CA2242913A1 - Hydroxamic acid based collagenase inhibitors - Google Patents

Abstract

Compounds of formula (I) wherein R and R3 are each independently hydrogen, alkyl, alkenyl, alkynyl or aryl; R1 is arylmethyl; and R2 is alkyl, alkenyl, aryl, cycloalkyl or cycloalkenyl, and pharmaceutically acceptable derivatives thereof, are useful in the treatment and prophylaxis of disorders in which the overproduction of sCD23 is implicated.

Description

CA 022429l3 l998-07-l3 W O 97/26257 PCT~EP97/00196 - HYDROXAMIC ACID BASED COLLAGENASE INHI~ITORS.

This invention relates to novel inhibitors of the formation of soluble human CD23 and their use in the tre~tment of conditions associated with excess production of soluble 5 CD23 (s-CD23) such as autoimmune disease and allergy.
CD23 (the low affinity IgE receptor FceRlI, Blast 2), is a 4S kDa type II integral protein e,.l"-,ssed on the surface of a variety of mature cells, including B and T lymphocytes, macrophages, natural killer cells, Langerhans cells, monocytes and platelets (DelPspe~s~e et al, Adv Irrununol, 49 [1991] 149-191). There is also a CD23-like molecule on eosinophils (Grangette et al, J Imm~nol, 143 [1989] 358~3588). CD23 has been implicated in the regulation of the immune response (Delecpe~s~ et al, Immunol Rev, 125 [1992] 77-97).
Human CD23 exists as two differentially regulated isoforms, a and b, which differ only in the amino acids at the intr~ce~ r N-terminus (Yokota et al, Cell, 55 [19883 611-618). In man the constitutive a isoform is found only on B-lymphocytes, whereas type b, inducible by IL4, is found on all cells capable of eAplcssing CD23.
Intact, cell bound CD23 (i-CD23) is known to undergo cleavage from the cell surface leading to the formation of a number of well-defined soluble fragments (s-CD23), which are produced as a result of a complex se~u.,l~ce of proteolytic events, the m~och~nism of which is still poorly understood (Bourget et al J Biol Chem, ~ [ 1994] 6g27-6930).
Although not yet proven, it is post~ d that the major soluble fragm~nt.c (Mr 37, 33, 29 and 2~ kDa) of these proteolytic events, all of which relain the C-~ermin~l lectin domain common to i-CD23, occur sequentially via initial forrnation of the 37 kDa fragment (~ PtelliPr et al, J
E~p Med, 172, [1990] 693-700). An alternative intracellular cleavage pathway leads to a stable 16 kDa fragment differing in the C-terminal domain from i-CD23 (Grenier-Brosette et al, Eur JImml~nol, ;~ [1992] 1573-1577).
Several ac~ivities have been ascribed to membrane bound i-CD23 in hunl:lnc, all of which have been shown to play a role in IgE regulation. Particular activities include: a) antigen presentation, b) IgE mPdi~ted eosinophil cytotoxicity, c) B cell homing to germinal centres of Iymph nodes and spleen. and d) downregulation of IgE synthesis (Delespesse et al, Adv Immlmol, 49, [1991] 149-191). The three higher molecular weight soluble CD23fr~ enLc (Mr 37, 33 and 29 kDa) have multifunction~l cytokine properties which appear to play a major role in IgE production. Thus, the excessive folmation ot s-CD23 has been W O 97/2~257 PCTAEP97/00196 irnplicated in the overproduction of IgE, Lhe h~llm~rk of allergic dice~ces such as extrinsic asthma, rhinitis, allergic conjuctivitis, eczem~ atopic dermatitis and anaphylaxis (Sutton and Gould, Nature, 366, [1993] 421 -428).
Other biological activities attributed to s-CD23 include the stimulation of B cell growth and the induction of the release of me~ tors from monocytes. Thus, elevated levels of s-CD23 have been observed in the serum of patients having B-chronic lymphocytic le~ mi~ (Sarfati et al, Blood, 71 ~ 1988~ 94-98) and in the synovial fluids of patients with rh~nm~tnid arthritis (Chomarat et al, Arthritis and Rheurnatism, ~ [1993] 234-242). That there is a role for CD23 in infl~rnm~tion is suggested by a number of sources. First, sCD23 10 has been reported to bind to extr~cell-)l~r receptors which when activated are involved in cell-me~i~tPd events of infl~mrn~ion Thus, sCD23 is reported to directly activate monocyte TNF, IL-I, and IL-6 release (Annant et al, vol 1~0, J.Exp. Med., 1005-1011 (lg94)). CD23 has been reported to interact with the B2-integrin adhesion molecules, COl lb and CDl lc on monocytelmacrophage (S. Lecoanet-Henchozetal, Immunity, vol 3; 119-125 (1995)) which 15 trigger NO2-, hydrogen peroxide and cytokine ( IL- 1, IL-6, and TNE~) release. Finally, IL-4 or IFN induce the eA~ ssion of CD23 and its release as sCD23 by human monocytes.Ligation of the membrane bound CD23 receptor with IgElanti-IgE irnmune complexes or anti CD23 mAb activates cAMP and IL-6 production and thromboxane B2 formation, demonstrating a receptor-me~i~tP-d role of CD23 in infl~mm~tion.
Rec~llce of these various pl~pe.lies of CD23, compounds which inhibit the formation of s-CD23 should have twofold actions of a) enhancing negative feedback inhibition of IgE synthesis by m:~int~ining levels of i-CD23 on the surface of B cells, and b) inhibiting the immunostimulatory cytokine activities of higher molecular weight soluble fM~mentc (Mr 37, 33 and 29 kDa) of s-CD23. In addition, inhibiLion of CD23 cleavage 25 should mitigate sCD23-induced monocyte activation and mediator formation, thereby reducing the infl~mm~ry response.
International Patent Application No. PCr/EP95/02693 (Smithkline Beecham plc) discloses that compounds which inhibit the action of matrix metalloproteases (eg collagenase, stromelysin and gel~tin~ce) are effective inhibitors of the release of human soluble CD23 30 transfected into m~mm~ n cell culture systems. Known inhibitors of matrix metalloprotease include the compounds described in the patent publications listed in the Table.

W O 97/26257 PCT~EP97/00196 TABLE
Patent puLli~ n Compounds ~ )se~ Specif~c compounds and methods of pr~p~r~tion-Example Nos.
US-A-4,595,700 lto8.
US-A-4,599,361 lto7.
GB-A-2 268 934 ltolO.
GB-A-2 272 441 lto5.
EP-A-0 231 081 lto8.
EP-A-0 236 872 lto28.
EP-A-0 262 053 Compounds of fonnula 1 to 15.
EP-A-0 273 689 (I)~ defined in c}aim 1, 1 to38.
EP-A-0 276 436 option~ly ~ fu~her lto44.
EP-A-0 274 453 subdefined in the lto8.
EP-A-0 320 118 descnption. 1 to5.
EP-A-0 489577 1 to 25.
EP-A-0 489579 lto4.
EP-A-0 497 192 lto80.
EP-A-0 498 665 lto27.
EP-A-0 520573 1 to 34.
EP-A-0 574 758 lto43.
EP-A-0 575 844 lto27.
EP-A-0 606 046 lto32.
EP-A-0 613 883 lto7.
EP-A-0 621 270 lto40.
WO 90/05716 lto38.
WO90/05719 lto26.
WO91/02716 lto17.
WO9V09563 Compounds of formula (1) or I to 21.
(2) as defined in cl~im 1.

