A) Starting material dibenzyl (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3- methylphenoxy)benzoyl]-L-leucyl)-L-glutamate was deprotected using the procedure described in Example 12, step A, to give the desired end product.

NMR (DMSO-d₆) δ 8.3 (d, IH); 8.1 (d, IH); 7.3 (d, IH); 7.0 (d, 2H); 6.9 (s, IH); 6.85 (d, IH); 4.5 (m, IH); 4.2 (m, IH); 3.55 (t, 4H); 3.3 (t, 4H); 2.3 (m, 5H); 2.0 (m, IH); 1.8 (m, IH); 1.7 (m, 2H); 1.5 (m, IH); 0.9 (d, 3H); 0.85 (d, 3H).

B) The starting material was made using an analogous procedure to that described in Example 12 but using dibenzyl L-leucyl-L-glutamate (Loukas, Spyros; Varoucha, Dido; Zioudrou, Christine; Streaty, Richard A.; Klee, Werner A; Biochemistry (1983), 22(19), 4567-73.) in place of dibenzyl N-( L-tert-leucvD-L-glutamate at step J. NMR (DMSO-d₆) δ 8.4 (d, IH); 8.3 (d, IH); 7.9 (d, 2H); 7.3 (m, 1 IH); 7.0 (d, 2H); 6.9 (s, IH); 6.85 (d, IH); 5.1 (s, 2H); 5.05 (s, 2H); 4.5 (m, IH); 4.4 (m, IH); 3.6 (t, 4H); 3.4 (t, 4H); 2.4 (m, 2H); 2.2 (s, 3H); 2.1 (m, IH); 1.9 (m, IH); 1.6 (m, 2H); 1.5 (m, IH); 0.9 (d, 3H); 0.8 (d, 3H).

Example 14

Preparation of (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyl]-L- norvalyl)-L-glutamic acid

A) Starting material dibenzyl (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3- methylphenoxy)benzoyl]-L-norvalyl)-L-glutamate was deprotected using the procedure described in Example 12, step A to give the desired end product.

NMR (DMSO-d₆) δ 8.3 (d, IH); 8.1 (d, IH); 7.9 (d, 2H); 7.3 (d, IH); 7.0 (d, 2H); 6.9 (s, IH); 6.85 (d, IH); 4.4 (m, IH); 4.2 (m, IH); 3.6 (t, 4H); 3.4 (t, 4H); 2.3 (m, 5H); 1.9 (m, IH); 1.8 (m, IH); 1.7 (m, 2H); 1.4 (m, 2H); 0.9 (t, 3H).

B) The starting material was made using an analogous procedure to that described in Example 12 but using N-Boc-L-norvaline in place of N-Boc- L-tert-leucine at step H. NMR (DMSO-d₆) δ 8.2 (d, IH); 7.3 (bs, 10H); 6.8 (d, IH); 5.1 (s, 2H); 5.05 (s, 2H); 4.4 (m, IH); 3.9 (m, IH); 2.4 (t, 2H); 2.1 (m, IH); 1.9 (m, IH); 1.5 (m, 2H); 1.3 (s, 9H); 1.2 (m, 2H); 0.8 (t, 3H).

Step J, product NMR (DMSO-d₆) 8.4 (d, IH); 8.3 (d, IH); 7.9 (d, 2H); 7.3 (m, 1 IH); 7.0 (d, 2H); 6.9 (s, IH); 6.85 (d, IH); 5.1 (s, 2H); 5.05 (s, 2H); 4.4 (m, 2H); 3.6 (t, 4H); 3.4 (t, 4H); 2.5 (t, 2H); 2.3 (s, 3H); 2.1 (m, IH); 1.9 (m, IH); 1.7 (m, 2H); 1.3 (m, 2H); 0.8 (t, 3H).

Example 15

Preparation of N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyI]-L-tert- leucine

Note this is the active drug corresponding with the prodrug of Example 12.

A) Starting material N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyl]- L-tert-leucine benzyl ester was deprotected using the procedure described in Example 12, step A to give the desired end product. NMR (DMSO-d₆) δ 8.0 (d, IH); 7.9 (d, 2H); 7.3 (d, IH); 7.0 (d, 2H); 6.9 (s, IH); 6.85 (d, IH); 4.3 (d, IH); 3.5 (t, 4H); 3.3 (t, 4H); 2.3 (s, 3H); 1.0 (s, 9H).

B) The starting material was made using an analogous procedure to that described in Example 12 but using L-tert-leucine benzyl ester (Deziel, Robert; Moss, Neil; Plante, Raymond. Preparation of antiherpes peptide derivatives having a ureido N-terminus. Eur. Pat. Appl., EP 560274 Al 930915.) instead of dibenzyl N-( L-tert-leucvl)-L-glutamate at step J.

NMR (DMSO-d₆) δ 8.2 (d, IH); 7.9 (d, 2H); 7.4 (bs, 5H); 7.3 (d, IH); 7.0 (d, 2H); 6.9 (s, IH); 6.85 (d, IH); 5.1 (d, 2H); 4.4 (d, IH); 3.6 (t, 4H); 3.4 (t, 4H); 2.3 (s, 3H); 1.0 (s, 9H).

Example 16

Preparation of N- [4-(4- [N,N-bis(2-chIoroethyl)amino] -3-methylphenoxy)benzoyl] -L- norvaline

Note this is the active drug corresponding with the prodrug of Example 14.

A) Starting material N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyl]- L-norvaline benzyl ester was deprotected using the procedure described in Example 12, step A to give the desired end product. NMR (DMSO-d₆) δ 8.5 (d, IH); 8.0 (d, 2H); 7.4 (d, IH); 7.1 (d, 2H); 7.0 (s, IH); 6.9 (d, IH); 4.4 (m, IH); 3.6 (t, 4H); 3.4 (t, 4H); 2.3 (s, 3H); 1.8 (m, 2H); 1.4 (m, 2H); 1.0 (t, 3H).

B) The starting material was made using an analogous procedure to that described in Example 12 but using L-norvaline benzyl ester (Handa, Balraj Krishnan; Johnson, William Henry; Machin, Peter James. Preparation of (hydroxylamino)acylpeptides as inhibitors of synovial collagenase. Eur. Pat. Appl., EP 236872 A2 870916) instead of dibenzyl N-C - tert-leucvl)-L-glutamate at step J.

NMR (DMSO-d₆) δ 8.6 (d, IH); 7.9 (d, 2H); 7.4 (bs, 5H); 7.3 (d, IH); 7.0 (d, 2H); 6.95 (s,

IH); 6.9 (d, IH); 5.1 (s, 2H); 4.4 (m, IH); 3.6 (t, 4H); 3.4 (t, 4H); 2.3 (s, 3H); 1.8 (m, 2H);

1.4 (m, 2H); 0.9 (t, 3H).

Example 17

Preparation of {N-[4-(4-[N,N-bis(2-chIoroethyl)amino]3-methylphenoxy)benzoyI]-L- alanyl}-3-(lH-l,2,3₅4-tetrazol-5-yl)methyl)-L-alanine

A) Starting material {N-[4-(4-[N,N-bis(2-chloroethyl)amino]3-methylphenoxy)benzoyl]- L-alanyl}-3-(lH-l,2,3,4-tetrazol-5-yl)methyl)-L-alanine methyl ester (0.15 g) was dissolved in 1,4-dioxane (15 ml), water (7 ml) and cone. HCl (7 ml) added. The solution was heated to 50 o for 1 h and then evaporated to dryness and azeotroped with toluene. The resulting residue was then chromatographed on silica gel eluting with 2% formic acid in ethyl acetate, azeotroped with toluene to give the desired end product, 0.1 g (70%). NMR (DMSO-d₆) δ 8.5 (d, IH); 8.1 (d, IH); 7.9 (d, 2H); 7.25 (d, IH); 7.0 (d, 2H); 6.7 (s, IH); 6.85 (d, IH); 4.5 (m, IH); 4.15 (m, IH); 3.55 (t, 4H); 3.35 (t, 4H); 2.85 (m, 2H); 2.25 (s, 3H); 2.15 (m, IH); 2.0 (m, IH); 1.35 (d, 3H).

