DIBENZODIHYDROPYRIDINECARBOXYLIC ESTERS AND THEIR USE IN CHEMILUMINESCENT ASSAY METHODS

Abstract: New dibenzodihydropyridinecarboxylic esters are provided which comply with formula (1) or (2), wherein: R<1> is substituted alkyl or cyano; R<2> and R<3> are independently hydrogen, alkyl, substituted alkyl, aryl or substituted aryl; R<4> is hydrogen, alkyl, substituted alkyl, aryl or substituted aryl; R<5> and R<6> are indenpendently alkyl, alkoxy or another substituent which is relatively resistant to oxidation; m and n are integers from 0 to 4; wherein, if m or n is at least 2, two groups R<5> or R<6> may be linked together; and Z is oxygen or sulphur. The new compounds are useful as chemiluminogenic reagents for detecting the presence of peroxidase activity at extremely low levels (10<-20> moles).

Description: Dibenzodihydropyridinecarboxylic esters and their use in chemilumineseent assay methods The cntιon relates to cl dropyι lιc esters, m particular aci idanccaibow hc cstcis, and to then use as a chemilumineseent substrate in pcroxidase assay methods Aromatic aci idane esters cais (McCapra, Ace Chem Res , 1976, 9 201) The chcmilumincsccncc of acπdanc esters proceeds in dipolar aprotic soK cnts upon addition of stiong base The mechanism is icportcd to ohc a icaction with gcn of the concsponding caibanion foimcd by the addition of the base EP-A-625510 discloses ccitain aio atic acndanc cstcis in particular p- hydroxyphcn> l 10-mcth latc which can be used as a pait of a signal reagent for the enzyme horseradish pcroxidase (HRP) The signal icagcnt also contained an enhancer c g para-iodophcnol, a chclating agent (EDTA), a nonio ic sui factant, and hydrogen pel oxide According to EP-A-625510. HRP can be detected with great sensitivity at an optimal pH langc of 8-9 Moic potent chemilumineseent polyfluoro- l esters of acid arc disclosed in WO 95/23971 and phenyl thiocstcis aic described in WO 95/28495 The optimum pH foi HRP is aiυund pH 5 Thcrcfoi c. it seems unsatisfactory that the optimum pH foi chcmilumincsccncc detection of HRP is at pH 8-9 This pH is presumably a co piomisc between the best pH foi the mc and the optimal pH for intermediate acπdinium estci chcmilumincsccncc. which occuis at basic pH's et al in Biolunnnescence and Che ihumnest enc e Fundamentals and Applied Aspects Proceedings of the 8th International on Biohtminescenc c and Chcmil mmescence Cambridge, September 1994 Ed Campbell. Kπcka, Stanlcs John Wiley and Sons 1994 199) The present im cntion aims at an cnzvmc a ssa\ and at reagents to be used therein, which allow a highci sensitn lty than the assav and lcagcnts of the pπoi art The present invention is based on the surprising cιy that certain aliphatic acndanc esters and phenanthridane cstcis aic capable of \ cry intense and prolonged chcmi- luminescence upon reaction with hydiogcn pcioxidc and ccitain cnhanccis This icaction proceeds a wide pH range (optimum pH range 6-8). and without the necessity of adding chelating compounds (EDTA) or surfactants. Moreover, these aliphatic esters produce a very low background luminescence, i.e. without enzyme. Thus the invention is concerned with dibcnzodihydropyridinccarboxylic ester havinε formula 1 or 2: wherein: R is substituted alkyl or cyano; R " and R" arc independently selected from hydrogen, alkyl. substituted alkyl, aryl and substituted aryl; R is selected from hydrogen, alkyl, substituted alkyl. aryl and substituted aryl; R" and R arc independently selected from alkyl, alkoxy, halogen, and other substitucnts which arc relatively resistant to oxidation: m and n