TABLE contd.

Patent pub!;~ on Compounds disclosed Specific co~ ,o~l...ls and methods of P~ lion-Example Nos.
WO 92113831 1 to 27.
WO 92/21360 1 to S.

3 I to X.
WO 93/14096 1 to 8.
WO 93/20047 1 to 14.
WO 93/24475 1 to 6.
WO 93124449 Compounds of formu}a 1 to 8.
WO 94/00119 (I) as defined in claim 1, 1 to 86.
WO 94~07481 optionally as further 1 to 15.
WO 94/12169 subdefined in the 1 to 24.
WO 94121625 description. 1 to 7.
WO 94/21612 1 to 116.
WO 94/24140 1 to S.
WO 94125434 1 to 7.
WO 94125435 Example 1.
WO g5104033 Examples 1 to 7.
WO 95104715 All examples.
WO 95/12603 All examples.

W O 97/262~7 PCT~P97/00196 According to the present invention, there is provided a compound of formula (I):
O R' o HON~ ~ NHR3 (I) S wherein R and R3 are each independently hydrogen, alkyl, alkenyl, aL~cynyl or aryl; Rl is arylmethyl; and R2 is alkyl, alkenyl, aryl, cycloalkyl or cycloalkenyl.
Alkyl, alkenyl and alkynyl groups referred to herein include straight and branched groups cont~ining up to six carbon atoms and are optionally substituted by one or more groups selected from the group concicting of aryl, heterocyclyl, (Cl 6)aL~ylthio, 10 (Cl 6)alkenylthio, (Cl 6~alkynylthio, arylthio, heterocyclylthio, (Cl 6)alkoxy, aryl(C 1 6)aL~coxy, aryl(C 1 6)alkylthio, amino, mono- or di-(C 1 6)aLkylamino, cycloalkyl, cyclo~lk~nyl, carboxy and esters thereof, hydroxy, and halogen.
Cycloalkyl and cycloaLkenyl groups referred to herein include groups having between three and eight ring carbon atoms and are optionally substi~lted as described 15 hereinabove for alkyl, aL~enyl and alkynyl groups.
When used herein, the term "aryl" incl~des phenyl and naphthyl such as 2-naphthyl.
Suitably any aryl group, including phenyl and naphthyl, may be optionally subs~i~u~Pd by up to five, preferably up to three substituents. Suitable subs~itllenLc include halogen, (C1 6)aLkyl, aryl(C1 6)alkyl, (C1 6)alkoxy, (CI 6)alkoxy(C1 6)alkyl, halo(Cl 6)aL~yl, 20 hydroxy, nitro, amino, mono- and di-N-(C l 6)alkylamino, acylamino, acyloxy, carboxy, carboxy salts, carboxy esters, carbamoyl, mono- and di-N-(C 1 6)alkylcarbamoyl, (C1 6)alkoxycarbonyl, aryloxycarbonyl, ureido, guanidino, sulphonylamino, aminosulphonyl, (C1 6)alkylthio, (Cl 6)alkyl sulphinyl (C1 6)alkylsulphonyl, heterocyclyl and heterocyclyl (C 1 6)aLkyl. In addition, tWO adjacent ring carbon atoms may be linked by 25 a (C3 s)alkylene chain, to form a carbocyclic ring.
When used herein the terms "heterocyclyl" and "he~erocyclic" suitably include, unless otherwise defined, aromatic and non-aromatic, single and fused, rings suitably con~ining up Lo four heteroatoms in each rin;" each of which is selected from oxygen, nitrogen and sulphur, which rings, may be unsubstituted or substituted by, for example, up to three s wo 97/262~7 PCT/EPg7l00196 s~1bsrin~ents. Each heterocyclic ring suitably has from 4 to 7, preferably 5 or 6, ring atoms.
A fused heterocyclic ring system may include carbocyclic rings and need include only one heterocyclic ring.
Preferably a substituent for a heterocyclyl group is selected from halogen, (C 1-6)alkyl, aryl(C 1 6)alkyl, (C I 6)alkoxy, (C I 6)alkoxy(C 1 6)alkyl, halo(C 1 6)alkyl, hydroxy, amino, mono- and di-N-~C 1-6)alkyl-aminot acylamino, carboxy salts, carboxy esters, carbamoyl, mono- and di-N-(CI 6)alkylcarbonyL aryloxycarbonyl, (Cl 6)alkoxycarbonyl(C 1 6)alkyl, aryl, oxy groups, ureido, guanidino, sulphonylamino, aminosulphonyl, (C 1 6)aLkylthio. (C 1 -6)alkylsulphinyl, (C 1 6)aL~cylsulphonyl, heterocyclyl 10 and heterocyclyl(Cl 6)aL~yl.
In a particular aspect of the invention, R is hydrogen or methyl, optionally subsdtuted by arylthio or heterocyclylthio; and/or Rl is a benzyl group; and/or R2 is a benzyl group; and/or R3 is hydrogen, methyl or benzyl.
According to a further aspect, the present invendon provides the use of a compound 15 of formula (I) for the production of a m~r~ic~ment for the treatrnent or prophyla~is of disorders such as allergy, infl~mm~tory disorders, and autoimmune dice~ce, in which the overproduction of s-CD23 is implicated.
In a further aspect the invention provides a method for the treatmen~ or prophylaxis of disorders such as allergy, infl~mm~tory disorders, and autoimmune ~ ce~ in which the 20 overproduction of s-CD23 is implicated, which method comprises the ~minictration of a compound of formula (I), to a human or non-human m:~mm~l in need thereof.
The invendon also provides a pharmaceutical composition for the treatment or prophylaxis of disorders such as allergy, infl~mm~tory disorders, and autoimmune ~ ~, in which the overproduction of s-CD23 is implicated which comprises a compound of formula 25 (I) and optionally a pharmaceutically acceptable carrier therefor.
Particular infl~mm~tory disorders include CNS disorders such as Al7h~imers disease, multiple sclerosis, and multi-infarct dementia, as well as the in~l~mm~ion mediated sequelae of stroke and head trauma.
It is to be understood that the pharmaceutically acceptable salts, solvates and other 30 pharrnaceutically acceptable derivatives of the compound of formula ~I) are also included in the present invention.

CA 022429l3 l998-07-l3 WO 97/26257 PCTrEP97/00196 Salts of compounds of formula (I) include for example acid addition salts denvedfrom inorganic or organic acids, such as hydrochlorides, hydrobromides, hydroiodides, p-toluen~sulrlhon~tes, phosphates. sulphates, Acet~tes trifluoroacetates, propionates, citrates, m~ res, fumarates, malonates, succinates, lactates, oxalates, tartrates and ben7O~tes Salts may also be formed with bases. Such salts include salts derived from inorganic or organic bases, for example alkali metal salts such as sodium or potassium salts, and organic amine salts such as morpholine, piperidine, dimethylamine or diethylamine salts.
It has surprisingly been found that the compounds of the present invention are potent and selective inhihitors of CD23 processirlE~ whilst exhibiting reduced collagenase inhibitory 10 activity in comparison with the above-m~ntioned compounds of the prior art.
The compounds of the invention may be prepared by use of any appropriate conventional m~thod, for example by analogy with the methods disclosed in patentpublications WO 90/05716, WO 93/24475, WO 94/21625, WO 95/19956, WO 90/05719, WO 91/02?16, WO 92113831, WO 93t20047, EP-A-0214639, EP-A-0236872, EP-A-15 0274453, EP-A-0489577, EP-A-0489579, EP-A-0497192, EP-A-0574758 and USP
4599361.
Accordingly, a further aspect of the invention provides a process for preparing a compound of formula (I) as defined hereinabove, which process comprises:
(a) deprotecting a compound of formula (II):
O R' O
XONH J~ ~JHR3 (II) wherein R to R3 are as defined hereinabove, and X is a protecting group such as benzyl or trimethylsilyl or W O 97/26257 PCT~P97/00196 (b) reacting a compound of formula (III):

o R' o HO J~ ~ NI~F13 R ~ R2 (III) wherein R to R3 are as defined hereinabove, with hydroxylamine or a salt thereof, or (c) reacting a compound of forrnula (IV):