B) The starting material was made using an analogous procedure to that described in Example 12 but using N-Boc-L-alanine instead of N-Boc- L-tert-leucine, 3-(lH-l,2,3,4- tetrazol-5-yl)methyl)-L-alanine methyl ester (Boyle, Francis Thomas; Crook, James William;

Matusiak, Zbigniew Stanley. Anti-cancer cyclopentaquinazolines. UK Pat. Appl., GB

2272217 A 1 940511) instead of dibenzyl L-glutamate and hydroxysuccinamide instead of

HOBT at step H. NMR (DMSO-dβ) δ: 8.2 (d, IH); 6.9 (d, IH); 4.36 (m, IH); 3.9 (m, IH); 3.6 (s, 3H); 2.9 (t,

2H); 2.2 (m, IH); 2.0 (m, IH); 1.3 (s, 9H); 1.2 (d, 3H).

Product from step I, NMR (DMSO-d₆) δ: 9.0 (d, IH); 8.2 (bs, 2H); 4.38 (m, IH); 3.9 (m, IH);

3.6 (s, 3H); 3.0 (t, 2H); 2.2 (m, IH); 2.1 (m, IH); 1.3 (d, 3H).

Product from step J, NMR (DMSO-d₆) δ 8.4 (t, 2H); 7.9 (d, 2H); 7.3 (d, IH); 7.0 (d, 2H); 6.9 (s, IH); 6.85 (d, IH); 4.45 (m, IH); 4.35 (m, IH); 3.6 (s, 3H); 3.59 (t, 4H); 3.3 (t, 4H); 2.95 (t,

2H); 2.25 (s, 3H); 2.2 (m, IH); 2.0 (m, IH); 1.35 (d, 3H).

Example 18

Preparation of (N-[4-(4-[N,N-bis(2-bromoethyl)amino]phenoxy)-3,5-dimethylbenzoyl]-L- alanyl)-L-glutamic acid

Starting material ditcrtbutyl (N-[4-(4-[N,N-bis(2-bromoethyl)amino]phenoxy)-3,5- dimethylbenzoyl]-L-alanyl)-L-glutamate (140 mg) was dissolved in dichloromethane (2 ml) and trifluoroacetic acid (2 ml) added. The mixture was stirred at ambient temperature for 2 h and evaporated to dryness to give the desired end product as an oil (140 mg). NMR (d₆-DMSO) δ: 8.3 (d, IH), 8.1 (d, IH), 6.6 (m, 4H), 4.5 (m, IH), 4.1 (m, IH), 3.6 (t, 4H), 3.5 (t, 4H), 2.1 (s, 6H), 2.3-1.8 (m, 4H), 1.3 (d, 3H). The starting material was prepared using analogous methodology to that described in

Example 1 but using 3,5-dimethyl-4-(4-nitrophenoxy)benzoic acid in place of 4-(4- nitrophenoxy)benzoic acid. The 3,5-dimethyl-4-(4-nitrophenoxy)benzoic acid was prepared as described by Rarick, Brewster and Daines J.A.C.S. 55 (1993), 1289-90 but using 3,5- dimethyl-4-hydroxybenzoic acid in place of 4-hydroxybenzoic acid. NMR of 3,5-dimethyl-4-(4-nitrophenoxy)benzoic acid, (d₆-DMSO) δ: 8.2 (d, 2H), 7.8 (s, IH), 7.0 (d, 2H), 2.1 (s, 6H).

Example 19

Preparation of N-[4-(4-[N,N-bis(2-bromoethyl)amino]phenoxy)-3,5-dimethylbenzoyl]-L- alanine

To a solution of starting material N-[4-(4-[N,N-bis(2-bromoethyl)amino]phenoxy)- 3, 5 -dimethylbenzoyl] -L-alanine tertbutyl ester (220 mg) in dichloromethane (4 ml) was added trifluoroacetic acid (4 ml). The mixture was allowed to stand at ambient temperature for 1 hour and then evaporated to dryness. The residue was azeotroped twice with ethyl acetate and evaporated to give the desired end product as an oil (315 mg).

NMR (CDC1₃ ) δ: 7.7 (s, 2H), 7.1-6.8 (m, 4H), 4.9 (m, IH), 3.8 (t, 4H), 3.4 (t, 4H), 2.1 (s, 6H ), 1.6 (d, 3H). The starting material was made using an analogous procedure to that described in the previous example but using alanine tert butyl ester hydrochloride in place of dibenzyl L- alanyl-L-glutamate hydrochloride.

Example 20 Preparation of (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-2-methylphenoxy)benzoyl]-L- alanyl)-L-glutamic acid

A) Starting material ditertbutyl (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-2- methylphenoxy)benzoyl]-L-alanyl)-L-glutamate (390 mg) was dissolved in 4 M hydrogen chloride in ethyl acetate (8 ml). The mixture was stirred at ambient temperature for 30 min then evaporated to dryness. The product was triturated with diethyl ether and the solid hydrochloride collected, 294 mg (91 %), m.p. 120-122°. NMR (d₆-DMSO) δ: 8.3 (d, IH), 8.1 (d, IH), 7.8 (d, 2H), 6.6-6.8 (m, 5H), 4.5 (m, IH), 4.2 (m, IH), 3.7 (s, 8H), 2.3-1.8 (m, 4H), 2.0 (s, 3H), 1.3 (d, 3H).

The starting material was prepared as follows. B) 4-(2-methyl-4-nitrophenoxy)benzoic acid was prepared as described by Rarick, Brewster and Daines J.A.C.S. 55 (1993 ) 1289-90 but using 5-fluoro-2-nitrotoluene in place of 4-fluoro-l -nitrobenzene.

NMR (d₆-DMSO) δ: 8.3 (d, IH), 8.1 (dd, IH), 8.0 (d, 2H), 7.2-7.0 (m, 3H), 2.3 (s, 3H).

The nitro group thereof was converted through to a bis(2-chloroethyl)amino group using the general route described in Example 1 but using lithium chloride in place of lithium bromide to give the desired starting material. Example 21

Preparation of N- [4-(4- [N,N-bis(2-chloroethy l)amino] -2-methylphenoxy)benzoyl] -L- alanine

A) To a solution of starting material N-[4-(4-[N,N-bis(2-chloroethyl)amino]-2- methylphenoxy)benzoyl] -L-alanine tert butyl ester (400 mg) in dichloromethane (4 ml) was added trifluoroacetic acid (4 ml). The mixture was allowed to stand at ambient temperature for lhour and then evaporated to dryness. The residue was azeotroped twice with ethyl acetate and evaporated to give the desired end product as an oil (400 mg).

NMR (CDC1₃ ) δ: 7.7 (s, 2H), 7.0-6.7 (m, 6H), 4.8 (m, IH), 3.8 (t, 4H), 3.6 (t, 4H), 2.2 (s, 3H), 1.6 (d, 3H).

B) The starting material was made using an analogous procedure to that described in previous example but using L-alanine tertbutyl ester hydrochloride in place of dibenzyl L- alanyl-L-glutamate hydrochloride and lithium chloride in place of lithium bromide.