are integers from 0 to 4; wherein, if m or n is at least 2. two groups R or R f) may be bound together; and Z is oxygen or sulphur. The dibcnzodihydropyridinccarboxylic esters according to the invention can be acridane-9-carboxylic esters having formula 1, or phcnanthridanc-6-carboxylic esters having formula 2. A key feature of the compounds according to the invention is that the α-carbon atom of the ester alkyl group is an aliphatic carbon atom. It may be substituted with various aliphatic or other groups, designated in the above formulae 1 and 2 by R 1 , R^ and R 3 . In the specification of the compounds according to the present invention, "alkyl" means straight or branched, saturated or unsaturatcd C ] -C-, 4 . preferably C j -C,-,, most preferably C, -C 4 alkyl, such as methyl, ethyl, propyl, isopropyl. butyl and decyl. "Substituted alkyl" means alkyl as defined above containing one or more substituents such as halogen, hydroxy, alkoxy, alkanoyl, carboxyl, alkoxycarbonyl, alkanoyloxy, aryl, aryloxy, aroyl, aroyloxy, cyano, nitro and the like, especially halogen, hydroxy, alkoxy and aryl; the substituents themselves may also be substituted, e.g. with halogen, alkyl or alkoxy. "Aryl" means phenyl, naphthyl and higher carbocyclic aryl, as well as hctcro- aryl such as furyl, thicnyl, pyrrol I, imidazolyl, thiazolyl, oxazolyl, pyridyl, pyrimidyl, quinolyl and the like. "Substituted aryl" means aryl as defined above containing one or more substituents as exemplified above for substituted alkyl; examples include chloro- phenyl, trifluoromcthylphcnyl, methoxyphenyl, mcthylcncdioxyphcnyl and hydroxy- pyridyl. In "alkoxy", the alkyl part may be as defined above; examples include mcthoxy, ethoxy, t-butoxy, 2-chloroctho.xy. 2-mcthoxycthoxy, hcxafluoroisopiopoxy, bcnzyloxy, 2-phenylcthoxy and the like. Substituents which arc relatively resistant to oxidation arc understood to comprise substituents that do not interfere with the enzymatic oxidation process, such as halogen, alkyl, haloalkyl. hydroxyalkyl, alko yalkyl, alko y, carboxyl, alkoxycarbonyl, alkanoyl, alkanoyloxy, cyano, nitro, carbamoyl, acylamino, sulpho, phospho, phenyl, pyridyl and other aryl groups, and the like. Preferably, at least one of R , R- and R ' is a group containing clcctron-with- drawing substituents such as halogen, nitro, sulpho, cyano, acyl, sulphonyi, carboxyl. A suitable group with electron-withdrawing substituents is haloalkyl, such as mono-, di- and trichloromcthyl, mono-, di- and trifluoromcthyl. tri- or pcntafluorocthyl, hepta- fluoropropyl and higher homologucs. Especially preferred arc trifluoron cthyl groups for R and optionally also for R". R may then be e.g. hydrogen, lower alkyl, haloalkyl, phenyl or halophcnyl. R 1 may also be cyano, in which case R- may be e.g. hydrogen, methyl, phenyl or trifluoromcthyl and R 1 e.g. hydrogen. The nature of . the substitucnt represented by R 4 is not very critical. For reasons of convenience, R 4 may be lower alkyl, especially methyl, but it may also be e.g. hydrogen, substituted alkyl (examples: carboxymcthyl, hydro.xycthyl, benzyl), phenyl or substituted phenyl. The substituents R 5 and R 6 arc preferably attached to the m- and/or p-positions with respect to the ring nitrogen, most preferably in the p-positions. Combinations of substituents R 5 or R 6 (m or n being a number of 2 oi higher) that may be bound together include C^-C^ alkylenc and other bivalent groups that, together with the bcnzo groups to which they arc attached, form an additional ring such as cyclopcnta ((R 5 ) τ = (CH j fe), cyclohcxa ((R 5 ) 2 = (CH 2 ) ), furo ((R 5 ) 2 = CH 2 -O-CH 2 ), dioxolo ((R 5 ) 2 = O-CH 2 -O), bcnzo ((R 5 ) 2 = CH=CH-CH=CH), pyrido ((R 5 ) 2 = N=CH-CH=CH or CH=N-CH=CH), and the like. Preferred groups R "1 and/or R arc chloro, fluoro, bro o, methoxy and mcthylcncdioxy, most preferred group R" is chloro. The dibcnzodihydropyridinccarboxylic esters according to the invention can be prepared by methods known per sc. A general synthesis scheme for the acridanc esters, which is further illustrated in the examples, can be as follows: A suitably substituted aniline (1) having the formula (R ) m C 6 H 5 _ n) ) NH 2 is converted to the acetanilidc (II). which is reacted with a suitably substituted phenvl bromide (III) having the formula (R ^C^H^^B in the presence of copper iodide to produce the diphenylaminc (IV) having the formula (R' C H ( _ m ) -NH-C f ,H (n _ 3) (R 6 ) n . This diphenylaminc (IV) is converted to the acridinc-9-carboxylic acid (V) by reaction with oxalic chloride in the presence of a Lewis acid. After esterification with the appropriate alcohol R^RYOH or thiol R'R 2 R λ CSH and N-alkylation, the 9-alkoxy- carbonyl- or 9-alkylthiocarbonyl-acridinium compound with formula 3 is obtained, which can then be reduced to produce the desired acridanc ester having formula 1. The synthesis of the phenanthridanc esters proceeds in an analogous way starting with optionally substituted 2-aminobiphcnyl. Acctylation and ring closure yields (substituted) 5-mcthylphenanthridinc. The υ-carhoxy group is introduced by reaction of (substituted) 5-mcthylphcnanthridinc with formaldehyde, followed by oxidation to give(substitutcd) 6-carboxy-5-mcth\ phcnanthridine. Esterification witht e appropriate alcohol or thiol, followed by N-alkylation yields the 6-alkoxycarbonyl- or 6-alkyl- thiocarbonyl-phcnanthridinium compound with formula 4. The invention furthermore relates to a method for assaying a peroxidasc, wherein a dibcnzodihydropyridinccarboxylic ester as described above is used as a chemilumineseent substrate. The method can be used for assaying the pcroxidase enzyme itself, or for assaying an a alytc labelled with the pcroxidase. The method is extremely sensitive in that pcroxidase lev els as low as 10 " -° moles can be reliably detected. In the method of the invention, the dibcnzodihydiOpyridinccarboxylic ester according to the invention is contacted with a sample containing the pcroxidase or the analyte labelled with the pcroxidase, and with a peroxide and optionally an enhancer so as to generate light. The light is most probably generated by an excited state of the dihydropyridonc resulting from the reaction with peroxide. The preferred pcroxidase is horseradish pcroxidase (HRP). The preferred peroxide is hydrogen peroxide; other suitable peroxides include urea peroxide, perborates and the like. The preferred enhancer is p-phenylphcnol, but other phenolic enhancers such as p-iodophcnol, p-thiazolyl- phenol, p-hydroxycinnamic acid, other substituted phenols and optionally substituted naphthols, hydroxy-bcnzothiazolcs. hydro.xy-bcnzoxazolcs and hydroxy-fluorcnoncs, can also be used. The pH to be used in the method of the invention can vary over a wide range, for example from 4 to 10. The preferred pH range is from 5 5 to 8, in particular from 6 to 7.5. The method is preferably performed in a buffered aqueous system. The dibenzodihydropyridinecarboxylic ester may be used in a concentration of 10 ~7 to 10 "4 molar, the enhancer in a similar or somewhat higher concentration (e.g. 10 "7 to 10 ~? molar). The peroxide may be added in a concentration of e.g. 10 ~5 to 10 ~2 . The enzyme may be present in the attomol ( 10 " mole) range or even lower. The presence of surfactants such as poly(cthylcnc o ides) or chelating agents such as EDTA is not necessary, and is therefore preferably dispensed with. The method of the invention can also be used to assay peroxides such as hydrogen peroxide which may be generated by the action of dehydrogenase or oxidase enzymes in solution, or to assay molecular oxygen in a solution in an aprotic solvent such as acctonitrile, nitromcthanc, dimethyl sulphoxidc and the like. The method of the inv ention can further be used for assaying hydiolytic enzymes such as phosphatascs and glycosidascs by using a phosphorylatcd or glyco- sylatcd phenol which, after hydiolytic cleavage of the phosphate or glycosyl group, serves as an enhancer in a pcroxidase reaction. The invention also relates to a kit for assaying pcroxidase activ ity comprising a dibcnzodihydropyridinccarboxylic ester as described above and optionally a peroxide, a phenolic enhancer and/or further constituents for performing the assay as described above. The kit may further contain an analyte, such as an antibody, labelled with a peroxidase enzyme such as HRP. If used for peroxide detection, the kit may also contain a pcroxidase as such. The invention also relates to novel dibenzopyridinium (acridinium and phen- anthridinium) compounds having formula 3 or 4: wherein R 1 to R 6 and Z are as defined above for compounds 1 and 2. The counterion X " may be any suitable, relatively inert ion. such as halidc. sulphate. R 7 -sulphonatc wherein R 7 is e.g. alkyl, haloalkyl or aryl, tctrafluoroboratc etc. Preferably R 1 is trifluoromcthyl or cyano, R ~ is trifluoromcthyl and R is hydrogen, trifluoromcthyl, phenyl or substituted phenyl. R ? and/or R ( ' arc preferably halogen or mctho.xy, and m and n arc preferably 0, 1 or 2, most preferably at least one of them is 1 . The dihydropyridinium salts can be used as a direct label for detecting the presence of analytcs such as antibodies, antigens, DNA-probcs and other biologically interesting substances as described in EP-A-324202. Suitable compounds 3 and 4 have substituents containing a moiety capable of chemically reacting with (bio)molcculc . Examples thereof arc carbo.xy, aldehyde, alcohol, amino or thiol functions that can be present in any one of R 1 to R f> . especially in R 4 . such as when R 4 is carboxymcthyl. EXAMPLES Synthesis of the acridan derivatives GZ-l/GZ-12. The compounds given in table I were synthesiscd. The general synthesis procedure was as follows: Acetanilide (JJ): The particular aromatic aminc 1 (20 g), acetic acid anhydride (20 ml), acetic acid (glacial, 20 ml) and zinc powder (0.1 g) were mixed and rcfluxcd. After 30 min. the solution was poured into 500 ml ice water and the precipitate was filtered and dried to obtain the acetanilide. Diphenyl amine (IV): The acetanilide II (13.5 g), the particular aromatic bromide III (25 g), potassium carbonate (13.2 g) and copper iodide (1.9 g) were heated (190 °C) and stirred overnight. After cooling to room temperature toluene was added and the precipitate filtered. The solution was concentrated and the excess of bromide removed by distillation under reduced pressurc.Thc residue was dissolved in cthanol (200 ml), potassium hydroxide (10.3 g) was added and the mixture rcfluxcd overnight. Ethanol was evaporated, the residue dissolved in dichloromcthanc, and washed with brine. The organic layer was dried over MgSO 4 and concentrated to obtain the crude diphenyl amine. Table 1 Acrιdιne-9-carboxylιc acid (GW) To a solution of oxalic chloride (5 g) in dichloiomcthanc, a solution of the diphenyl amine IV (5 g) in dichloromcthanc was added diopwise and rcfluxed for 30 m . The solution was concentrated (50 %) and aluminium ti ichloiidc (8 g) added in portions The mixture was rcfluxed foi 45 nun and the solv ent cv poiatcd To this icsiduc hydio- chloric acid in ice watci (1 molai ) was added and the led colouicd piccipitatc filtered The precipitate was dissolv ed in potassium hv ioxide ( 10 ''< m watci ). icfluxcd overnight and poured into hydrochloπc acid in ice watci (5 molai) The v cllow acπdinc- 9-caιboxylic acid (GW) was filtered, washed with watci and di icd Acridιne-9-carboxylιc estet (GX) ' The acridιnc-9-carboxylιc acid GW (] g) was mixed with thionv l chloride (20 ml) and refluxed until a clear solution was obtained The solution was concentrated and the excess thionvl chloride cocv apoiatcd with toluene To the icsiduc pyiidinc (20 ml), dimethylaminopyridinc (1 g) and an excess of the pai ticulai alcohol (oi thiol) R 1 R 2 R 3 COH (-SH) were added The mixtuic was stu icd o ci ghi at loom tcmpciaturc, poured into hydrochloric acid in ice watci (1 molar) and cxti acted with dichloromcthanc The organic layer was washed with watci , dried ovci MgSO 4 and concentrated The crude product was purified by flash-chiomatographv (66% hcxancs/cthyl acetate) lO-Methylacrιdιnιum-9-carboxylιc estei tπfluoi o ethanesulfonalc (GY) The acridιnc-9-carboxylιc cstci GX (100 g) was dissolv ed in dichloiomcthanc (5 ml) To this solution an excess of mcthv l tiifluoiomcthanesulfonaic WM added The mixture was stirred ov ernight at room tcmpciatuic Dicthv l cthci was added and the obtained precipitate was filtered, vvashed with dicthv l cthci and dried 10-Methylacridane-9-carboxylate (GZ) " The lO-methylacridinium-9-carboxylιc cstci tiifluoromcthancsulfonatc GY (100 mg) was dissolved in dichloromcthanc (100 ml) To this solution pcichloi ic acid (20 drops) and zinc powder (6 g) were added The mixtuic was placed in an ultrasonc bath for three hours and filtcied The solution was washed with watci ( 1 x) and hydrochlonc acid (2x, 0.1 molar), dried ov ci MgSO 4 and conccntiatcd Example 1. Synthesis of GZ-1. Hexafluoroisopropyl acndπιe-9- rboxylate (GX- 1) 1H NMR (acctone-d 6 ) ό 7.2 (m, 1 H), 7 8-8 3 ( . 8H) 9-Hexafluoroisopropoxycarbonyl- ]0-methyl(icritliniunι triflitorometh tiesulfonate (GY-1) 1H NMR (acctone-d 6 ) δ 5.3 (s, 3H), 7.4 (m, I H), 8.2-9.2 ( , 8H). 13 C NMR (acetone-d f) ) δ 38.3, 41 .4, 70.4. 120.5. 121 .6, 124.2. 125.3, 127.8. 131.6, 141.2, 144.1, 146.4, 149.4, 163.4, 165.2. Hexafluoroisopropyl 10-meτhylacridane-9-carbo.xylate (GZ- 1) 1H NMR (acctonc-d ) δ 3.5 (s, 3H). 5.4 (s. I H) 6.2 (m, I H ). 6.9-7.4 (m. 8H). Example 2. Synthesis of GZ-2. 2, 7 -Dimethoxycicridine -9-carboxylic acid (GW-2) ] H NMR (acctonc-d 6 ) δ 4.0 (s, 6H). 7.1 -8.2 (m, 6H) exafluoroisopropyl 2, 7 -dimethoxycicridine -9-carboxylaie (GX-2) 1H NMR (CDC1 3 ) δ 4.0 (s, 6H), 6.4 (m, I H), 7.0-8.1 (m, 6H). 9-Hexafluoroi.sopropoxycarboιιyl-2. 7-dimcthoxy- 10-nιeihylacridinium trifluoro- methanesulf nate (GY-2) 1H NMR (acctone-d 6 ) δ 4.1 (s. 6H ), 5.2 (s. 3H ), 7.2 (d, 2H-acr). 7.3 (m, I H), 8.1 (dd, 2H-acr), 9.0 (d, 2H-acr). Hexafluoroisopropyl 2, 7-dimcthυxy-10-methyUtι ι idane -9-carboxyUιte (GZ-2) 1H NMR (acctone-d f ) δ 3.4 (s. 3H), 3.7 (s. 6H). 5.3 (s .I H ). 6.2 (m. I H), 6.8-7.0 (m, 6H). Example 3. Synthesis of GZ-3. 2,3 -Dimethoxycicridine -9 -carboxylic acid (GW-3) 1H NMR (DMSO-d 6 ) δ 3.9 (s, 3H), 4.0 (s. 3H). 