O R' O
HONH ~¢N~,HR3 (IV) wherein Rl to R3 are as defined hereinabove, with a thiol to give a compound of formula (I) wherein R is methyl substituted by alkylthio, arylthio, araL~cylthio, or heterocyclylthio, or (d) converting a compound of formula (I) to a different compound of formula (I) as defined hereinabove.
Compounds of formulae (II), (m) and (IV) are novel and form a further aspect of lS the invention.
Compounds of formula (II) can be prepared from compounds of formula (III) by reaction with a protected hydroxylamine. Compounds of formula (III) can be prepared by hydrolysis of a compound of fonnula (V):

O R' o y ~ ~NHR3 R ~ R2 (V) wherein R to R3 are as defined hereinabove, and Y is a protecting group such as t-butyl.
Suitable protecting groups for a hydroxarnic acid are well known in the art and 2~ include benzyl, trimethylsilyl, t-butyl and t-butyldimethylsilyl.

Suitable protecting groups for a carboxylic acid are well known in the art and include t-butyl, benzyl and methyl.
Compounds of formula (V) can be prepared by reduction of a compound of formula (VI): ' s Y-a J~j~ ~ NHR3 O R
-(VI) wherein R1 to R3 and Y are as defined hereinabove, and Z is a group such that ZCH2- is R.
Compounds of formula ~V) can also be prepared by reacting a compound of 10 formula (VII):

Y-O
R O
(VII) wherein R, Rl and Y are as defined hereinabove, with a compound of formula (Vm):
o NH2 ~
'r NHR3 (VIII) wherein R2 and R3 are as defined hereinabove, or an activated derivative thereof.

W O 97/26257 PCTAEP97/OOlg6 Compounds of formula (lII) can also be prepared by reacting a compound of formula (IX):

O R O
HO ~ ~ NHR3 (IX) wherein Rl to R3 are as hereinabove defmed, with a thiol to give a compound of formula (I~I) wherein R is methyl substituted by aLkylthio, arylthio, aralkylthio, or heterocyclylthio.
Compounds of formula (IX) can be prepared by hydrolysis of a compound of formula (X):
O R' o y ~ ~NHR3 (X) wherein Rl to R3 and Y are as defined hereinabove.
Compounds of forrnula (X) can be prepared by reacting a compound of formula ~XI):

~'~'~

(XI) wherein Rl and Y are as defined hereinabove, with a compound of formula (VII~) as defined hereinabove, or an activated derivative thereof.
The starting materials and other reagents are available commercially or can be syn1hesiced by well-known and conventional methods.
The isomers, including stereoisomers, of ~he compounds of the present invention may be prepared as mixtures of such isomers or as individual isomers. The individual isomers may be prepared by any appropriate method, for example individual stereoisomers may be prepared by stereospecific chemic~l synthesis starting from chiral substrat~s or by separating mixtures of diastereoisomers using known methods. In a preferred aspect, the invention provides compounds of formula (IA):
s O R O
HONI~ ~ NHR3 R ~ R2 (lA) It is plefc--~d that the compounds are isolated in subst~nti~lly pure form.
As stated herein an inhibitor of the formation of soluble human CD23 has useful medi~l p~pe.lies. Preferably the active compounds are a~imini.ctered as pharrn~eu~ lly acceptable compositions.
The compositions are preferably adapted for oral ~dnninictration. However, they may be adapted for other modes of a~lminictration, for example in the form of a spray, aerosol or other convention~l method for inh91~tion for treating respiratory tract disorders; or p~nte.~l a~minictration for patients suffering from heart failure. Other alternative modes of ~tlminictration include sublingual or transdermal ~<lminictration.
The compositions may be in the form of tablets, c~ps~ c, powders, granules, lo~nges, suppositories, reconctitut~ble powders, or liquid preparations, such as oral or sterile parenteral solutions or suspensions.
In order to obtain concictency of ~minictration it is preferred that a composition of the invention is in the forrn of a unit doce.
Unit dose pres~nt~tion forms for oral ~mini.ctration may be tablets and c~psulesand may contain conventional excipients such as binding agents, for exarnple syrup, acacia, gelatin, sorbitol, trag~nlh, or polyvinylpyrrolidone; fillers, for example 2~ lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycollate or microcrystalline cellulose; orpharmaceutically acceptable wetting agents such as sodium lauryl sulphate.
The solid oral compositions may be prepared by conventional methods of bl~ in~, filling or tabletting. Repeated blending operations may be used to distribute W O 97126257 PCT~P97100196 the active agent throughout those composiLions employing lar~e quan~ities of fillers.
Such operations are of course conventional in the art. The tablets may be coatedaccording to methods well known in normal pharrn~neutical practice, in particular with an enteric coating.
Oral liquid preparations may be in the form of, for example, emulsions, syrups, or elixirs, or may be presented . s a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for ex,qmpl~ sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, 10 hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hy~u~yl er-7oqte or sorbic acid; and if desired conventional flavouring or colouring 15 agents.
For parenteral a~minictration~ fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, and, depen~ling on the concentration used, can be either suspended or dissolved in the vehicle. In p,ep~ing solutions the compound can bedissolved in water for injecdon and filter sterilized before filling into a suitable vial or 20 ampoule and sealing. Advantageously, adjuvants such as a local ~n~~~thPtic, ap~t;se.vddve and buffering agents can be dissolved in the vehicle. To çnh~nce the stability, the cornposition can be frozen after filling into the vial and the water removed under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle instead of being dissolved, and 25 sterili~tion cannot be accomplished by filtration. The compound can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition lo facilitate uniformdistribution of the compound.
Compositions of this invention may also suitably be presented for a~mini.~traLion 30 to the respiratory tract as a snuff or an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case the particles of active compound suitably have diameters of less W O 97/26257 PCTAEr97/00196 than 50 microns, preferably less than 10 microns for exarnple diameters in the range of 1-50 microns, 1-10 microns or 1-5 microns. Where appropriate, small amounts of other anti-as~hm~tirS and bronchodilators, for example sympathomimetic amines such as isopren~line, isoetharine, salbutamol, phenylephrine and ephedrine; x~nthine 5 derivatives such as theophylline and aminophylline and corticosteroids such as prednisolone and adrenal stimulants such as ACrH may be included.
The compositions may contain from 0.1% to g9% by weight, preferably from 10-60% by weight, of the active material, depen~in~ upon the method of ~minictration. A preferred range for inhaled ~tlminictr~tion is 10-99%, especially 60-99%, for example 90, 95 or 99%.
Microfine powder formulations may suitably be ~lminictPred in an aerosol as a metered dose or by means of a suitable breath-activated device.
Suitable metered dose aerosol fonn~ tions comprise conventional propellants, cosolvents, such as ethanol, sllrfPrt~ntc such as oleyl alcohol, lubricants such as oleyl 15 ~ ohol, desi< c~ntc such as c~lrinm sulrh~tP and density modifiers such as sodium chloride.
Suitable solutions for a nebuli~r are isotonic sterilised solutions, optionally buffered, at for example bet~en pH 4-7, co~ ining up to 20mglml of compound but more generally 0.1 to lOmg/ml, for use with standard nebulisadon equipmpnt An effecdve amount will depend on the reladve efficacy of the compounds of the present inventdon, the severity of the disorder being treated and the weight of the sufferer. Suitably, a unit dose form of a composidon of the invendon may containfrom 0.1 to lOOOmg of a compound of the invendon (0.001 to lOmg via inhaladon) and more usually &om 1 to 500mg, for example I to 25 or 5 to 500mg. Such compositions may be a~iminic~Pred from 1 to 6 times a day, more usually from 2 to 4 times a day, in a manner such that the daily dose is from lmg to lg for a 70 kg human adult and more particularly from S to 500mg. That is in the range of about 1.4 x lo-2 mglkglday to 14 mglkglday and more pardcularly in the range of about 7 x 10-2 mglkglday to 7 mglkg/day.
The following examples illustrate ~he invendon but do not limit it in ~ny way.