Example 22

Preparation of (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)benzoyl]-L- alanyl)-L-glutamic acid

A) Starting material ditertbutyl (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3- methoxyphenoxy)benzoyl]-L-alanyl)-L-glutamate (250 mg) was dissolved in dichloromethane (3 ml) and trifluoroacetic acid (3 ml) added. The mixture was stirred at ambient temperature for 90 min then evaporated to dryness. The residue was dissolved in ethyl acetate, hydrogen chloride (5 M) in ethyl acetate (8 ml) added and the desired end product was precipitated as a solid hydrochloride salt which was collected, 170 mg (81 %).

NMR (d₆-DMSO) δ: 8.5 (d, IH), 8.1 (d, IH), 7.9 (d, 2H), 7.1 (m, 3H), 6.8 (s, IH), 6.5 (d,

IH), 4.5 (m, IH), 4.2 (m, IH), 3.8 (s, 3H), 3.6 (t, 4H), 3.4 (t, 4H), 2.3-1.8 (m, 4H), 1.3 (d,

3H). The starting material was prepared as follows.

B) 4-(3-methoxy-4-nitrophenoxy)benzoic acid was prepared as described in Jacquet et al, Heterocycles, 34, 1992, 2301-2311. The nitro group thereof was converted through to a bis(2- chloroethyl)amino group using the general route described in Example 1 but using lithium chloride in place of lithium bromide.

Example 23

Preparation of N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)benzoyI]-L- alanine

To a solution of starting material N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3- methoxyphenoxy)benzoyl]-L-alanine tertbutyl ester (113 mg) in dichloromethane (2 ml), trifluoroacetic acid (2 ml) was added. The mixture was allowed to stand at ambient temperature for 1 hour and then evaporated to dryness. The residue was azeotroped twice with ethyl acetate and 5 M hydrogen chloride in ethyl acetate (8 ml) added. The desired end product was precipitated as a solid hydrochloride salt, 76 mg (77 %). NMR (CDCI₃) δ: 7.7 (d, 2H), 7.0 (d, 2H), 6.6 (m, 3H), 4.8 (m, IH), 3.8 (s, 3H), 3.6 (s, 8H), 1.5 (d, 3H).

The starting material was made using an analogous procedure to that described in the previous example but using alanine tertbutyl ester hydrochloride in place of dibenzyl L- alanyl-L-glutamate hydrochloride and lithium chloride in place of lithium bromide in the final step.

Example 24

Preparation of (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)-3,5- dimethylbenzoyl] -L-alanyl)-L-glutamic acid

A) Starting material ditertbutyl (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3- methoxyphenoxy)-3,5-dimethylbenzoyl]-L-alanyl)-L-glutamate (250 mg) was dissolved in dichloromethane (3 ml) and trifluoroacetic acid (2 ml) added. The mixture was stirred at ambient temperature for 90 min then evaporated to dryness. The residue was dissolved in ethyl acetate, hydrogen chloride (5 M) in ethyl acetate added and the desired end product was precipitated as a solid hydrochloride salt which was collected, 148 mg (72 %). NMR (d₆-DMSO) δ: 8.5 (d, IH), 8.1 (d, IH), 7.7 (s, 2H), 7.0 (d, IH), 6.6 (d, IH), 6.0 (m, IH), 4.5 (m, IH), 4.2 (m, IH), 3.7 (s, 3H), 3.5 (t, 4H), 3.3 (t, 4H), 2.3-1.8 (m, 4H), 2.0 (s, 3H), 1.3 (d, 3H).

The starting material was prepared as follows. B) 4-(4-nitro-3-methoxyphenoxy)-3,5-dimethylbenzoic acid was prepared as described by Rarick et al J.A.C.S. 55 (1993) 1289-90 but using 4-fluoro-2-methoxy-l -nitrobenzene in place of 4-fluoro-l -nitrobenzene and 4-hydroxy-3,5-dimethylbenzoic acid in place of 4- hydroxybenzoic acid.

NMR (d₆-DMSO) δ: 7.9 (d, IH), 7.8 (s, 2H), 6.9 (s, IH), 6.2 (dd, IH), 3.9 (s, 3H), 2.1 (s, 6H). The nitro group thereof was converted through to a bis(2-chloroethyl)amino group using the general route described in Example 1 but using lithium chloride in place of lithium bromide in the final step.

Example 25

Preparation of N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)-3,5- dimethylbenzoyl]-L-alanine

To a solution of starting material N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3- methoxyphenoxy)-3,5-dimethylbenzoyl]-L-alanine tertbutyl ester (240 mg) in dichloromethane (2 ml), trifluoroacetic acid (2 ml) was added . The mixture was allowed to stand at ambient temperature for 1 hour and then evaporated to dryness. The residue was azeotroped twice with ethyl acetate and hydrogen chloride (5 M) in ethyl acetate added. The desired end product was precipitated as a solid hydrochloride salt, 150 mg (67 %). NMR (d₆-DMSO) δ: 8.6 (d, IH), 7.7 (s, 2H), 7.0 (d, 2H), 6.6 (s,lH), 6.0 (dd, IH), 4.5 (m, IH), 3.7 (s, 3H), 3.5 (t, 4H), 3.3 (t, 3H), 2.1 (s, 6H), 1.4 (d, 3H).

The starting material was made using an analogous procedure to that described in previous example but using alanine tertbutyl ester hydrochloride in place of dibenzyl L- alanyl-L-glutamate hydrochloride and lithium chloride in place of lithium bromide in the final step. Example 26

Preparation of (N-[4-(4-[N,N-bis(2-bromoethyl)amino]phenoxy)benzoyl]-L-leucyl)-L- glutamic acid

5 Starting material dibenzyl (N-[4-(4-[N,N-bis(2-bromoethyl)amino]phenoxy)benzoyl]-

L-leucyl)-L-glutamate (300 mg) was dissolved in 48 % hydrogen bromide in acetic acid (10 ml). The mixture was stirred at ambient temperature for 3 h, evaporated to dryness and azeotroped with toluene to give an oil. The oil was triturated with diethyl ether to yield the desired end product as an orange solid hydrobromide salt (175 mg).

10 NMR (d₆-DMSO) δ: 8.3 (d, IH), 8.1 (d, IH), 6.8-6.6 (m, 6H), 4.5 (m, IH), 4.2 (m, IH), 3.7 (t, 4H), 3.5 (t, 4H), 2.4 (t, 2H), 2.3-1.8 (m, 5H), 0.9 (m, 6H).

The starting material was prepared using analogous methodology to that described in Example 1 but using dibenzyl L-leucyl-L-glutamate (Loukas, Spyros; Varoucha, Dido; Zioudrou, Christine; Streaty, Richard A.; Klee, Werner A; Biochemistry (1983), 22(19),

15 4567-73.) instead of dibenzyl L-alanyl-L glutamate up to step D thereof. Bromine (0.19 ml) was added to a solution of triphenylphosphine and imidazole in dichloromethane (10 ml). A solution of product from step D, dibenzyl (N-[4-(4-[N,N-bis(2-hydroxyethyl)amino]- phenoxy)benzoyl]-L-leucyl)-L-glutamate (660 mg), in dichloromethane (3 ml) was then added and the reaction mixture stirred at ambient temperature overnight. Insoluble material

20 was filtered off and the filtrate evaporated to dryness. The residue was flash column chromatographed, eluting with isohexane/ ethylacetate (2:1) to yield the desired starting material as an oil.

NMR (CDC1₃) δ: 7.7 (d, 2H), 7.3 (m, 10H), 6.9 (m, 4H), 5.2 (s, 2H), 5.1 (s, 2H), 4.6 (m, 2H), 3.8 (m, 4H), 3.5 (m, 4H), 2.3-1.6 (m, 7H), 0.9 (m, 6H).

