THIOL-REACTIVE CROSS-LINKING REAGENTS

Abstract: Thiol-reactive cross-linking reagents which are highly specific for sulphydryl groups and react with thiols at an excellent rate. The reagents are substituted 2-cyclopenten-1,4-diones of formula (I), wherein X is a -(CH2)n- or -(CH2)mCH=CH- group (where n is an integer 1, 2 or 3 and m is zero or an integer 1); Y is a bridging group; R is a reactive functional group; and R<1> is a C1-4 alkyl group. Reagents of formula (I) may be used to cross-link proteins, such as antibodies, and other molecules to effector molecules such as drugs, toxins, enzymes and chelated radioactive metal atoms.

Description: THIOL-REACTIVE CROSS-LINKING REAGENTS FIELD OF THE INVENTION This invention relates to a new class of thiol-reactive cross-linking reagents, the members of which each comprise at least one thiol-reactive functional group covalently linked to at least one further reactive functional group, and to processes for their preparation. In particular, the invention relates to substituted 2-cyclopenten-l,4-diones, to processes for their preparation, and to their use as cross-linking reagents. BACKGROUND TO THE INVENTION Cross-linking reagents are well known, and a considerable range of such reagents is available commercially. In broad terms, a cross-linking reagent comprises two or more reactive functional groups covalently linked together. The covalent linkage may be direct, but in many cases the reactive functional groups are spaced apart by respective covalent attachment to a bridg.ng group. The reactive functional groups may be the same or different, (so-called homo- or hetero-functional cross-linking reagents). One application of cross-linking reagents is in the production of conjugate compounds in which a polypeptide or protein is covalently attached to another chemical entity. Examples of such conjugate compounds include an antibody covalently attached to an effector molecule, for example a drug or a toxin, or a signal producing chemical entity such as an enzyme or a chelated radioopaque or radioactive metal atom; and an enzyme covalently attached to an antigenic analyte. There is considerable interest in the use of thiol-reactive cross-linking reagents for linking thiol groups in proteins, especially antibodies or antibody fragments, to effector molecules. Heterofunctional reagents which have a thiol-reactive functional group covalently linked to a different functional group such as an amino-reactive activated ester have been used for this purpose. Thus, in general the heterofunctional cross-linking reagent is first reacted with the effector molecule to which it is desired to attach a protein. This may be done, for example, by reacting amino groups on the effector molecule with the amino reactive functional group on the cross-linking reagent. Next, free sulphydryl groups on the protein are reacted with the thiol-reactive functional group on the cross-linking reagent to yield the desired conjugate compound. Heterobifunctional thiol-reactive cross-linking reagents (that is, heterofunctional cross-linking reagents with two reactive functional groups) are known which employ maleimides as the thiol-specific functional groups. A particular example of such a reagent is m-maleimido-benzoyl-N-hydroxysucccinimide ester (MBS) which has been used to prepare conjugate molecules comprising antibody or antibody fragments and enzymes or cytotoxic compounds [O'Sullivan, M.J. et. al. (1979), Anal. Biochem. , 100, 100-108; Freytag, J.W. et al, (1984), Clin. Chem. , 30., 417-420; Youle, R.J. et aJ, (1984), PNAS USA, 7_7_, 5483-5486]. Various derivatives of maleimide-based heterobifunctional cross-linking reagents are also known.The maleimide reagents are however very reactive and readily hydrolyse to malea ic acid. They are, in general, of low specificity for sulphydryl groups, particularly when used at the pH and in the stoichiometric amounts that are often necessary for the preparation of conjugate molecules, to ensure that the resulting molecule has only one of each conjugated group. Heterobifunctional thiol-reactive cross-linking reagents are also known which employ α-halo acid derivatives