W O 97/26257 PCT~EP97100196 BIOLOGICAL TEST METHODS

re 1: The ability of test compounds to inhibit the release of soluble CD23 was investig~ted by use of the following procedure.

RPMI 8866 Cell membrane CD23 cl~..~r..tg~ ac~vity assay:

Plasma membr~nes from RPMI 8866 cells, a human Epstein-Barr virus transformed B-cell line (Sarfati et al., Immunology 60 t19871 539-547) e~ cssing high levels of CD23 are purified using an aqueous extraction method Cells resuspended in homogeni7~ti-~P
buffer (20mM HEPES pH 7.4, 150 mM NaCI, 1.5 mM MgCl2, 1 mM Dl'r) are broken by N2 cavitation in a Parr bomb and the plasma membrane fraction mixed with other membranes is recovered by centrifugation at 10,000Xg. The light pellet is resuspended in 0.2 M pot~s~nm phosph~t~-, pH 7.2 using 2 ml per 1-3 g wet cells and the nuclear pellet is discarded. The membranes are further fracdonated by par~itioning between Dextran 500 (6.4% wlw) and polyethylene glycol (PEG) S000 (6.4% wtw) (ref), at 0.25 M sucrose in a total of 16 g per 10-15 mg membrane proteins [Morre and Morre, BioTechniques 7t 94~957 (1989)]. The phases are separated by brief centrifugation at 1000Xg and the PEG (upper) phase is collected, diluted 3-5 fold with 20 mM potassium phosphate buffer pH 7.4, and centrifuged at 100,000Xg to recover membranes in that phase. The pellet is resuspended in phosphate-buffered saline and consists of 3-4 fold enriched plasma membranes as well as some other cell membranes (e.g. Iysosomes, Golgi). The membranes are aliquoted and stored at -80~C. Fractionation at 6.6 % Dextran/PEG yields plasma membranes enriched 10-fold.
The fractionated membranes are incubated at 37~C for times up tO 4 hrs to produce fr~gmPntc of CD23 which ~re separated from the membrane by filtration in 0.2 micron Durapore filter plales (Millipore) after quenching the assay with S uM Preparation 1 from P
30994. sCD23 released from the membrane is deterrnined using the EIA kit from The Binding Site (Birmingham, UK) or a similar one ulili7.ing MHM6 anti-CD23 mAb [Rowe et al., Int. J. Cancer, 29, 373-382 (1982)} or another anti-CD23 mAb as the capture antibody in a sandwich ELA.. The amount of soluble CD23 made by 0.5 ug membrane protein in a total W 097~62~7 PCTAEP97~00196 volume of 50 ul phosphate-buffered. saline is measured by EL~ and compared to the amount made in ~Lhe presence of various concentrations of inhibitors. Inhibitors are prepared in solutions of water or dimethylsulfoxide (DMSO) and the final DMSO concentration is not more than 2 %. IC50's are determined by curve f1tting as the concentration where 50 %
inhibition of producLion of sCD23 is observed relative to the difference in sCD23 between controls incubated without inhibitor.

Procedure 2: The ability of test compounds to inhibit coll~gen~ce was investigated using the following procedure.
Collagenase inhibition assay:

The potency of compounds to act as inhibitors of coll~g~-n~ce was determined by the method of Cawston and Barrett (Anal. Biochem. 99, 340-345, 1979), hereby incorporated by 15 reference, whereby a 1 mM solution of the inhibitor being tested or dilutions thereof, was incubated at 37 ~C for 18 h with coll~g~n and human recombinant coll~n~ce, from synovial fibroblasts cloned, expressed and purified from E. Coli, (buffered with 150 mM
Tris, pH 7.6, containing 15 mM calcium chloride, 0.05% Brij 35, 200 mM sodium chlo- de and 0.02% sodium azide). The collagen was acetylated 3H type 1 bovine collagen prepared 20 by the method of Cawston and Murphy (mPthr)~ls in Enzymology 80, 711,1981) The samples were centrifuged to sediment undigested collagen and an aliquot of the radioactive supernatant removed for assay on a scintill~tion counter as a measure of hydrolysis. The coll~gPn~ce activity in the p-~sence of lmM inhibitor, or dilution thereof, was compared to activity in a control devoid of inhibitor and the results reported as that concentration 25 effecting 50% of the collagenase (ICso).

WO 97/26257 PCT~EP97/00196 ExamDle 1 N-(2-(R)-Benzyl-4-hydroxyaminos-lccinyl)-(S)-phenyl~ nin~-N'-benzylamide ~Ph ~ ~ Ph HOHNOC O ~
Ph a) 2-Benzylidene-4-tert-butoxysuccinic acid ~CO2H
Bu'02C

A solution of methyl 2-benzylidene-4-tert-buto~ysuccinate (2g,7.2Smmol) in me.~h~nol (20ml) was treated with a solution of sodium hydroxide (2M,7.25ml,14.5mmol) and the mixture stirred for 16h at 20~C. The reaction was concentrated under vacuum and water (20ml) added.The aqueous solution was extra~ted with ether (2x20ml), acidified with 2M
hy~uehloric acid and extracted again with ether (3x25ml). llle ether from acidic extraction was dried ~MgSO4), filtered and evaporated to give the acid as a white solid (1.74g,92%).
mp 110-115~C.

dH (CDC13) 1.46 (9~,s), 3.47 (2H,s), 7.3~ (SH,m), 7.94 (lH,s), 12.58,br s).

b) N-(2-Benzylidene-4-tert-butoxysuccinyl)-(S)-phenyl~l~ntne-N'-benzylamide ,~ Ph ~ Ph ButO2C O Ph W O 97~6257 PCT/EP97100196 A solution of 2-benzylidene-4-tert-butoxysuccinic acid (524mg,2mmol), (S)-phenyl~l~nine-N'-benzylamide hydrochloride ~580mg,2mmol), l-hydroxybenzotriazole hydrate (486mg,3.6mmol) and diisopropylethylamine (0.35ml ~mmol) in DMF (20ml) was treated at 0~C with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (424mg,~ ~mmol) S and the reaction s~irred for lh at 0~C and 16h at 20~C. Following evaporation, tne residue was partitioned between saturated sodium hydrogen carbonate (25ml) and dichlorometl-~ne (SOml). The organic layer was washed with sodium hydrogen carbonate (2x25ml), 10% citric acid (2x25ml) and brine (25ml) and dried (MgS04). Filtration and evaporation gave a residue which was chromatographed on silica (chloroforrn-me~h~nol 100:1) to give the title compound as an off-white foam (850mg,85%).