25 Example 27

Preparation of N- [4-(4- [N,N-bis(2-bromoethyl)amino] phenoxy)benzoyl] -L-leucine

Starting material N-[4-(4-[N,N-bis(2-bromoethyl)amino]phenoxy)benzoyl] -L-leucine tertbutyl ester (1.25 g) was dissolved in 48 % hydrobromic acid in acetic acid (10 ml). The mixture was stirred at ambient temperature for 1 h and evaporated to dryness to give an oil. The oil was triturated with diethyl ether to give the desired end product as a brown solid, hydrobromide salt (340 mg). NMR (d₆-DMSO) δ: 8.4 (d, IH), 7.85 (d, 2H), 6.9 (m, 4H), 6.25 (d, 2H), 4.5 (m, IH), 3.7 (m, 4H), 3.5 (m, 4H), 1.7-1.5 (m, 3H ), 0.9 (m, 6H).

The starting material was prepared using analogous methodology to that described in Example 2 but using L-leucine tertbutyl ester at step B thereof and using the following bromination methodology. Bromine (1.8 ml) was added to a solution of triphenylphosphine (8.9 g) and imidazole

(2.31 g) in dichloromethane (70 ml) at ambient temperature. To this mixture a solution of N- [4-(4-[N,N-bis(2-hydroxyethyl)amino]phenoxy)benzoyl]-L-leucine tertbutyl ester (4.1 g) in dichloromethane (20 ml) was added, stirred at ambient temperature for 3 hours, insoluble material filtered off and the filtrate evaporated to dryness. The residue was chromatographed, eluting with iso-hexane/ ethyl acetate (4:1) to yield the desired starting material as an oil. NMR (CDC1₃) δ: 7.8 (d, 2H), 6.9-6.7 (m, 6H), 6.4 (d, IH), 4.3 (m, IH), 3.7 (m, 4H), 3.4 (m, 4H), 1.7-1.5 (m, 3H), 1.3 (s, 9H), 0.9 (m, 6H). Example 28

Preparation of (N- [4-(4- [N,N-bis(2-chloroethyl)amino] - 1 -naphthyloxy)benzoyl] -L- alanyl)-L-glutamic acid

A) Starting material ditertbutyl (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-l- naphthyloxy)benzoyl]-L-alanyl)-L-glutamate (43mg) was stirred in trifluoroacetic acid (4 ml) for 30 minutes. The TFA was removed in vacuo and chloroform was used to azeotrope off remaining TFA. To the gum produced was added diethyl ether and HCl/ diethyl ether (a few drops). The mixture was stirred for a few minutes, isohexane added and the majority of the solvent was pipetted off. The solid was washed with more isohexane which was again pipetted off and the remaining solvent was removed in vacuo to give the desired end product as a solid, 37 mg (96 %). NMR (DMSO-d₆) δ: 8.36 (d, IH); 8.34 (d, IH); 8.12 (t, IH); 7.92 (d, IH); 7.88 (d, 2H); 7.59 (t, IH); 7.52 (t, IH); 7.44 (d,lH); 7.12 (d, IH); 7.04 (d, 2H); 4.48 (m, IH); 4.20 (m, IH); 3.66 (t, 4H); 3.54 (t, 4H); 2.26 (q, 2H); 1.87 (m, IH); 1.77 (m, IH); 1.29 (d, 3H). The starting material was prepared as follows.

B) End Product from Example 29 (190 mg) in dry DMF (6 ml) with HOBT hydrate (66 mg) was stirred in an ice/salt bath at -5° for 10 minutes. EDCI (84 mg) was added and the mixture was stirred for 10 minutes, the temperature of the mixture was then allowed to rise to room temperature and stirred for 1 hour. Ditertbutyl glutamate hydrochloride (100 mg) was added in DMF (4 ml), then triethylamine (52 μl) and the mixture was stirred overnight. The DMF was removed in vacuo and the solid formed was dissolved in ethyl acetate and water. The organic layer was washed twice with sodium bicarbonate solution, water, 0.5 M citric acid, water and saturated brine solution. The organic layer was dried and the solvent removed in vacuo. The residue formed was chromatographed on silica gel eluting with 3 % ethyl acetate/methylene chloride and then 5 % ethyl acetate/methylene chloride to give the desired starting material, 48 mg (18 %).

NMR (DMSO-d6) d: 8.38 (d, IH); 8.36 (d, IH); 8.11 (d, IH); 7.88 (t, 3H); 7.57 (t, IH); 7.50 (t, IH); 7.42 (d, IH); 7.10 (d, IH); 7.01 (d, 2H); 4.48 (m, IH); 4.09 (m, IH), 3.60 (d, 4H); 3.54 (d, 4H); 2.24 (m, 2H); 1.89 (m, IH), 1.72 (m, IH); 1.34 (s, 18H); 1.29 (d, 3H).

Example 29

Preparation of N- [4-(4- [N,N-bis(2-chloroethyI)amino] -1 -naphthyloxy)benzoyl] -L-alanine

A) Starting material N-[4-(4-[N,N-bis(2-chloroethyl)amino]- 1 -naphthyloxy)benzoyl]-L- alanine tertbutyl ester (270 mg) was stirred in trifluoroacetic acid (4 ml) for 30 minutes. The mixture was evaporated to a gum which was dissolved in ethyl acetate and HCl/Diethyl ether was added. The mixture was stirred for a few minutes and the solid formed was filtered off, washed with diethyl ether and dried under vacuum to give the desired end product as a solid, 37mg (14%). NMR (DMSO-d₆) δ: 8.57 (d, IH); 8.39 (d, IH); 7.92 (d, IH); 7.89 (d, 2H); 7.60 (m, 2H); 7.44 (d, IH); 7.12 (d, IH); 7.06 (d, 2H); 4.38 (t, IH); 3.64 (d, 4H); 3.56 (d, 4H); 1.37 (d. 3H). The starting material was prepared as follows.

B) 1 -Methoxy-4-nitronaphthalene (Aldrich, 10.16 g) and lithium chloride (8.48 g) were weighed into a flask. Dry DMF (100 ml) was added and the flask flushed with nitrogen. The mixture was stirred and heated at 160° for 10 .5 hours. The mixture was cooled to room temperature and 10 % NaOH (300 ml) and water (100 ml) was added. The mixture was extracted with diethyl ether (500 ml). The aqueous layer was adjusted to pHl with hydrochloric acid and the solution extracted twice with ethyl acetate. The combined organics were washed with saturated brine and evaporated. The crude product was dissolved in hot methanol (150 ml), filtered and evaporated. The solid was triturated in methylene chloride, filtered and dried under vacuum to give l-hydroxy-4-nitronaphthalene 3.84 g (41%). NMR (DMSO-dβ) δ 11.9 (s, IH); 8.64 (d, IH); 8.38 (d, IH), 8.30 (d, IH); 7.77 (t, IH); 7.63 (t, IH); 6.96 (d, IH).

C) 1 -Hydroxy-4-nitronaphthalene (2.0 g) was dissolved in methylene chloride (40 ml) at -15°. Triethylamine (4.4 ml) was added and then trifluoromethanesulphonic anhydride (2.7 ml) in methylene chloride (10 ml). The mixture was stirred for 30 minutes and then diluted with methylene chloride (100 ml), washed with saturated sodium bicarbonate solution (50 ml), saturated brine (50 ml), dried and evaporated . The solid was then dissolved in methylene chloride, filtered through a pad of silica and the filtrate was evaporated give 1- (trifluoromethylsulfonyloxy)-4-nitronaphthalene, 2.99 g (88%). NMR (DMSO-dβ) δ: 8.44 (d, IH); 8.40 (t, IH); 8.14 (dd, IH); 7.98 (d, IH); 7.96 (d, IH); 7.90 (d, IH).