7.2-8.1 (m. 6 H). Hexafluoroisopropyl - 2,3 -dimcthoxyacridine -9- carboxylate (GX-3) l NMR (acctonc-d 6 ) δ 4.0 (s, 3H), 4.1 (s, 3H), 7.1 (s, l H-acr). 7.2 (m, I H), 7.5-8.2 (m, 5H-acr). 9-Hexafluoroisoρropc)xycarbonyl-2.3-dimetho.xy- ]0-methylacridinium trifliioromethanesulfonate (GY-3) 1H NMR (acctone-d 6 ) δ 4.2 (s, 3H), 4.4 (s, 3H). 5.1 (s, 3H ). 7.3 (s, l H-acr), 7.4 (m, IH), 8.1 -8.9 (m, 5H-acr). Hexafluoroisopropyl 2, 3 -dimcthoxy- 10-mcthylacridanc - -carboxylate (GZ-3) *H NMR (acctone-d () ) δ 3.5 (s, 3H), 3.8 (s, 3H). 3.9 (s. 3H). 5.3 (s. I H ), 6.2 (m. I H), 6.8-7.3 (m, 6H). Example 4. Synthesis of GZ-4. 2-Fluoroacridine-9-carboxylic acid (GW-4) 1H NMR (CDC1 3 ) δ 7.7-8.4 (m, 7H). Hexafluoroisopropyl 2-fluoroacridine-9 -carboxylate (GX-4) 1H NMR (CDC1 3 ) δ 6.3 (m, I H). 7.5-8.4 (m, 7H). 13 C NMR (CDC1 3 ) δ 67.5 (m), 106.5 (d). 1 18.3. 122.4 (d), 122.5 (d). 122.9, 123.7, 128.9, 130.4 (d), 133.3 (d), 146.0, 148.0 (d), 159.4, 163,1 , 1 4.2. 9-Hexafluoroisopropoxycarbonyi -2-fluoro - 10-methvlacrιduιιuιn trifluoromethanc - sulf nate (GY-4) 1H NMR (acctone-d 6 ) δ 5.3 (s, 3H), 7.3 (m, I H ), 7.9-9.3 (m, 7H). Hexafluoroisopropyl 2-fluoro-lO-methylacridane - -carboxylate (GZ-4) 1H NMR (CDC1 3 ) δ 3.4 (s, 3H). 5.1 (s. I H). 5.5 (m, I H), 6.9-7.4 (m, 7H). 13 C NMR (CDC1 3 ) δ 33.4 (d), 48.8 (d), 76.3 ( ). 1 12.9. 1 13.8 (d), 1 15.4 (2xd), 118.0, 1 19.9, 120.0, 121 .0 (q). 122.2. 128.9 (d), 138.8. 142.2, 155.8, 159.3, 168.0. Example 5. Synthesis of GZ-5. 2-Chloroacridine-9-carboxylic acid (GW-5) 1H NMR (DMSO-d 6 ) δ 7.7-8.3 (m, 7H). Hexafluoroisopropyl 2-chloroacridιne - -carboxylate (GX-5) ] H NMR (CDC1 ) δ 6.3 (m, I H), 7.7-8.3 (m, 7H). 13 C NMR (CDC1 ) δ 67.6 (m ). 1 18.3. 122.6. 124.0. 128.9. 130.5. 130.8, 131 .6. 131.9, 146.8. 148.6, 164,1 . 9-Hexafluoroisopropoxycarbonyl -2 -chloro- JO-methylacridiniitm trifliioromethane - sulfonate (GY-5) 1H NMR (acctonc-d 6 ) δ 5.3 (s, 3H), 7.4 (m. I H ). 8.3-9.2 (m, 7H). Hexafluoroisopropyl 2-chloro- 10-methylacridcιne-9-carboxylate (GZ-5) ] H NMR (acctone-d 6 > δ 3.4 (s, 3H). 5.5 (s, I H), 6.2 (m. I H), 7.0-7.5 (m. 7H). Example 6. Synthesis of GZ-6. 2,3,6, 7-Tetramethoxyacridine-9-carb<λxyli( acid (GW-6) 1H NMR (DMSO-d ) δ 3.8 (s, 6H), 3.9 (s, 6H). 7.3 (s. 2H). 7.4 (s.2H). Hexafluoroisopropyl 2,3,6, 7-tetramethoxyacridine - 9-ιarbυxylatc (GX-6) ! H NMR (CDCI 3 ) δ 4.0 (s, 6H), 4.1 (s. 6H ). 6.4 (m. I H). 7.1 (s. 2H). 7.5 (s, 2H). 9-Hexafluoroisopropoxycarbonyl-2, 3.6, 7- tetramethoxy - 10 - methylacridinium tri - fluoromethanesulfonate (GY-6) 1H NMR (acctonc-d 6 ) δ 4.1 (s, 6H), 4.3 (s. 6H). 5.0 (s. 3H), 7.2 (s, 2H), 7.3 (m, IH), 8.1 (s, 2H). Example 7. Synthesis of GZ-8. a, a-Bis(trifluoromethyl)benzyl 2-chloroacridine- 9-carboxyUιie (GX-H) 1H NMR (CDC1 3 ) δ 7.5-8.3 (m, 12H). 9-[a, a-Bis(trifluoromethy!)benzyloxycarbonyl/-2-chlor( - 10- methylacridinium trifluoromethanesulfonate (GY-8) 1H NMR (acetonc-d 6 ) δ 5.3 (s, 3H), 7.1 -9.3 (m, 12H). a, a-Bis(trifluoromethyl)benzyl 2-chloro- 10-methylucridane -9 -carboxylate (GZ-S) 1H NMR (CDC1 3 ) δ 3.39 (s. 3H), 5.07 (s. I H). 6 8-7.4 (m. 12H) 1 C NMR (CDC1 3 ) δ 165.6, 141 .8, 140.9, 129.9, 129.2. 129.0, 128.7, 128.5, 128.2, 127.8, 126.8, 126.3, 126.0, 125.7, 123.5, 121 .1 , 120.6, 1 19.3, 1 18.5. 1 15.0, 1 14.5, 1 14.0, 112.9, 49.8, 33.2 Example 8. Synthesis of GZ-9. , a-Bis(trifluoromethyl)bcnzyl 2.3-dimeιhoxyacridιne -9-ι arboxylate (GX-9) l NMR (CDCI 3 ) δ 4.0 (s, 3H), 4.1 (s, 3H). 7.5-8.2 (m, 1 I H). 9- [a, cx-Bis(trifluoromethyl)benzyloxyc arbonylJ-2.3-dimetlιoxy - 10 -methylacridinium trifluoromethanesulfonate (GY-9) l H NMR (acctonc-d 6 ) δ 4.1 (s, 3H ). 4.4 (s, 3H). 5.1 ( s, 3H ). 