dH (CDC13) 1.40 9H,s), 3.25 (2H,m), 3.32 (IH,d,J=16.5Hz), 3.50 (lH,d,J=16.5Hz), 4.40 (2H,m), 4.80 (lH,dd,J=6.9,7.2Hz), 6.75 (2H,m), 7.26 (16H,m).

c) N-(2-(RJS)-Benzyl-4-tert-butoxysuccinyl)-(S)-phenyl~l~nin~-N'-benzylamide ~,Ph ~ . ~ Ph Bu'02C O Ph A solution of N-(2-benzylidene-4-tert-butoxysuccinyl)-(S)-phenylalanine-N'-'oenzylamide (790mg,1.58mmol) in ethanol (SOml) was hydrogenated at atmospheric pressure for 4.5h.
The reaction was filtered, evaporated under reduced pressure and the residue chromatographed on silica (chloroform-methanol 100:1) to give the desired material as a white solid and a 1:1 mixture of diastereoisomers (660mg,83%) mp 95-100~C.
dH (CDC13) 1.25 + 1.41 (9H,2 x s), 2.3-3.~ (7H,m), 4.05-4.70 (2H,m), 4.60-4.~0 (lH,m), 5.33-6.02 (2H,m), 6.7-7.4 (lSH).

W O 97t26257 PCT~EP97/00196 d) N-(2-(R/S)-Benzyl-4-hydroxysuccinyl)-(S)-phenyl~l~ninP-N'-benzylamide ~,Ph ~ ~ Ph HO2C O Ph Asolutionof N-(2-(R/S)-benzyl-4-tert-butoxysuccinyl)-(S)-pheny~ nine-N~-benzyl~mide (650mg,1.3mmol) in dichlorometh~nP- (15ml) was treated with trifluoroacetic acid (15ml) and sdrred at 20~C for 4h. The reaction was evaporated and co-evaporated with toluene.
Trituration of the residue with ether afforded the acid as a white solid and a 1: 1 mixture of diastereoisomers (470mg,81%) mpl72-5~C.
dH [(CD3)2S0~ 2.01-3.03 (7H,m), [4.22 (d,J=5.8Hz) + 4.27 (d, J=5.4Hz), total 2H], 4.53 (lH,m), 7.02-7.31 ~lSH,m), [8.18 (t,J=5.8Hz) + 8.27 (t,J=5.9Hz, total lH], [8.22 (d,J=8.3Hz) + 8.35 (d,J=8.5Hz), total lH], 12.1 (lH,br s,C02H).

e) N-(2-(R)-Benzyl-4-hydroxy~minosuccinyl)-(S)-phenyl~l~ninP-N'-benzylamide ~Ph ~ _ ~ Ph HOHNOC O ~ Ph 20 Asolutionof N-(2-(RJS)-benzyl-4-hydroxysuccinyl)-(S)-phenyl~ ninP-N'-benzylamideasa 1: 1 mixture of diastereoisomers (250mg,0.56mmol), diisopropylethylamine (0.2ml,1.12mmol) and O-trimethylsilylhydroxylamine (300mg,2.8mmol) in dichlorometh~ne ~lOml) was cooled to 0~C and treated with bromo-tris-pyrrolidinophosphonium hP~r~fluorophosphate (PyBroP) (160mg,0.67mmol) under argon. The reaction was stirred for lh at 0~C and 16h at 20~C. The organic layer was washed with brine (2xlOml), dried (MgS04) and evaporated to a sticky solid. This residue was subjected to preparative HPLC

using a VYDAC protein and peptide Clg reverse-phase column, elu~ing with a 1:1 mixture of 0.1%TFA in H2O: 70% acetonitrile in 0.1% TFA in H2O at 20ml min~l.
The first eluting diastereoisomer (9.7min) was shown to be N-(2-(R)-benzyl-4-hydroxy~minosuccinyl~-(s)-phenylAl~nine-Nl-benzylamide (50mg,19%) mp lg5-7~C.

dH [(CD3)2SO] 1.88 (lH,dd,J=6.9,14.9Hz), 2.09 (lH,dd,J=7.5,14.9Hz), 2.72-3.05 (SH,m), 4.21 (2H,m), 4.47 (lH,m), 7.23 (ISH,m), 8.18 (2H,m), 8.70 (lH,br s), 10.35 (lH,br s).

The second eluting diastereoisomer (12.5min) was shown to be N-(2-(S)-benzyl-4-hydroxy~minos~ccinyl)-(S)-pheny~ nine-N~-~enzylamide (30mg,12%) mp 203-5~C.

~Ph N~V~N~Ph HOHNOC O ~ Ph dH ~(CI)3)2SO] 1.92 (lH,dd,J=6.1,15.1Hz), 2.23 (lH,dd,J=8.8,15.0Hz), 2.35-2.75 (3H,m), 2.98 (2H,m), 4.27 (2H,m), 4.45 (lH,m), 7.21 (lSH,m), 8.33 (lH,d,J=8.5Hz), 8.4S (lH,m), 8.71 (lH,s), 10.42 (lH,s).

W O 97J26257 PCT~P97100196 -F.Y~mDIe 2 N-(2-(R)-Benzyl-4-hydroxyaminosuccinyl)-(S)-phenyl~l~nin~-N'-methylamide f~ Ph ~ -~ NHMe HOHNOC O ~
Ph a) 4-(S~-Benzyl-3-(3-phenylpropionyl)-2-oxazoli-linone ~ ~I~
~N _~O

Bn To a solution of 4-(S)-benzyl-2-oxazolidinone ( 23.9g, 135mmol) in tetrahydrofuran (250ml) at -65~C was added dropwise n-BuLi (1.6M in hPX~nPS lOlml, 178mmol). The res~llt~n~
orange solution was s~irred for 30 minl~tPs then a solution of 3-phenylpropionyl chloride ( 25g, 148mmol~ in tetrahydrofuran (40ml) was added dropwise. After l.Sh the reaction was 15 poured into ice-cold 2M hydrochloric acid (200ml) and extracted with ethyl acetate (3xl50ml). The combined organic extracts were washed with 2M hydrochloric acid (150ml), sodium hydrogen carbonaoe solution (150ml) and brine (lOOml). Afoer drying ~MgSO~) and evaporation the residue was clyst:~lli.ced from ether to give the title compound as white needles mp 109-111 ~C. dH(CDCl3) 2.75 (lH,dd,J=13.5,10Hz), 2.96-3.07(2H,m);3.17-3.38 (3H,m), 4.14 (lH,s), 4.16 (lH,d,J=2), 4.60-4.70 (lH,m), 7.15-7.35 (lOH,m).