D) Product from step C (7.5 g) was dissolved in dry DMF and lithium bromide (20.28 g) was added. The mixture was heated and stirred at 100^° under nitrogen for 6 hours, the temperature reduced to 60^° and the mixture stirred overnight. The DMF was removed in vacuo and the residue was dissolved in water and extracted twice with ethyl acetate. The organics were washed twice with water and saturated brine then evaporated in vacuo. The residue was dissolved in methylene chloride and filtered through a pad of diatomaceous earth and washed through with 25 % methylene chloride/isohexane (2 x 100 ml) to give l-bromo-4- nitronaphthalene, 3.68 g (62.7%). NMR (DMSO-d₆) δ: 8.34 (t, 2H); 8.18 (d, IH); 8.08 (d, IH); 7.86 ( d, 2H). E) 4-hydroxybenzoic acid (Aldrich, 0.15 g) was dissolved in methanol (10 ml) and potassium tertiary butoxide (0.25 g) was added. The mixture was evaporated to dryness. The crude product from step D (185 mg) was dissolved in DMF (20 ml) and added to the dipotassium salt of 4-hydroxybenzoic acid. The mixture was heated to 140^° with stirring under nitrogen for 3.5 hours. The reaction mixture was cooled and poured into water (150 ml), acidified with 2M hydrochloric acid, extracted into ethyl acetate (150ml), washed twice with water, brine, dried and the solvent removed in vacuo. The solid was triturated with hot water and filtered to give 1 -benzoyl-4-nitronaphthalene, 227 mg (43%). NMR (DMSO-d₆) δ: 8.58 (d,lH); 8.38 (d, IH); 8.36 (d, IH); 8.04 (d, 2H); 7.90 (t, IH); 7.74 (t, IH), 7.32 ( d, 2H); 7.06, (d,lH). F) Product from step E (1.50 g) was dissolved in dry DMF (60 ml) and HOBT hydrate (0.82 g), EDCI (0.98 g), triethylamine (0.68 ml) and L-alanine tertbutyl ester hydrochloride were added. The mixture was stirred at room temperature overnight. The DMF was removed in vacuo and the residue partitioned between ethyl acetate and water. The organics were washed twice with saturated sodium bicarbonate solution (50 ml), water (50 ml), 0.5 M citric acid, water and saturated brine, dried and evaporated to give N-[4-(4- nitronaphthyloxy)benzoyl]-L-alanine tertbutyl ester, 1.14 g (54 %).

NMR (DMSO-dβ) δ: 8.72 (d, IH); 8.59 (d, IH); 8.40 (d, IH); 8.38 (d, IH); 8.02 (d, 2H); 7.90 (t, IH); 7.79 (t, IH); 7.35 (d, 2H); 6.94 ( d, IH); 4.34 (q, IH); 1.40 (s, 9H); 1.38 (d, 3H). G) Product from step F was dissolved in ethyl acetate (30 ml) and the flask flushed with nitrogen. 10 % Palladium on carbon (50 % moist with water, 0.22 g) suspended in ethyl acetate (10 ml) was added. The mixture was stirred under an atmosphere of hydrogen for 4 hours. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate was evaporated to give N-[4-(4-amino-l-naphthyloxy)benzoyl]-L-alanine tertbutyl ester, 0.95 g (93 %). NMR (DMSO-dβ) δ: 8.46 (d, IH); 8.10 (d, IH); 7.79 (d, 2H); 7.64 (d, IH); 7.40 (d, IH); 7.39 (d, IH); 7.03 (d, IH); 6.86 (d, 2H); 6.66 (d, IH); 5.70 (s, 2H); 4.28 (q, IH); 1.38 (s,9H); 1.30 (d, 3H).

H) Product from step G (0.95 g) was dissolved in acetic acid (20 ml) and the mixture was stirred whilst adding water (15 ml). Ethylene oxide was passed through the mixture until 3.5 g had been added. The mixture was then stirred overnight and the solvent was removed in vacuo. The residue was partitioned between saturated sodium bicarbonate solution and ethyl acetate The organic layer was washed twice with water, brine, dried and the solvent removed in vacuo. The residue was chromatographed on silica gel eluting with 9: 1 ethyl acetate/ methylene chloride and then ethyl acetate to give N-[4-(4-[N,N-bis(2-hydroxyethyl)amino]- 1 -naphthyloxy)benzoyl] -L-alanine tertbutyl ester, 0.72 g (62 %). NMR (DMSO-dβ) δ: 8.54 (d, IH); 8.40 (d, IH); 7.86 (d, 3H); 7.50 (q, 2H); 7.32 (d, IH); 7.14 (d, IH), 7.00 ( d, 2H); 4.49 (t, 2H); 4.29 (q, IH); 3.48 (dd, 4H), 3.24 (dd, 4H); 1.39 (s, 9H); 1.32 (d, 3H).

I) Product from step H (360 mg) was dissolved in methylene chloride (8 ml) and was stirred in an ice/salt bath at 0°. Triethylamine (310 μl) was added and then methanesulphonyl chloride (141 μl) was added dropwise over 3-4 minutes, keeping the temperature below 5°. After addition was complete the mixture was stirred at 0° for 5 minutes and then for lhour at room temperature. The mixture was diluted with methylene chloride and washed in turn with ice cold water, ice cold sodium bicarbonate solution and saturated brine. The organic layer was dried and evaporated to give N-[4-(4-[N,N-bis(2-mesyloxyethyl)amino]-l- naphthyloxy)benzoyl] -L-alanine tertbutyl ester, 452 mg (95 %).

NMR (CDClj) δ 8.36 (d, IH); 8.09 (d, IH); 7.80 (d, 2H); 7.59 (t, IH); 7.52 (t, IH); 7.35 (d, IH); 7.04 (d, 3H); 6.74 (d, IH); 4.66 (q, IH); 4.26 (t, 4H); 3.66 (t, 4H); 2.92 (s, 6H); 1.52 (s, 9H); 1.48 (d, 3H). J) Product from step I (422 mg) was dissolved in DMF (10 ml) and anhydrous lithium chloride (280 mg) was added. The mixture was heated and stirred at 50° for 3.5 hours. The DMF was removed in vacuo and the residue was taken up in ethyl acetate and washed three times with water, saturated brine, dried and evaporated. The residue was chromatographed on silica gel eluting with 1 % ethyl acetate/methylene chloride to give the desired starting material, 284 mg (82 %).

NMR (DMSO-d₆) δ: 8.54 (d, IH); 8.38 (d, IH); 7.92 (d, IH); 7.86 (d, 2H); 7.59 (t, IH); 7.52 (t, IH); 7.43 (d, IH); 7.13 (d, IH); 7.04 (d, 2H); 4.29 (q, IH); 3.62 (t, 4H); 3.54 (t, 4H); 1.38 (s, 9H); 1.33 (d, 3H).

Example 30

Preparation of {N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyl]-L- alanyl}-L-aspartic acid

A) Trifluoroacetic acid (5 ml) was added to a solution of starting material di-tert-butyl {N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyl]-L-alanyl}-L-aspartate (310 mg, 0.46 mmol) in dichloromethane (5 ml). The solution was kept for 18 h at ambient temperature and evaporated to dryness. The residue was triturated with 1 :1 diethyl ether/isohexane to yield a white solid. The solvent was decanted off and the solid was vacuum dried to yield the desired end product, 335 mg.