as the thiol specific functional group [see for example U.S. Patent Specif cation No. 4671958] .Whilst less reactive than the maleimide reagents, these are readily hydrolysed to the corresponding α-hydroxy acid derivatives and their tautomers. The aldehyde tautomers are essentially non-specific and reduce the overall specificity of the a-halo acid derivatives for thiol groups. The low specificity of the above known heterobifunctional thiol-reactive cross-linking reagents can result in the production of complex mixtures of reaction products. The complex mixtures arise from non-specific reaction of the thiol reacting group with non-thiol groups of the protein or the linked chemical entity. For example, the known thiol reacting groups commonly exhibit competitive reactivity for amines which can lead to complex polymerisation products. Unwanted side reactions are particularly disadvantageous where the linked chemical entity possesses secondary amines such as saturated ring nitrogens (e.g. in macrocyclic chelating ligands). Not only are these mixtures difficult to separate, but where the conjugate compound is for _i_n vivo use, absolute purity is of paramount importance. Thiol-reactive cross-linking reagents in which the thiol-reactive functional group is a vinyl group conjugated with an aromatic nitrogen heterocycle have also been described [International Patent Specification No. WO 88/05433]. SUMMARY OF THE INVENTION We have now found a new class of thiol-reactive cross-linking reagents in which the thiol-reactive functional group is a substituted cyclopentenedione group. The compounds according to the invention have a markedly improved specificity for sulphydryl groups compared with known maleimide thiol-reactive cross-linking reagents, and also advantageously react with thiols at an excellent rate. Thus according to one aspect of the invention we provide a compound of formula (1): wherein X is a -(CH„) - or -(CH_) CH=CH-group (where n is an integer c n Δ m 1, 2 or 3 and is zero or an integer 1); Y is a bridging group; R is a reactive functional group; and R is a C .alkyl group. The group X in compounds of formula (1) may be -CH -, - . ~W . - , -(CH ) -, -CH=CH- or -CH CH=CH-. In the compounds of formula (1), the bridging group represented by Y may be varied within a wide range of possibilities without affecting the overall usefulness of the compounds. Thus in general Y may be any divalent organic radical, providing it is substantially free of functional groups likely to interfere in any cross-linking reaction undertaken with a compound of formula (1). The group Y in compounds of formula (1) may thus be an optionally substituted aliphatic hydrocarbyl chain optionally interrupted by one or more heteroatoms selected from -0- or -S- or one or more 2 2 -N(R )- (where R is a hydrogen atom or a C alkyl group), l—o 2 2 -C0N(R )-, -N(R )C0-, cycloaliphatic, aromatic, or heteroaromatic groups. Thus, for example, Y may be an optionally substituted straight or branched C alkylene, C alkenylene or C alkynylene chain, optionally interrupted by one or more -0- or -S- atoms or C c β cycloalkylene, (e.g. cyclopentylene or cyclohexylene) , C, ,„ D—O 0—1 aromatic ( e . g . phenylene , or substituted phenylene) , C 2 2 heteroaro atic (e.g. furanyl, pyridyl) , -N(R )-, -C0N(R )- or 2 -N(R )C0- groups. Examples of substitutents which may be present on the chain Y include halogen atoms, e.g. fluorine, chlorine, bromine, or iodine atoms or a group selected from C. ,alkoxy (e.g. methoxy or ethoxy) , l—o 3 4 3 hydroxy, nitro, -N(R )(R ), [where R is a hydrogen atom or a C, , 1—6 _t alkyl group and R is a C. , lkyl group, e.g. -NHCH. or -N(CH_)„], 1—0 _ J £ or substituted amido, e.g. a group of formula -(CH„) CON(R )(R ) . 