wo 97/262s7 PCT/EPg7/00196 ~ b) 4-(S)-Benzyl-3-(2-~tert-butoxycarbonylmethyl]-3-phenyl)propionyl-2-oxazolidinone ~0 ~
V~ N o ~ ~J
tBu02C Bn To a solution of 1,1,1-3,3,3-hPx~methy~ si~ ne (9.6ml, 45mmol) in tetrahydrofuran (60ml) at -20~C was added dropwise n-BuLi (1.6M in hex~nes, 28.3ml, 45mmol). After 45 minut~S the solution was cooled to -75~C and a solution of 4-(S)-~enzyl-3-(3-phenylpropionyl)-2-oxazolidinone (lOg, 32mmol) in tetrahydrofuran ~60ml~ was added dropwise. The solution was stirred for lh then allowed to warm to -50~C over the next hour.
After cooling to -75~C tert-butyl bromo~cel~tP (7.8ml, 48mmol) in tetrahydrofuran (50ml) was added dropwise and the solution stirred for 1 h prior tO warming to -20~C over a further 2.5h. The reaction was then poured into saturated ammonium chloride solution (lOOml) and extracted with ether (3xlOOml). The combined organic extracts were washed with 2M
hyd~ocllloric acid (SOml), sodium hydrogen carbonate solution (50ml) and brine (SOml).
After drying (MgSO,) and evaporation the yellow oil was triturated with etherfhexane (1:4) to yield the title compound as a white solid (12.3g, 90%) mp 111.5-112.5~C. dH(CDCl3) 1.4 ~9H,s), 2.37 (lH,dd,J=17,4Hz), 2.64 (lH,dd,J=13,9Hz), 2.72 (lH,dd.J=14,10Hz), 2.85 (lH,dd,J=17,11Hz), 3.01 (lH,dd,J=13,6Hz), 3.31 (lH,dd,J=13.5,3Hz), 3.93 (lH,t,J=6Hz), 4.07 (lH,dd,J=9,2.5Hz), 4.44-4.57 (2H,m), 7.1X-7.37 (lOH,m).

W O 97126257 PC~EP97/00196 c) 2-(R)-Benzyl-4-tert-butoxysuccinic acid Ph f ~ CO2H
Bu'02C
s A solution of 4-(S)-benzyl-3-(2-[tert-butoxycarbonylmethyl]-3-phenyl)propionyl-2-oxazolidinone (5.5g,13mmol) in 3: 1 THF/water (200ml) was treated with 27.5% hydrogen peroxide (9.65ml,78mmol) at 0~C and stirred for lOmin before the addition of a solution of lithium hydroxide ~1. lg,26mmol) in water (lOOml). The reaction was stilTed for 30min at 10 0~C and the excess peroxide destroyed by the addition of 1.5M sodium s~llrhite solution (llg,87mmol) in water (58ml). The solution was buffered to pH 9 with sodium hydrogen carbonate and the THF evaporated. The residue was partitioned between water (300ml~ and dichlorometh~n~ (300ml) and the aqueous layer washed with dichlorometh~n~ (2x20~ml).
The aqueous layer was ~oidifi~d with lM HCI and e~lla~,~d with ethyl acetate (3x200ml).
is Drying of the organic layer (MgSO,,), filtration and evaporation gave the required acid as a clear oil (3.4g,100%).

dH(CDCI3) 1.42 (9H,s), 2.35 (lH,dd,J=4.7,16.8Hz), 2.56 (lH, dd, J=8.7,16.8Hz), 2.77 (lH,dd,J=10.4,15.4Hz), 3.09 ~2H,m), 7.17-7.34 (SH,m), acid proton not observed.

d) N-(2-(R)-Benzyl-4-tert-butoxysuccinyl)-(S)-phenyl~ nine-N'-methylamide ~ NHMe Bu O2C O Ph W O 97126257 PCT~EP97/00196 2~ Benzyl-4-tert-butoxysuccinic acid (lg,3.78mmol), (s)-pheny~ nine-N~-methylamide hydrochloride (812mg,3.78mmol), N,N-diisopropylethylamine (0.66ml,3.78mmol), 1-hydroxybenzotriazole hydrate (919mg,6.8mmol), and 1-(3-dime~hylaminopropyl)-3-ethylcarboAiimide hydrochloride (797mg,4.16mmol) in DMF (70ml) were treated as in S es~m~le 1 b) to give the ~ille compound as a white solid following recryst~lli.cation from ethyl acetate-hexane (1.15g,72%).

dH (CDCl3) 1.43 (9H,s), 2.38 (IH,dd,J=4.4,16.8Hz), 2.57 (3H,d,J=4.7Hz), 2.53-2.99 (SH,m) 3.15 (lH,dd,J=5.8,13.8Hz), 4.54 (lH,m), 5.33 (lH,br m), S.90 (lH,br d,J=8Hz), 7.12-7.31 10 (lOH,m).

e) N-(2-(R)-Benzyl-4-hydroxysuccinyl)-(S)-phenyl~l~ninP-N'-methylamide ~Ph ~ ~ NHMe HO2C O Ph N-(2-(R)-Benzyl-4-tert-butoxysuccinyl)-(S)-pheny~ nin~-N'-methylamide (1.02g,2.41mmol) and TFA (lOml) in dichloromethane (lOml) were treated according to e~ample 1 d) to give the required ~cid as a white solid (830mg,94%).
dH [(CD3)2SO] 2.03 (lH,dd,J=5.5,16.5Hz), 2.33 (lH,dd,J=8.4,16.6Hz), 2.50 (3H,d,J=4.6Hz), 2.56 (lH,m), 2.75-2.g9 (4H,m), 4.39 (lH,m), 7.14-7.27 (lOH,m), 7.47 (lH,m), 8.17(lH,d,J=8.2Hz), 12.07 (lH,br s).

W O 97/26257 PCT~EP97/00196 f) N-(2-~R)-Benzyl-4-hydroxyarninosuccinyl)-(S)-phenyl~ ninP-N'-methylamide O
~ ~Nl tMe HOHNOC O -~

S A solution of N-(2-(R)-benzyl~-hydroxysuccinyl)-(S)-phenyl~l~nin~-~'-methylamide ~0.8g,2.17mmol) in THF (50ml) was cooled to -20~C and treated with N-methylmorpholine (0.29ml,2.61mmol) and isobutylchloroformate (0.34ml,2.61mmol) and the reaction stirred for lh at -20~C. O-TMS-hydroxylarnine (2.2ml,20.9mmol) was added dropwise, the reaction allowed to warm to ambient temperature and stirred for 16h. After that time, the reaction was 10 evaporated and partitioned be~ween ethyl acetate and 10% citric acid. The organic layer was washed with brine and dried (MgSO,). Filtration and evaporation gave a solid which was recryst~lliced from ethyl acetate-methanol to give the title compound as a white solid (300mg,36%) mp 188- 190~C.

dH [(CD,)2SO] 1.91 (lH,dd,J=7.2,15.0Hz), 2.06 (lH,dd,J=7.2,15.0Hz~, 2.50 (4H,m), 2.75 (2H,m), 2.95 (2H,m~, 4.31 (lH,m), 7.06-7.27 (lOH,m), 7.38 (lH,m), 8.11 (lH,d,J=8.2Hz), 8.73 (lH,s), 10.4 (lH,s~.

W 097t26257 PCTrEP97/00196 ~ E~h~ Jle 3 N-(2-(R)-Benzyl-4-hydroxyamino-3-(S)-[2-thienothiomethyl]succinyl)-(S)-S phenyl~l~nin~mide.

O ~[30 HONI l~b' ~NIl2 ~S~ ~1 S~

a) Benzyl 3-(R)-phenyllactate.