NMR (Me₂SO-d₆) δ: 1.31 (3 H, d, CH₃ of ala), 2.25 (3 H, s, ArCH₃), 2.64 (2 H, m, CH₂CO₂), 3.33 (4 H, t, CH₂CH₂C1), 3.56 (4 H, t, CH2CH2C1), 4.50 (2 H, m, 2 x CH), 6.88 (1 H, dd, ArH), 6.92 (1 H, d, ArH), 7.03 (2 H, d, ArH), 7.28 (1 H, d, ArH), 7.89 (2 H, d, ArH), 8.15 (1 H, d, CONH), 8.38 (1 H, d, CONH); ms 552 [M-H]^"; Found: C,45.7%; H, 4.4%; N, 5.2%. C₂₅H₂₉C1₂N,0₇ requires: C,45.7%; H, 4.3%; N, 5.4% The starting material was prepared as follows.

B) A mixture of Z- Ala (1.10 g, 4.93 mmol), aspartic acid ditertbutyl ester hydrochloride (1.26g, 4.48 mmol) and HOBT (685 mg, 4.48 mmol) in dichloromethane (30 ml) was stirred at 0° under nitrogen. N-methylmorpholine (983 μl, 8.96 mmol) and 2-(lH-benzotriazole-l- yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (1.87 g, 4.93 mmol) were added. Stirring was continued for 1 h at 0° and for 18 h at ambient temperature . The reaction mixture was diluted with ethyl acetate (50 ml) and washed with 10 % citric acid, aqueous sodium bicarbonate solution and brine. The solution was dried and evaporated to a gum which was purified by chromatography eluting with 0-15% ethanol/dichloromethane to yield ditertbutyl Z-L-alanyl-L-aspartate as an almost colourless gum. 1.85 g. NMR (Me₂SO-d₆) δ: 1.20 (3 H, d, CH₃ of Ala), 1.35 (9 H, s, Bu'), 1.37 (9 H, s, Bu'), 2.58 (2 H, m, CH₂CO₂), 4.05 (1 H, m, CH), 4.45 (1 H, m, CH), 5.00 (2 H, s, CH₂Ph), 7.34 (5 H, m, ArH), 7.40 (1 H, d, CONH), 8.10 (1 H, d, CONH).

C) A solution of product from step B (450 mg, 1.0 mmol) in ethyl acetate (10 ml) was stirred with 10% palladium on carbon catalyst (80 mg) in an atmosphere of hydrogen for 3 hr. The catalyst was filtered off and the filtrate was evaporated to give crude ditertbutyl L-alanyl- L-aspartate which was used in the next step without purification.

D) Oxalyl chloride (100 μl, 1.14 mmol) was added to a stirred solution of 4-{4-[N,N- bis(2-chloroethyl)amino]-3-methylphenoxy}benzoic acid (210 mg, 0.57 mmol; see International Patent Application WO 97/07769, published 6-Mar-97) and DMF (1 drop) in dichloromethane (5 ml). After 1 hr the solution was evaporated and kept at 0.1 mm Hg for 2 h to give the acid chloride. This was dissolved in dichloromethane (10 ml) and added to the product from step C generated above. N-methylmorpholine (125 μl, 1.13 mmol) and 1- hydroxybenzotriazole hydrate (20 mg) were added and the mixture was stirred overnight. The resulting solution was washed with water, dried and evaporated to dryness. The crude product was purified by chromatography on silica, eluting with 0-3 % ethanol/dichloromethane to give the desired starting material as a white foam, 320 mg.

NMR (Me₂SO-d₆) δ: 1.32 (3 H, d, CH₃ of ala), 1.36 (18 H, s, 2 x Bu'), 2.25 (3 H, s, ArCH₃), 2.58 (2 H, m, CH₂C0₂), 3.33 (4 H, t, CH₂CH₂C1), 3.56 (4 H, t, CH2CH2C1), 4.48 (2 H, m, 2 x CH), 6.85 (1 H, dd, ArH), 6.91 (1 H, d, ArH), 7.01 (2 H, d, ArH), 7.28 (1 H, d, ArH), 7.89 (2 H, d, ArH), 8.15 (1 H, d, CONH), 8.38 (1 H, d, CONH).

Example 31

Preparation of (N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]-2-methylphenyl)-benzoyl]-L- alanyl)-L-glutamic acid

A) Starting material ditertbutyl (N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]-2- methylphenyl)-benzoyl]-L-alanyl)-L-glutamate (480 mg) was dissolved in trifluoroacetic acid (8 ml). The mixture was stirred at room temperature for 30 minutes. Excess trifluorofluoroacetic acid was removed in vacuo and the residue dissolved in chloroform and evaporated. The crude product was dissolved in ethyl acetate (2 ml) and diethyl ether (15 ml) and then 45 % hydrogen bromide in acetic acid (0.23 ml) added. The white solid produced was filtered under vacuum and dried to give the desired end product (307 mg) as a hydrobromide salt.

NMR (DMSO-dβ): 1.34 (d, 3H); 1.80 (m, IH); 1.96 (m, IH); 2.22 (s, 3H); 2.30 (t, 2H); 3.59 (t, 4H); 3.80 (t, 4H); 4.23 (q, IH); 4.49 (q, IH); 6.62 (d, IH); 6.64 (s, IH); 7.08 (d, IH); 7.36 (d, 2H); 7.88 (d, 2H); 8.10 (d, IH); 8.44 (d, IH).

The starting material was prepared as follows.

B) 2-Bromo-5-nitrotoluene (6.48 g, 30 mM), 4-carboxyphenylboronic acid (4.97 g, 30 mM) and anhydrous potassium carbonate (8.28 g, 60 mM) were added to a mixture of ethanol (309 ml) and water (31 ml). A suspension of 10 % Pd/C catalyst (2.98 g) in ethanol was added. The mixture was stirred and refluxed for 22 h. Water was added and the mixture heated and then filtered hot through diatomaceous earth and washed with hot ethanol /water (1 :1). The filtrate was evaporated, re-dissolved in water and the solution extracted twice with diethyl ether. The aqueous layer was acidified with hydrochloric acid to pH 1.0. The precipitated solid was filtered, washed with water and dried at 60° under vacuum, to give 4-(2- methyl-4-nitrophenyl)benzoic acid (5.89 g) as a yellow solid. NMR (DMSO-d₆) δ: 2.33 (s, 3H); 7.5 (m, 3H); 8.03 (d, 2H); 8.1 (dd, IH); 8.22 (d,lH).

C) Product from step B was converted to di-tert-butyl N-[4-(4-[N,N-bis-(2- hydroxyethyl)amino]-2-methylphenyl)-benzoyl]-L-alanyl-L-glutamate using analogous methodology to that described in Example 1 up to step D thereof but using 4-( 4-nitro2- methylphenyl ) benzoic acid in place of 4-( 4-nitrophenoxy ) benzoic acid.

D) Product from step C (600 mg, 1.36 mM) was dissolved in dry dichloromethane (20 ml) and triphenylphosphine (1.422 g, 5.42 mM), imidazole (369 mg, 5.42 mM) and carbon tetrabromide (1.779 g, 5.42 mM), each being added in 1 portion to this solution. The reaction was stirred at room temperature for 4 hours and the solution evaporated. The crude mixture was purified by chromatography on silica eluting with 15 % ethyl acetate/isohexane to give the desired starting material (552mg) as a gum.

Example 32

Preparation of N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]-2-methylphenyI)-benzoyI]-L- alanine

A) The desired end product was prepared in an analogous manner to the methodology described in Example 31 except that L-alanyl-L-glutamic acid di-tert-butyl ester was replaced with L-alanine tert-butyl ester.