2 n [where n is zero or an integer 1 to 4 inclusive, R is a hydrogen atom or a C, , alkyl group, e.g. methyl and R is an optionally l—o substituted C, alkyl group], l—o When R is a sustituted alkyl group it may be for example an alkyl group substituted by one or more amino or cyclic amino groups. The reactive functional group represented by R in compounds of formula (1) may be any group capable of reacting with a thiol, amino, carboxyl, hydroxyl, aldehyde, aromatic or heteroaromatic group. Aromatic groups include, for example, phenolic groups. Heteroaromatic groups include, for example, imidazolyl groups. Thus, R may be, for example, a halogen atom, for example a chlorine, bromine or iodine atom, or a group selected from -SH, -NH 7 , hydrazine (-NHNH ) or a derivative thereof, [for example -N(CH )NH , -NHCONHNH , -NHCSNHNH , or phenyl hydrazine], -NCO, -NCS, -NHOH, -COR , [where R is a halogen atom such as a chlorine or bromine atom, or a N , C alkoxy, e.g. methoxy, J l—o C, .„ aryloxy (e.g. nitrophenyloxy or dinitrophenyloxy) , imidyloxy o—1_ (e.g. succinimidyloxy) or i idazolyoxy group] imide, e.g. maleimide, 1 2 1 2 a vinyl group of formula Het -C(Het )=CH (where Het and Het , which may be the same or different, is each a nitrogen containing heterocyclic group, e.g. a pyridyl group, or Het is a nitrogen 2 containing heterocyclic group and Het is a hydrogen atom), for example a vinyl pyridyl group of formula or a dione of formula (where R is as defined previously). The group R in compounds of formula (1) may be for example a methyl, ethyl, propyl (e.g. n-propyl) or butyl (e.g. n-butyl) group. In general, in compounds of formula (1) the bridging group Y is preferably an optionally substituted c n _- ι0 alkylene, C alkenylene or C_ Q alkynylene chain optionally interrupted by one or more -0- or -S- atoms or cyclohexylene, phenylene, -NH-, -N(CH )-, -CONH- or -C0N(CH )- groups. Particularly useful substituents which may be present on chains of this type include substituted amido groups, particularly groups of formula -(CH.) C0N(R 5 )(R 6 ). I n R in compounds of formula (1) is preferably a group -COR where R is C 1 _ 6 alkoxy, especially methoxy, C 6 _ 12 &ryloxy, especiall nitrophenyloxy or dinitrophenyloxy, imidyloxy, especiall succinimidylox ; or Particularly useful compounds of formula (1) are those wherein R i -COR and R is nitrophenyloxy or succinimidyloxy, or wherein R is R in compounds of formula (1) is preferably methyl, One particular group of compounds according to the invention has th formula (1) wherein X is a -(CH„) - or -(C_ι ' _) CH=CH- group (where e. n z m is an integer 2 or 3 and m is zero or an integer 1) ; Y is a covalen bond or a bridging group; R is a reactive functional group, and R is a C alkyl group. Particularly useful compounds according to the invention are: 2-(3-Carboxyproρan-l-yl)-2-methyl-4-cyclopenten-l,3-dione, succinirαide ester and substituted derivatives thereof, 2-(3-Carboxypropan-l-yl)-2-methyl-4-cyclopenten-l,3,-dione, p-nitrophenyl ester and substituted derivatives thereof, and l,2-Ethanediylbis[2-(3-aminocarbonylρropan-l-yl)-2-methyl -4-cyclopenten-l,3-dione] and substituted derivatives thereof _ Substituted derivatives of the above compounds include those having substituents as described earlier for the chain Y. Compounds of formula (1) are useful for cross-linking molecules, such as proteins, for example antibodies or antibody fragments, to effector molecules such as drugs, toxins and chelated radioopaque or radioactive metal atoms, or to other proteins, carbohydrates or other biologically occuring molecules, to form conjugate compounds for use in diagnosis or therapy In use, the compounds of the invention may be reacted with the molecules to be cross-linked using sequential reactions and methods analogous to those described for the use of known hetero- or homo-functional thiol-reactive cross-linking reagents. Thus the functional group R in compounds of formula (1) may first be reacted with an effector molecule, protein, carbohydrate or other biologically occurring molecule as described above, to form a conjugate in which the thiol specific cyclopentenedione group is left available for reaction with a thiol group in a second molecule such as a protein. Compounds of formula (1) may be prepared by the following processes wherein the groups X, Y, R and R are as defined above except where stated otherwise. To avoid undersirable side-reactions in some of the reactions described below, it may be necessary to protect functional groups where these are present in starting materials and