,3 -HO CO2Bn A soludon of 3-(R)-phenyllactic acid (20.381g, 123mmol) in THF (160ml) was treated with triethylamine (23.5ml, 17.02g, 168.5mmol) and benzyl bromide (16.2ml, 23.18g, 20 135.3mmol) and the mixture stirred at reflux for Ih. After cooling the precipitate was removed by filtration and the filtrate evaporaled. The residue was taken up in ethyl acetate, washed with saturated sodium hydrogen carbonate, lM HCl, brine, and dried (MgSO4).
Filtration and evaporation gave the title compound as a yellow oil ~24.086g, 76%).

wo 97J26257 PCT/EP97/00196 .
-dH [(CD3)2SO] 2.84 (lH, dd, J=7.97, 13.7~ Hz), 2.97 (lE~, dd, J=5.5, 13.75 Hz~, 4.26-4.34 (lH, m), 5.09 (2H, s), 5.62 (IH, d, J=6.32 Hz), 7.16-7.43 (1OH, m) b) Dibenzyl 2-(R)-benzyl-3-tert-butoxycarbonylsuccinate ~[3 ~nO2C~c02Bn tBu Sodium hydride (60% dispersion in mineral oil, 3.37g, 84.3mmol) was added to a solution of benzyl tert-butyl malonate (21.1g, 84.4mmol) in DMF (165ml) at 0~C. The solution was allowed to warm to room temperature and stirred, under argon, for lh.
Triflic anhydride (21.85ml, 36.6g, 130.2 mmol) was added in one portion to a solution of benzyl 3-(R)-phenyllactate(24g, 93.75mmol) and pyAdine (7.09ml, 86.8mmol) in dichloromethane (220ml) at 0~C. The solution was stirred for 1 h under argon. The mixture was washed with ice cold water, brine, dried (MgSO4), and filtered.

The filtrate was added to the DMF solution of malonate at 0~C and the solution allowed to warm to ambient temperature and stirred for 16h. Followin, evaporation, the residue was dissolved in ethyl acetate, washed with saturated sodium hydrogen carbonate solution, 1 HCl, brine and dried (MgSO4). Filtration and evaporation gave a residue which was 25 chromatographed on silica (~ % ethyl acetate-hexane) to give the title compound as an oil (24.67g, 60~.

W 097/26257 PCT~P97/00196 dH (CDC13) 1.35 (9H, d, J=11.55 Hz) 2.84-3.02(2H, m), 3.38-3.48 (lH, m), 3.68 (lH, dd, J=3.7, 9.5 Hz), 4.86-5.19 (4H, m), 7.03-7.37 (15H, m) 5 c) 2-(R~-Benzyl-4-tert-butoxy-3-ethenylsuccinic acid.

q~CO2H
tBu02C

A soludon of dibenzyl 2-(R)-benzyl-3-tert-butox~c~lonylsuccinate(14.64g, 30mmol) in isopropyl alcohol ~90ml) was treated with 10% Pd/C and hydrogenated at atrnospheric plGs~ ~ The reaction was filtered and the filtrate treated with piperidine (8.91ml, 90mmol) and fo~ ehyde ~37%, 36.63ml, 450mmol) and stirred at ambient temperature overnight.
15 Following evaporation the residue was dissolved in ethyl acetate, washed with lM HCl, brine, and dried (MgSO4). Filtration and evaporadon gave the dtle compound as a colourless oil ~7.17g, 87%).

dH ~CDC13) 1.48 ~9H, s), 2.96 ~lH, dd, J=8.25,14.02 Hz), 3.26 (lH, dd, J=7.98, 14.02 Hz), 20 3.75 (l~I, t, J=7.98 Hz), 5.55 (lH, s), 6.23 (lH, s), 7.14-7.30 (SH, m), acid proton not seen.

W O 97/26257 PCT~P97/OOlg6 d) N-(2-(R)-Benzyl-4-tert-butoxy-3 ethenylsuccinyl~-(s)-pheny~ nin~mide.

~0 tBUO2C~h~N~NH2 O

S ~

A solution of 2-(R)-benzyl-4-tert-butoxy-3-ethenylsuccinic acid (7.083g, 25.63mmol), (S)-phenyl~l~nin~mi~e hydrochlonde (4.68g,23.3mmol), diisopropylethylamine (3.97ml, 23.3mmol) and l-hydroxybenzolliazole hydrate (7.16g, 46.4mmol) in DMF (80ml) wastreated at 0~C with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.35g, 27.96mmol) and the reaction stirred for lh at 0~C and 16h at arnbient temperature. Following evaporation, the residue was dissolved in dichloromethane, washed with 10% citric acid, ~hlrat~d sodium hydrogen bicarbonate, and dried (MgS04). Filtration and evaporation gave 15 a residue which was chromatographed on silica (gradient elution hexane-ethyl acetate 7:3 to 3:7) to give the title compound as a white solid (5.83g, 60%).

dH [(CD3)2SO3 1.39 (9H, s), 2.68-2.79 (2H, m), 2.92-3.05 (2H, m), 3.72 (lH, t, J=7.42 Hz), 4.38-4.47 (lH, m), 5.44 (lH, s), 5.94 (IH, s), 7.03-7.25 (12H, m), 7.90 (lH, d, J=8.25 Hz).

wo 97/26257 PCT/EPg7/00196 e) N-(2-(R)-Benzyl-4-hydroxy-3~thenylsuccinyl)-(S)-phenyl~nin~mide.

~0 HO2C ~ H ~ NH2 ~3 A solution of N-(2-(R)-benzyl-4-tert-butoxy-3-ethenylsuccinyl~-(S)-phenyl~l~nin~mi(1e (2.1 lg, Smmol) in dichloromethane (50ml) was treated with trifluoroacetic acid (SOml) and 10 sdrred at ambient temperature for 3h. The reacdon was evaporated and co-evaporated with tol~lene. Trituradon of the residue with ether afforded the title compound as a white solid (1.59g, B7%).

dH [(CD3)2SO] 2.70-2.78 (2H, m), 2.92-3.05 (2H, m), 3.77 (lH, t, J=7.42 Hz), 4.36-4.44 (lH,m), 5.48 (lH, s), 6.02 (lH, s), 7.04-7.24 (12H, m), 7.87 (lH, d, J=8.53 Hz), 12.59 (lH, br. s).

W O 97/26257 PCT~P97/00196 f) N-(2-(R)-Benzyl-4-hydroxy-3-(S)-[2-thienothiomethyl]succinyl)-(S)-phenyl~l~nin~mide.