NMR (DMSO-d₆ ) δ : 1.38 (d, 3H); 2.20 (s, 3H); 3.60 (t, 4H); 3.80 (t,4H); 4.42 (q, IH); 6.63 (d, 2H); 7.08 (d, IH); 7.37 (d, 2H); 7.88 (d, 2H); 8.61 (d, IH).

Example 33 Preparation of (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyl]-O³- methyl-L-seryl)-L-glutamic acid

A) Starting material dibenzyl (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3- methylphenoxy)benzoyl]-0³-methyl-L-seryl)-L-glutamate (0.13 g) was dissolved in ethyl acetate (15 ml) and 10% palladium on carbon (50% moist with water, 0.1 g) added. The mixture was stirred under an atmosphere of hydrogen for 18 h. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate evaporated to give the desired end product as a yellow foam 0.07 g (70%).

NMR (DMSO-dβ) δ: 8.38 (d, IH); 8.2 (d, IH); 7.9 (d, 2H); 7.3 (d, IH); 7.0 (d, 2H); 6.9 (s, IH); 6.85 (d, IH); 4.7 (m, IH); 4.2 (m, IH); 3.6 (d, 2H); 3.5 (m, 4H); 3.35 (m, 4H); 3.23 (s, 3H); 2.23 (m, 5H); 2.0 (m, IH); 1.8 (m, IH).

The starting material was prepared as follows. B)-G) These steps were carried out as described in Example 12. H) N-Boc-O3-methyl-L-serine (0.5 g, Bachem) was dissolved in DMF (20 ml) and dibenzyl L-glutamate (1.3 g) added, followed by HOBT (0.34 g), EDCI (0.48 g) and N- methylmorpholine (0.28 ml). The mixture was stirred at RT under an inert atmosphere for 15 h. The mixture was evaporated to dryness and the residue partitioned between 1 M citric acid and ethyl acetate. The organics were washed with saturated sodium hydrogen carbonate, dried and evaporated to dryness to give dibenzyl N-(N-Boc-O3-methyl-L-seryl)-L-glutamate as an oil, 1.2 g (99%). NMR (DMSO-dβ) δ: 8.3 (d, IH); 7.3 (m, 10H); 6.8 (d, IH); 5.1 (s, 2H); 5.05 (s, 2H); 4.4 (m, IH); 4.2 (m, IH); 3.4 (m, 2H); 3.2 (s, 3H); 2.4 (t, 2H); 2.0 (m, IH); 1.9 (m, IH); 1.4 (s, 9H). I) Product from step H (1.2 g) was dissolved in HCl in ethyl acetate (3M, 25 ml) and stirred for 4 h. The solution was evaporated to dryness and then azeotroped with toluene to give dibenzyl N-(0 -methyl-L-seryl)-L-glutamate, 1.2 g. NMR (DMSO-d₆) δ 8.9 (d, IH); 7.35 (s, 10H); 5.1 (s,2H); 5.05 (s, 2H); 4.4 (m, IH); 3.6 (m, 2H); 3.2 (s, 3H); 2.5 (m, 2H); 2.1 (m, IH); 1.8 (m, IH).

J) Product from step G (0.158 g) was dissolved in DMF (20 ml) and product from step I

(0.22 g) added, followed by HOBT (0.064 g), EDCI (0.091 g) and N-methylmorpholine (0.052 ml). The mixture was stirred at RT under an inert atmosphere for 15 h. The mixture was evaporated to dryness and the resulting residue was chromatographed on silica gel eluting with 1.1 hexane/ethyl acetate to give the desired starting material as an oil, 0.14 g (42%). NMR (DMSO-dβ) δ: 8.45 (d, IH); 8.0 (d, IH); 7.9 (m, 1 IH); 7.0 (d, 2H); 6.9 (m, 2H); 5.1 (s, 2H); 5.05 (s, 2H); 4.7 (m, IH); 4.4 (m, IH); 3.6 (t, 4H); 3.4 (t, 4H); 3.2 (s, 3H); 2.4 (t, 2H); 2.1 (m, lH); 1.9 (m, IH). Example 34

Preparation of N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyl]-O³- methyl-L-serine

A) Starting material benzyl N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3- methylphenoxy)benzoyl]-O3-methyl-L-serine was deprotected using the procedure described in Example 12, step A to give the desired end product. NMR (DMSO-dβ) δ 8.5 (d, IH); 7.9 (d, 2H); 7.3 (d, IH); 7.0 (d, 2H); 6.9 (s, IH); 6.85 (d, IH); 4.6 (m, IH); 3.7 (m, 2H); 3.6 (t, 4H); 3.4 (t, 4H); 3.3 (s, 3H); 2.2 (s, 3H).

B) The starting material was made using an analogous procedure to that described in Example 1 but using 0 -methyl-L-serine benzyl ester instead of dibenzyl N-(03-methyl-L- seryl)-L-glutamate at step J. NMR (DMSO-dβ) δ 8.7 (d, IH); 7.9 (d, 2H); 7.35 (bs, 5H); 7.3 (d, IH); 7.0 (d, 2H); 6.9 (s, IH); 6.85 (d, IH); 5.2 (d, 2H); 4.7 (m, IH); 3.7 (m, 2H); 3.6 (t, 4H); 3.4 (t, 4H); 3.3 (s, 3H); 2.3 (s, 3H).

Example 35 Characterisation of Intermediates used in preparation of the end products of Examples 18-27 & 31-32

The following tables set out the characterisation data obtained. Solvent A = CDC1₃ Solvent B = DMSO-d_Λ

4^ 4. UJ UJ to to T © un O l ©

Scheme 1 Synthesis of mustard prodrugs ( X = DIRECT BOND, -OCH₂-, -CH₂- )

Reagents : a) Ala-Glu(0-t-Bu)₂, EDCI, hydroxybenzotriazole, N-methylmorpholine, DMF b) H₂, 10% Pd/C, Ethyl acetate c) Ethylene oxide/ aq HO Ac, d) (MeSO₂)₂0, CH₂C1₂, Et₃N, e) LiBr, DMF, f) TFA, CH₂C1₂ g) P(Ph)₃/I₂/imidazole h) TMSI Scheme 2 Synthesis of mustard prodrugs ( X = DIRECT BOND, R = eg. CH₂CH(Me)₂ or X = O, R = Me)

0₂N

6 Reagents : a) Ala-Glu(0-CH₂Ph)₂, EDCI, hydroxybenzotriazole, N-methylmorpholine, DMF b) H₂, 10% Pt/C, EtOAc c) Ethylene oxide/ aq HO Ac, d) (MeS0₂)₂0, CH₂C1₂, Et₃N, e) LiBr, DMF, f) H₂, 10% Pd/C, EtOAc. Scheme 3. Synthesis of mustard drugs (X =DIRECT BOND, -OCH₂-, -CH₂- )

Reagents: a) H₂N-CH(R)-C0₂-t-Bu, EDCI.HCl, HOBT, Et3N, b) H2, 30%Pd/C. EtOAc, c) Ethylene oxide/ aq HOAc, d) (MeSO₂)₂O, CH₂C1₂, Et₃N, e) LiBr, DMF, f) TFA, CH₂C1₂ g) P(Ph)₃/I₂/IMIDAZOLE h) TMSI

Scheme 4

(a) Diphenylphosphoryl azide, dioxan, benzyl alcohol, reflux; (b) 2N aq NaOH, EtOH then HCl;

(c) ala-glu di-t-butyl ester, EDCI.HCl, N-methylmorpholine, 1 -hydroxybenzotriazole, DMF;

(d) H₂, 10% Pd/C, EtOAc. Scheme 5

\

(a) Diphenylphosphoryl azide, Et3N, t-BuOH, reflux; (b) 3M HCl, EtOAc; (c) ethylene oxide, Aq HOAc: (d) 2N aq NaOH, H20; (e) HCl; (f) Alanine t-butyl ester.HCI, EDCI.HCl, 1 -hydroxybenzotriazole, N-methylmorpholine, DMF Scheme 6

^<r ~CK

Reagents :- a) Alanine Benzyl ester, EDCI, DMF; b) 10%Pd/C, H₂ ; c) Ala-Glu-dibenzyl ester, EDCI, DMF.