also desired in the final product, but are not required to participate in the reaction in question. Conventional protection and deprotection procedures may be used. Thus, for example, compounds of formula (1) may be prepared reaction of an intermediate dione of formula (2) with a phenylselenenyl halide, e.g. phenylselenenyl chlorid followed by oxidation using an oxidising agent such as hydrog peroxide or periodate. The reaction may be performed in an organic solvent such as ester, e.g. ethyl acetate, or an ether, e.g. a cyclic ether such a tetrahydrofuran, or mixtures thereof, at any suitable temperature e.g. at 0 -50 C, such as 10 -30 C. In another process, compounds of formula (1) in which R is a grou - -CCOORR mmaayy bbee pprepared by reaction of a corresponding carboxylic aci of formula (3) or an activated derivative thereof (e.g. a halide or anhydride) wit an alcohol R OH optionally in the presence of a coupling agent, suc as dicyclohexylcarbodiimide, in a solvent, such as halogenate hydrocarbon e.g. dichloromethane, at a temperature in the rang o o 0 -50 C. This reaction is particularly suitable for the preparation o compounds of formula (1) where R is COR and R is aryloxy o imidyloxy. Intermediates of formula (3) are new compounds and form a furthe aspect of the invention. In a further process, compounds of formula (1) in which Y is a 2 2 2 bbrriiddggiinngg ggrroouupp iinntteerrrruupptteedd bbyy aa --CC0ONN((RR ))-- aanndd -N(R )C0- group may be prepared by reaction of an acid of formula (4) 2 3 with an acid RY C0 ? H in the presence of a diamine H NY NH (where 1 2 3 Y , Y , and Y together form the bridging group Y) . The reaction is preferably performed in the presence of a coupling reagent, e.g. dicyclohexylcarbodii ide, in a solvent such as a halogenated hydrocarbon, e.g. dichloromethane, at a temperature, for example, between 0 -50 C. Intermediates of formula (2) in which X is a -(CH„) -CH=CH- group 2 m may be prepared by reaction of an aldehyde of formula (5) with a ylide, for example a phosphorous ylide of formula 8 8 (R ),P=CH_-Y-R (where R is an aryl, e.g. pheinnyyll ggrroouupp]) in a solvent such as a halogenated hydrocarbon, e.g. dichloromethane, at a suitable temperature, e.g. 0° to 30°C. The ylides for use in this reaction may be prepared using standard procedures from appropriate readily available starting materials. Aldehydes of formula (5) may be prepared by ozonolysis of a dione of formula (6) using ozone in a solvent such as dicloromethane at a low o tteemmppeerraattuurree,, ee..gg.. --7788 CC,, followed by treatment with a reducing agent such as dimethyl sulphide. Diones of formula (6) are either known compounds or may be prepared by methods analogous to those used for the preparation of the known compounds. Intermediates of formula (2) in which X is a -(CH„) - group and n is a integer 2 or 3 may be prepared by hydrogenation of a corresponding compound of formula (2) in which X is -(CH„) CH=CH- for example using hydrogen in the presence of palladium or platinum on a support such as carbon in a solvent such as an ester, e.g. ethyl acetate. Intermediates of formula (2) in which X is -CH -, Y is a covalent bond and R is a group -COR may be prepared by ozonolysis of a dione of formula (6) wherein m is an integer 1 as described earlier followed by oxidation to an intermediate acid (i.e. a compound where R is -C0_H) using an oxidising agent such as chromic acid or a silver complex such as Ag(NH_) ? , and subsequent reaction with an alcohol R OH as described previously for the preparation of compounds of formula (1) from compounds of formula (3). Other intermediates of formula (2) in which X is -CH„-, Y is a covalent bond, and R is other than -COR may be prepared from the corresponding compounds wherein R is -COR or CO.H by conventional methods for modification of carboxyl or esterified carboxyl groups such as are described herein. Intermediate acids of formulae (3) and (4) may be prepared from a corresponding ester [e.g. a compound of formula (1) where R is -COR and R is a C, c alkoxy group] by treatment with an acid, for example l—o trifluoroacetic acid, in a solvent such as dichloromethane. DESCRIPTION