~ O
HO2C ~ NH2 S ~
S~ W
s \J

Asolutiono~N-(2-(R)-benzyl 1-hydroxy-3-elhenylsuccinyl)-(S)-phenyl~l~nin~mi~e(1.48g, 3.9mmol) in meth~lol (20ml) was treated with 2-mercaptothiophene (5.07ml) at ambient 10 temperature and heated under renux under argon for 18h. The reaction was evaporated and co-eYaporated with toluPnp Trituration of the residue with ether afforded the title compound as a white solid (1.63g, 869~o).

dH ~(CD,~2SO] 1.95 (lH, dd, J=3.03, 12.92 Hz), 2.28-2.75 (6H, m), 2.98 (lH, dd, J=4, 13.5 Hz), 4.32-4.41 tlH, m), 6.32 (lH, s), 6.91-7.27 (13H, m), 7.60 (lH, dd, J=1.37, 5.22 Hz), 8.19 (lH, d, J=8.53Hz), 12.70 (lH, br. s) W O 97/26257 PCT~EP97/00196 g) N-~2-(R)-Benzyl-4-hydroxyamino-3-(S)-~2-thienothiomethyllsuccinyl)-(S)-phenyl~l~nin~mi~

HONH ~~ NH2 ~S ~
S~ W
s \J

A solution of N-(2-(R)-benzyl-4-hydroxy-3-~S)-[2-thienothiomethyl]succinyl)-(S)-phenyl~l~nin~nide(l.445g, 3mmol) in THF (25ml) and DMF (lOml), at -20~C, under argon, 10 was treated with N-methylmorpholine (0.42ml, 3.6mmol) and isobutylchloroformate (0.48ml, 489mg, 3.6mmol). After stirring at -20~C for lh the solution was treated dropwise with O-trimethylsilylhydroxylamine (2.6ml, 2.53g, 21mmol) and stirred at ambienttemperature for 16h. Following evaporation the residue was dissolved in ethyl acetate and washed with 10% citric acid, brine and dried (MgS04). After filtration, evaporation and 15 trituration with ether the solid was heated in ethyl acetate and the residue filtered to give (after repetition of this a number of times) the title compound as a white solid (25mg).

dH [(CD3)2SOJ 1.81 (lH, dd, J=2.3, 12.5 Hz), 2.25-2.66 ~6H, m), 2.97 (lH, dd, J=3.7, 13.S
Hz), 4.25-4.33 ~lH, m), 5.89 (lH, s), 6.85 (lH, s), 6.96-7.24 (12H, m), 7.56 (lH, dd, 20 J=1.2,5.4Hz), 8.13 (lH, d, J=B.52 Hz), 8.98~1H, s), 10.71 (lH, s).

W 097/26257 PCT~EP97/00196 -ExamDIe 4 N-(2-(R)-Benzyl-4-hydroxyamino-3-(s)-[2-thienothiomethyl]succinyl)-(s)-pheny~ nin~
N'-methylamide O ~Ph HOHN ~ ~NHMe S Ph S~

a) N-(2-(R)-Benzyl 4-hydroxy-3-(S)-[2-thienothiomethyl]succinyl)-(S)-phenyl~l~nine-~-methylamide HO2C~ ~Nl IMe ~S ~ ~Ph S~

Prepared according to Example 3f but using N-(2-(R)-benzyl-4-hydroxy-3-ethenylsuccinyl)-(S)-phenyl~ ine-N'-methylamide (200mg,0.53mmol), to give the titie compound as a white 15 solid (lOOmg,38%~

dH ~(Cl),),SO] 1.94 (lH,dd,J=3.2,12.8Hz), 2.28-2.72 (6H,m), 2.44 (3H,d,J=4.7Hz), 2.93 (lH,dd,J=4.1,13.5Hz), 4.37 (lH,m), 6.79 (IH,m), 7.01-7.27 (12H,m), 7.60 (lH,dd,J=l.5,5.lHz), 8.23 (lH,d,J=8.8Hz), 12.7 (lH,br s).

CA 022429l3 l998-07-l3 b) N-(2-(R~-Benzyl-4-hydroxyamino-3-(S)-[2-thienothiomethyl]succinyl)-(S)-phenylalanine-N'-methylamide HOHN~
_ ~S ~Ph S~

Prepared according to Example 3g affording the title compound as a white solid (35%).

dH [tCD,)2SO] 1.83 (lH,d,J=lO.SHz), 2.30 (lH m), 2.39 (3H,d,J=4.7Hz), 2.50-2.63 (SH,m), 2.94 (lH,dd,J-4.1,13.7Hz), 4.33 (lH,m), 6.45 (lH,m), 7.00 (4H,m), 7.17 (8H,m), 7.56 (lH,dd,J=1.4,5.2Hz), 8.20 (lH,d,J=8.2Hz), 8.97 ~lH,br s), 10.70 (lH,br s).

PCT~EP97/00196 .

Activity Data Compound CD23 proteinase inhibition Coll~e~ce inhibition % at 1uM IC50 uM

Example 3 105 0.93 Example 4 85 0.37 Comparative Example* 96 0.005 *The comparative eY~mrle was Ex~nple 2 of WO90/05719, the compound of formula:
~ O
R~N -J~HN
I lOHNOC O Ph 20 wherein R is CH2S-(2-thienyl) and R I is methyl.

Claims (10)

Claims ,

1. A compound of formula (I):

wherein R and R3 are each independently hydrogen, alkyl, alkenyl, alkynyl or aryl; R1 is arylmethyl; and R2 is alkyl, alkenyl, aryl,cycloalkyl or cycloalkenyl, or a pharmaceutically acceptable derivative thereof.

2. A compound of formula (IA):

wherein R to R3 are as defined in claim 1, or a pharmaceutically acceptable derivative thereof.

3. A compound according to claim 1 or 2 wherein R is hydrogen or methyl, optionally substituted by arylthio or heterocyclylthio; and/or R1 is a benzyl group; and/or R2 is a benzyl group; and/or R3 is hydrogen, methyl or benzyl.

4. A compound selected from the group consisting of N-(2-(R)-Benzyl-4-hydroxyaminosuccinyl)-(S)-phenylalanine-N'-benzylamide, N-(2-(R)-Benzyl-4-hydroxyaminosuccinyl)-(S)-phenylalanine-N'-melhylamide, N-(2-(R)-Benzyl-4-hydroxyamino-3-(S)-[2-thienothiomethyl]succinyl)-(S)-phenylalaninamide, and N-(2-(R)-Benzyl-4-hydroxyamino-3-(S)-[2-thienothiomethyl]succinyl)-(S)-phenylalanine-N'-methylamide.

5. Use of a compound according to any preceding claim for the production of a medicament for the treatment or prophylaxis of disorders in which the overproduction of s-CD23 is implicated.

6. A method for the treatment or prophylaxis of disorders in which the overproduction of s-CD23 is implicated, which method comprises the administration of a compoundaccording to any one of claims 1 to 4, to a human or non-human mammal in need thereof.

7. A pharmaceutical composition for the treatment or prophylaxis of disorders in which the overproduction of s-CD23 is implicated which comprises a compound according to any one of claims 1 to 4, and optionally a pharmaceutically acceptable carrier.

8. A process for preparing a compound according to any one of claims 1 to 4, which process comprises:
(a) deprotecting a compound of formula (II):

wherein R to R3 are as defined in claim 1, and X is a protecting group such as benzyl or trimethylsilyl or (b) reacting a compound of formula (III):

wherein R to R3 are as defined in claim 1, with hydroxylamine or a salt thereof, or (c) reacting a compound of formula (IV):

wherein R1 to R3 are as defined in claim 1, with a thiol to give a compound of formula (I) wherein R is methyl substituted by alkylthio, arylthio, aralkylthio, or heterocyclylthio, or (d) converting a compound of formula (I) to a different compound of formula (I).

9. A compound of formula (II), (III), or (IV) as defined in claim 8.

10. A compound of formula (V):

wherein R to R3 are as defined in claim 1, and Y is a protecting group such as t-butyl; or a compound of formula (VI):

wherein R1 to R3 and Y are as defined hereinabove, and Z is a group such that ZCH2 is R;

or a compound of formula (IX):

wherein R1 to R3 are as hereinabove defined; or a compound of formula (X):

wherein R1 to R3 and Y are as defined hereinabove.