Claims

1. A compound of Formula la

wherein A is

and A is optionally substituted with upto 4 substituents independently selected from fluoro, chloro, bromo, C╬╣_-4alkyl or C╬╣_-4alkoxy; X is a direct bond, CH₂ or oxygen;

1

Y and Y are independently selected from chloro, bromo, iodo, -0-SO₂-C_1-4alkyl and -O- SO₂-phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from C_)-4alkyl, halo, cyano or nitro; R is hydrogen, fluoro, chloro, bromo, C_]-4alkyl or C_1-4alkoxy; p is 0-4 wherein values of R may be the same or different and when p is less than 4 remaining substituent values are hydrogen;

R is C_1-6alkyl, hydroxyC_1-6alkyl, phenylC_1-6alkyl, C_1-4 alkoxy C_1-4alkyl, phenylC_1-4alkoxyC_1-4alkyl, C_1- alkylthioC_1-4alkyl, phenylC_1-4alkylthioC╬╣_-4 alkyl or carbamoylC _-4alkyl; or an enantiomer, diastereoisomer, an in-vivo hydrolysable ester, a pharmaceutically acceptable salt or a carboxypeptidase activatable prodrug thereof with the proviso that the following compounds and salts thereof are excluded N-(4-{ 4- [N,N-bis-(2-chloroethyl)-amino] -3 -methyl -phenoxy }-benzoyl)-L- alanine, N-(4-{4-[N,N-bis-(2-chloroethyl)-amino]-phenoxy}-benzoyl)-L-alanine, N-[N-(4-{4-[N,N- bis-(2-chloroethyl)-amino]-3 -methyl-phenoxy } -benzoyl)-L-alanyl] -L-glutamic acid and, N-[^-(4-{4-|T^,N-bis-(2-chloroethyl)-amino]-phenoxy}-benzoyl)-L-alanyl]-L-glutamic acid.

2. A compound according to claim 1 wherein A is optionally substituted phenyl, X is oxygen, Y¹ and Y² are independently selected from chloro, bromo or iodo, p = 0 or p = 2 and R¹ is methyl, and R² is C╬╣-₆alkyl.

3. A compound according to claim 1 which is a carboxypeptidase activatable prodrug of Formula

wherein A is

and A is optionally substituted with upto 4 substituents independently selected from fluoro, chloro, bromo, C_1-4alkyl or C_1- alkoxy; X is a direct bond, CH or oxygen; V is NH or O; r is 0-2, provided that when V is O then r is zero;

Y and Y are independently selected from chloro, bromo, iodo, -O-S0₂-C_{1 -4}alkyl and -O- S0₂-phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from C_]-4alkyl, halo, cyano or nitro;

W is COOH or lH-l,2,3,4-tetrazol-5-yl;

R¹ is hydrogen, fluoro, chloro, bromo, C_1-4alkyl or C_1-4alkoxy; p is 0-4 wherein values of R may be the same or different and when p is less than 4 5 remaining substituent values are hydrogen;

R is C_1-6alkyl, hydroxyC_1-6alkyl, phenylC╬╣_-6alkyl, C╬╣_-4alkoxyC_1-4alkyl, phenylC_1-4alkoxyC_1-4alkyl, C╬╣_-4alkylthioC_)- alkyl, phenylC_1- alkylthioC_1-4alkyl or carbamoylC_]-4alkyl; or an enantiomer, diastereoisomer, pharmaceutically acceptable salt, in-vivo hydrolysable 0 ester or solvate thereof with the proviso that the following compounds and salts thereof are excluded, N-[N-(4-{4-[N,N- bis-(2-chloroethyl)-amino]-3-methyl-phenoxy}-benzoyl)-L-alanyl]-L-glutamic acid and

N-|T^-(4-{4-[ ,N-bis-(2-chloroethyl)-amino]-phenoxy}-benzoyl)-L-alanyl]-L-glutamic acid . 15

4. A compound according to claim 3 wherein W is COOH.

5. A compound according to any one of claims 3-4 wherein X is oxygen.

0 6. A compound according to any one of claims 3-5 wherein Y' and Y² are independently selected from chloro, bromo or iodo.

7. A compound according to any one of claims 3-6 wherein p = 0 or p = 2 and R¹ is methyl.

25

8. A compound according to any one of claims 3-7 wherein R² is Ci^alkyl.

9. A compound according to any one of claims 3-8 wherein V is oxygen.

30 10. A compound according to any one of claims 3-9 wherein r is 1.

1 1. A compound according to any one of claims 3-10 wherein A is optionally substituted phenyl.

12. Any one of the following compounds or a pharmaceutically acceptable salt thereof: (N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanyl)-L-glutamic acid;

(N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)benzoyl]-L-alanyl)-L-glutamic acid;

(N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanyl)-L-glutamic acid; (N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)benzoyl]-L-alanyl)-L-glutamic acid; and

(N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)-3,5-dimethylbenzoyl]-L- alanyl)-L-glutamic acid.

13. The compound (N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L- alanyl)-L-glutamic acid or a pharmaceutically acceptable salt thereof.

14. Any one of the following compounds or a pharmaceutically acceptable salt thereof: N-[4-(4-[N,N-bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanine ; N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)benzoyl]-L-alanine; N-[4-(4-[N,N-bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanine ;

N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)benzoyl]-L-alanine; and N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methoxyphenoxy)-3,5-dimethylbenzoyl]-L-alanine.

15. A process for preparing a compound of the Formula I or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof which process comprises a) deprotecting a compound of the Formula II, or

Formula II b) deprotecting a compound of the Formula Ila,

COOPr²

Formula Ha

1 2 wherein Pr and Pr independently represent hydrogen or carboxy protecting groups which may be the same or different, other variable groups are as defined in claim 3, and wherein any other functional group is optionally protected with the proviso there is at least one protecting group and optionally, if desired, forming a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof.

16. A pharmaceutical composition comprising a compound as defined in any one of claims 1-14 together with a pharmaceutically acceptable diluent or carrier.

17. A compound as defined in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for use as a medicament.

18. A matched two component system designed for use in a host in which the components comprise:

(i) a substantially non-immunogenic first component that is a targeting moiety capable of binding with a tumour associated antigen, the targeting moiety being linked to a mutated form of CPB enzyme capable of converting a prodrug as defined in any one of claims 3-13 into an antineoplastic drug and;

(ii) a second component that is a prodrug as defined in any one of claims 3-13 convertible under the influence of the enzyme to the antineoplastic drug, the prodrug not being significantly convertible into antineoplastic drug in the host by natural unmutated host enzyme.

19. A system according to claim 19 wherein the enzyme is [A248S,G251T,D253K]HCPB and the targeting moiety is humanised CDR grafted 806.077 antibody.

20. A method for the delivery of a cytotoxic drug to a site which comprises administering to a host a first component, which first component comprises an antibody or fragment thereof capable of binding a given antigen, the antibody or fragment thereof being conjugated to a mutant CPB enzyme capable of converting a compound as defined in any one of claims 3-13 or pharmaceutically acceptable salt thereof into a cytotoxic drug; followed by administration to the host of a second component, which second component comprises a compound as defined in any one of claims 3-13 or a pharmaceutically acceptable salt thereof convertible under the influence of the enzyme to the cytotoxic drug.