7.5-8.9 (m, 1 1 H). Example 9. Synthesis of GZ-10. Hexafluoroisopropyl 3 -mcthoxyacridine -9 -carboxylate (GX- 10) 1H NMR (CDC1 3 ) δ 4.0 (s, 3H), 6.3 (m, I H), 7.3-8.2 ( , 7H). 9-Hexafluoroisopropoxycarbonyl-3-methoxy-10-methylacridiniuιnrifluoromethane- sulfonate (GY- 10) 1H NMR (acctone-d 6 ) δ 4.4 (s, 3H), 5.0 (s, 3H). 7.3 (m. I H). 7.7-9.0 (m, 7H). Example 10. Synthesis of GZ-1 1. Perfluoro-t -butyl 2-chloroacridine-9-carbox\ Uιte (GX- 11) 1H NMR (CDC1 3 ) δ 7.6-7.9 (m, 5H ), 8.2-8.3 (m. 2H). 9-Perfluoro - 1 - butoxycarbonyl -2-ch loro - 10- ethylacridiniuii i trifluoromethanc - sulfonate (GY-]]) 1H NMR (acctonc-d 6 ) 6 5.3 (s. 3H), 8.2-9.3 (m. 7H). Perfluoro-t -butyl 2-chIoro- 10-methyla richιne-9-carboxylate (GZ- 11) 1H NMR (CDC1 3 ) δ 3.4 (s, 3H), 5.1 (s, I H), 6.9-7.4 (m, 7H). 1 C NMR (CDC13 δ 163.9, 141 .7, 140.8, 129.3, 128.9, 128.5, 125.7, 121 .4, 121.2, 119.7, 117.8, 117.1, 1 14.0, 1 13.0, 49.5, 33.1 Example 11. Chemiluminescence measurements using GZ-8. Chemilumincsccnce (CL) measurements were performed in lumacuvcttcs using a Lumac Biocounter M2010. A chcmiluminogcnic signal reagent consisted of a solution of phenylphenol (9* 10 "5 M ), GZ-8 (4* 10 "(> M ) in PBS buffer (pH 7.2. 0.01 M). To this solution was added 250 attomolcs of HRP. The lumacuv cttc was placed in the luniinomcter and the CL reaction was started by the addition of hydrogen peroxide (4* 10 -4 M). The CL reaction was followed over a period of 22 minutes. The resulting CL kinetic profile is shown in figure 1 . Example 12. HRP calibration curve using GZ-8. In a similar experiment as described in example 12, different amounts of HRP (0-250 amol) were incubated with phenylphenol ( * 1 ()"5 M), GZ-8 (4* 10 " '' M) in PBS buffer (pH 7.2, 0.01 M). To all the tubes was added hydrogen peroxide (4* 10 -4 M). After 16 minutes the CL was measured. The CL-counts, corrected for background, were plotted against the amounts of HRP to obtain a HRP calibration curv e. An example of such a calibration curve is shown in figure 2. Exam ple 13. Chemilumineseent measurements using GZ-11. In a similar experiment as described in example 12. a kinetic curve for the CL-rcaction of HRP (25 amol) with GZ-1 1 was constructed. The results arc shown in figure 3. When comparing figures 1 and 3, it can be seen that the use of GZ- 1 1 as part of the CL-signal reagent causes a much more rapid rise of the CL with time. Within 2 minutes the curve reaches half of the maximal value. The results arc shown in figure 3. Example 14. HRP calibration curve using GZ-1 1. A HRP calibration curve was constructed using the same procedure as described in example 13. This gave the calibration curve as shown in figure 4. Example 15. Comparison of chemiluminescence of GZ-8 and GZ-11 with prior art compound Compound 5c of WO 95/23971 (2,3,6-trifluorophcnyl 10-mcthylacridan-9-carboxylatc, also denoted as Lumigcn PS-3) was synthesized according to the general synthesis procedure starting from 9-acridinccarboxylic acid and 2.3,6-trifluorophcnol. 1H NMR (CDCI 3 ): 3.44 (s, 3H), 5.29 (s, I H). 6.76-7.4 (m. 1 H). The CL measurements of GZ-8 and GZ- 1 1 were performed as described in examples 12 and 14, respectively, using 25 amol of HRP for GZ-8 as well. The CL measurements of PS-3 were performed using a signal reagent containing PS-3 (0.05 mM), para-iodophcnol (1.1 mM), Twccn 20 (0.5%), EDTA (1 mM) and H 2 O 2 (0.4 mM) in Tris buffer (0.1 M, pH 8). Upon addition of 25 amol of HRP, the CL was recorded over a period of 17 minutes. The results arc shown in figure 5, wherein S/B denotes signal to background ratio.