OF SPECIFIC EMBODIMENTS The following Examples illustrates the invention. All temperatures Intermediate 1 2 Allyl-2-methylcvclopentan-l,3-dione 2-Methylcyclopropan-l,3-dione (0.22 mole) was dissolved in sodiu hydroxide solution [NaOH (0.22mole) in water (176 ml)] and allyl bromide (0.26 mole) was added slowly. The reaction mixture was heated to 60 for 3 days, cooled and extracted into CH Cl . The CH Cl extracts were combined, washed with saturated NaHCO , dried (MgSO ) , filtered and concentrated in vacuo. The resulting yello oil was distilled using a 20cm vigreux column. The title compound (yield 69%) was collected at 101-103 (water pump pressure). Intermediate 2 2-Formylmethyl-2-methylcyclopentan-l,3-dione Intermediate 1 (0.07 mole) was dissolved in CH.Cl. (20ml) and ozonised at -78 . Upon completion of ozonolysis, excess dimethyl sulphide was added and the reaction mixture was left to warm to room temperature. The reaction mixture was concentrated ij± vacuo and used without further purification for the preparation of Intermediate 3. Intermediate 3 (a) 2-(3-t-Butoxycarbonylprop-2-en-l-yl)-2-methylcyclopentan-l,3-dion Intermediate 2 (0.062 moles) was dissolved in CH„C1„ (20ml), cooled to 0 andtert-butoxycarbonylmethylenetriphenyl- phosphorane (0.074 mole) in CH Cl (50ml) was added slowly. The reaction mixture was then stirred at room temperature overnight and concentrated in vacuo. Purification using flash column chromatography was carried out (solid phase = silica gel; eluent = 1:1 diethyl ether:40-60 petroleum ether) to give the title compound (yield 54%). (b) 2-(3-Methyoxγcarbonylprop-2-en-l-yl)-2-methylcvclopentan-1.3-dion The title compound (yield 60%) was prepared in a similar manner, from Intermediate 2 except the ylide used was methoxycarbonylmethylenetriphenylphosphorane (0.074 mole) Intermediate 4 (a) 2-(3-t-Butoxycarbonylproρan-l-yl)-2-methylcγcloρentan-l,3-dione Intermediate 3(a) (0.037 mole) was dissolved in ethyl acetate (250ml) and a catalytic amount of palladium on activated carbon was added. The reaction mixture was then placed in the Parr Hydrogenator. When the uptake of hydrogen had ceased, the reaction mixture was filtered and concentrated in vacuo to give the title compound in quantitative yield. (b) 2-(3-Methoxycarbonylpropan-l-γl)-2-methylcyclopentan-1.3-dione The title compound was prepared in quantitative yield in a similar manner from Intermediate 3(a). Intermediate 5 2-(3-Carboxypropan-l-γl)-2-methyl-4-cyclopenten-1.3-dione _3 The compound of Example 1(a) (4.76 x 10 mole) was dissolved in _3 CH ? C1 2 lOπtl) and trifluoroacetic acid (9.52 x 10 mole) was added. The reaction mixture was refluxed for 10 hours after which time it was concentrated in. vacuo. Purification was carried out using flash column chromatography (solid phase = silica gel, eluent = 1:1 diethyl ether: 0-60 petroleum ether to 5:1 diethyl ether:40-60 petroleum ether) to give the title compound (yield = 78%) ra.p. 110°-111°. Analysis Found: C, 61.64; H, 6.30% C 10 H 12°4 Requirest C, 61.21; H, 6.16% Example 1 ( a) 2-( t-Butoxycarbonylpropan-l-yl ) -2-methyI-4-cyclopenten-l , 3-dione Intermediate 4(a) (0.018 mole) was dissolved in ethyl acetate (40ml) and phenylselenenyl chloride (0.026 mole) was added. The reaction mixture was stirred at room temperature overnight. It was then washed with water and tetrahydrofuran (20ml) was added to the ethyl acetate layer. The reaction was cooled to 0 and 10% H-0- (2.9 equivalents) was added. After stirring at room temperature for 2 hours, the reaction mixture was washed with water and then saturated with NaHCO„ . The ethyl acetate layer was then dried (MgSO, ), filtered and concentrated vacuo. Purification by flash column chromatography was then carried out (solid phase = silica gel; eluent - 1:1 diethyl ether:40-60 petroleum ether) to give the title compound (yield 64%). (b) 2-(3-Methoxycarbonylpropan-l-yl)-2-methyl-4-cyclopenten-l,3-dione The title compound (yield 41%) was prepared in a similar manner from Intermediate 4b) Example 2 (a) 2-(3-Carboxypropan-l-yl)-2-methyl-4-cyclopenten-l,3-dione. succinimide ester _3 Intermediate 5 (1.53 x 10 moles) was reacted with _3 