Claims: Claims 1. Dibcnzodihydropyridinccarboxylic ester having formula 1 or 2, wherein: R 1 is substituted alkyl or cyano, R' and R" are independently hydrogen, alkv , substituted alkyl, aryl or substituted aryl; R 4 is hydrogen, alkyl, substituted alkv i. aryl or substituted aryl: R" and R 6 arc independently alkyl, alko v or anothci substitucnt which is relatively resistant to oxidation; m and n are integers from 0 to 4: wherein, if m oi n is at least 2, two groups R s or R 6 may be linked together; and Z is oxygen or sulphur. 2. Dibcnzodihydropyridinccarboxylic ester according to claim 1 , wherein R 1 is a haloalkyl group, especially a trifluoromcthyl group. 3. Dibcnzodihydropyridinccarbox iic ester according to claim 2, wherein R 2 is a haloalkyl group, especially a trifluoromcthyl group. 4. Dibcnzodihydropyridinccarbo.xviic ester according to any one of claims 1 -3, wherein R is hydrogen, alkyl, haloalkyl, phenyl or halophcnyl. 5. Dibenzodihydropyridinecarboxylic ester according to any one of claims 1-4, wherein R 5 and R 6 are independently methoxy, chloro and/or fluoro, and m and n are independently 0 or 1. 6. Dibcnzodihydropyridinccarboxviic ester according to any one of claims 1-5, wherein Z is oxygen. 7. Method for assaying a pcroxidase. wherein a dibcnzodihydropyridinccarboxylic ester according to any one of claims 1 -6 is used as a chemilumineseent substrate. 8. Method according to claim 7, wherein a pH of 6 - 7.5 is used. 9. Method according to claim 7 or 8, wherein a phenol, in particular p-phenyl- phenol, is used as an enhancer. 10. Kit for assaying pcroxidase activity, comprising a dibenzodihydropyridine- carboxylic ester according to any one of claims 1 -6. and optionally a peroxide, a phenolic enhancer, and optionally comprising a pcroxidase. 11. Dibcnzopyridinium compound having formula 3 or 4. wherein R 1 is substituted alkyl, especially haloalkyl. or cyano: R' and R are independently hydrogen, alkyl, substituted alk , especially haloalkyl, aryl or substituted aryl; R is hydrogen, alkvi, substituted alk i, aryl or substituted aryl: R and R 6 arc independently alkvi, alko v. halogen or another substitucnt which is relatively resistant to oxidation; m and n are integers from 0 to 4; wherein, if m or n is at least 2. two groups R 5 or R f> may be linked together, preferably at least one of m and n being 1 or 2; and Z is oxygen or sulphur.

Inventors: JACQUEMIJNS MARJORIE, ZOMER GIJSBERT, JACQUEMIJNS, MARJORIE, ZOMER, GIJSBERT
Applicants: JACQUEMIJNS MARJORIE, NEDERLANDEN STAAT, ZOMER GIJSBERT, DE STAAT DER NEDERLANDEN, VERTEGENWOORDIGD DOOR DE, JACQUEMIJNS, MARJORIE, ZOMER, GIJSBERT
Application year: 1997
Main classification: C07D219
IPC codes: C07D219, C07D221, C12Q1, G01N21