N-hydroxysuccinimide (1.84 x 10 mole) using CH_C1 ? as solvent. The reaction mixture was stirred at room temperature for 2 hours. It was then filtered to remove precipitated urea a nd the resulting CH-C1 solution was concentrated jji vacuo to yield the crude succinimide ester, which was purified by flash column chromatography (solid phase = silica gel, eluent 1:3 diethyl ether: CH„C1 2 ) to give the title compound (yield 51%). . p. , 10 _.4. o - -10 Λ 6 ,o Analysis Found: C, 57.24; H, 5.25: N, 4.68% C, .H, -NO, Requires: C, 57.33; H, 5.15; N, 4.77% 14 15 o (b) 2-(3-Carboxypropan-l-yl)-2-methyl-4-cvclopenten-l,3 dione. p-nitrophenyl ester The crude ester was prepared from Intermediate 5 in a similar manner. Precipitation of the crude ester from ZW Z using ether followed by trituration of the resulting solid in ether gave the title compound (yield 39%) . m.p. 97°-98° Analysis Found: C, 60.71; H, 4.98; N, 4.53% C,_,H, c N0, Requires: C, 60.56; H, 4.76; N, 4.41% lb ID o Example 3 1,2-Ethanediylbis [2-(3-aminocarbonylpropan-l-yl)-2-methyl-4- cyclopenten-1,3-dione] _3 Intermediate 5 (2.0 x 10 mole) was dissolved in CH Cl (10ml) -3 and ethylene diamme (1.0 x 10 mole) was added. This was _3 followed by the addition of dicyclohexylcarbodiimine (2 x 10 mole) . The reaction mixture was stirred at room temperature for 19 hours, then filtered and concentrated ij. vacuo. The resulting brown oil was purified by flash column chromatography (solid phase = silica gel, eluent = 9:1 CH^Cl : methanol) to give the title compound. (Yield 26%). o o m.p. 99 -101 Analys i s Found : C, 63 .03 ; H, 6.99 ; N , 6.5% C 22 H 28 N 2°6 Re< 3 u ires : c « °3 - 44 ; H , 6. 77 ; N, 6.72%

Claims: CLAIMS 1. A compound of formula (1) wherein X is a -(CH„) - or -(CH„) CH=CH-group (where n is 2 n 2 m an integer 1, 2 or 3 and m is zero or an integer 1); Y is a bridging group; R is a reactive functional group; and R is a C . alkyl group. A compound according to Claim 1 wherein Y is an optionally substituted aliphatic hydrocarbyl chain optionally interrupted by one or more heteroatoms selected from -0- 2 2 or -S- or one or more -N(R )- (where R is a hydrogen atom or a C, ^ alkyl group), -C0N(R )-, -N(R )C0-, 1—0 cycloaliphatic, aromatic, or heteroaromatic groups. A compound according to Claims 1 or 2 wherein R is a group capable of reacting with a thiol, amino, carboxyl, hydroxyl, aldehyde, aromatic or heteroaromatic group. A compound according to Claim 3 wherein R is a halogen atom, or a group selected from -SH, -NH , hydrazine (-NHNH ) or a derivative thereof, -NCO, -NCS, -NHOH, -COR , [where R is a halogen atom or a H , C, .alkoxy, _i l—o c _ . . ar l° χ or imidazolyoxy group] imide, a vinyl group 0—12 of formula Het 1 -C(Het 2 )=CH, (where 1 2 Het and Het , which may be the same different, is each a nitrogen containing heterocyclic group, or Het is a 2 nitrogen containing heterocyclic group and Het is a hydrogen atom) , or a dione of formula (where R is a C, .alkyl group) A compound according to Claim 4 wherein R is a group -COR' where R is a C. .alkoxy, C, , aryloxy, imidyloxy, group 2-(3-Carboxypropan-l-yl)-2-methyl-4-cyclopenten-l,3-dione, succinimide ester and substituted derivatives thereof. 10 2-(3-Carboxyproρan-l-yl)-2-methyl-4-cyclopenten-l,3,-dione, p-nitrophenyl ester and substituted derivatives thereof l,2-Ethanediylbis[2-(3-aminocarbonylpropan-l-yl)-2-methyl -4-cyclopenten-l,3-dione] and substituted derivatives thereof 9. A compound according to Claim 1 wherein the reactive functional group R is covalently linked to an effector molecule or a protein, carbohydrate or other biologically occurring molecule, and the thiol specific cyclopentenedione group is available for reaction with a thiol group. 10. A process for the preparation of a compound of formula (1) which comprises reacting a dione of formula (2) with a phenylselenenyl halide, and oxidising the resulting product.

Inventors: MANN JOHN, TITMAS RICHARD CHARLES DOMINIC, MANN, JOHN, TITMAS, RICHARD, CHARLES, DOMINIC
Applicants: CELLTECH LTD, CELLTECH LIMITED
Application year: 1989
Main classification: C07C59
IPC codes: A61K51, C07C51, C07C59, C07C205, C07C233, C07C235, C07C323, C07D213