8-(ω-Aminoalkyl)theophyllines and Their Use in Preparing

Bioconjugate Chem. , 1997, 8 (4), pp 611–616. DOI: 10.1021/bc9700648. Publication Date (Web): July 29, 1997. Copyright © 1997 American Chemical Soc...
0 downloads 0 Views 221KB Size
Download PDF
Bioconjugate Chem. 1997, 8, 611−616

611

8-(ω-Aminoalkyl)theophyllines and Their Use in Preparing Fluorescently Labeled Derivatives for Applications in Immunoassay Fulya Yahioglu,† Colin W. Pouton, and Michael D. Threadgill* School of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom. Received September 10, 1996X

Reaction of alkane-1,ω-diamines with 6-chloro-1,3-dimethylpyrimidine-2,4-dione under carefully controlled conditions gives 6-(ω-aminoalkylamino)-1,3-dimethylpyrimidine-2,4-diones, which can be readily separated from traces of products of disubstitution after benzyloxycarbonyl protection. A sequence of nitrosation at the pyrimidine 5-position, thermal cyclization, and deprotection affords 8-(ω-aminoalkyl) derivatives of theophylline, an important drug in the treatment of asthma and related diseases. These 8-(ω-aminoalkyl)theophyllines can be coupled to fluorescein-5-isothiocyanate and to dansyl chloride, giving fluorescent derivatives of theophylline with applications in automated immunoassay of the drug in biofluids using the fluorescence capillary fill device.

INTRODUCTION

Theophylline (1; Scheme 1) is a drug that is commonly used as a bronchospasmolytic agent in the prevention and treatment of asthma, apnoea, and obstructive lung diseases and is an inhibitor of cyclic nucleotide phosphodiesterases (1-3). Drug-induced toxicity occurs at plasma concentrations not far above those required for therapy, and plasma concentrations vary greatly among individuals given the same dose. Rapid and accurate measurement of its concentration in biofluids is therefore necessary (2, 3). Current methods for the determination of theophylline in blood include ELISA (4), radioimmunoassay (5), and HPLC (6), although not all of these completely fulfill criteria of speed, ease, and low cost of assay. The fluorescence capillary fill device (FCFD) (710) overcomes these drawbacks. To develop an immunoassay for theophylline in whole blood using this device, we required fluorescent derivatives of theophylline that have good immunoreactivity with appropriate anti-theophylline antibodies. To be able to optimize both the photoproperties of the fluorophore and the length and lipophilicity of the link between theophylline and the fluorophore, a series of 8-(ω-aminoalkyl)theophyllines and related ethers was sought. Thus, a variety of widely used fluorescent groups bearing isothiocyanate and sulfonyl chloride electrophiles could be attached. EXPERIMENTAL PROCEDURES

Chemical Synthesis. IR spectra were recorded on a Perkin-Elmer 782 spectrophotometer as potassium bromide disks, unless otherwise stated. NMR spectra were recorded using JEOL JNM GX270 and JNM EX400 spectrometers of samples in (CD3)2SO, except where stated. Chemical shifts were measured relative to tetramethylsilane as internal standard. Mass spectra were obtained using a VG7070E analytical mass spectrometer using electron impact (EI) or fast atom bombardment (FAB) techniques in the positive ion mode. Melting points are uncorrected. Dioxane refers to 1,4-dioxane. Chromatographic separations were carried out using * Author to whom correspondence should be addressed (email [email protected]). † Present address: Department of Clinical Biochemistry, The London Hospital Medical College, Turner Street, London E1 2AD, U.K. X Abstract published in Advance ACS Abstracts, June 1, 1997.

S1043-1802(97)00064-5 CCC: $14.00

Sorbsil C60 (0.040-0.063) silica gel. Solvents were evaporated under reduced pressure. Solutions in organic solvents were dried over magnesium sulfate. Experiments were conducted at ambient temperature unless otherwise stated. Chemicals were purchased from Aldrich Chemical Co. (Gillingham, U.K.), Sigma Chemical Co. (Gillingham, U.K.), and Maybridge Chemicals (Tintagel, U.K.). Phenylmethyl N-[[[6-(1,1-Dimethylethoxy)carbonyl]amino]hexyl]carbamate (3). Phenylmethyl chloroformate (610 mg, 3.6 mmol) in CH2Cl2 (6 mL) was added during 30 min to 1,1-dimethylethyl N-(6-aminohexyl)carbamate hydrochloride (2) (900 mg, 3.6 mmol) and Et3N (720 mg, 7.1 mmol) in CH2Cl2 (12 mL). The mixture was stirred for 24 h and was filtered. The filtrate was washed twice with water and once with hydrochloric acid (2 M) and dried. The solvent was evaporated to afford 3 as a white crystalline solid (960 mg, 77%): mp 190 °C; IR 3340, 1680, 1550 cm-1; NMR δ (CDCl3) 1.44 (17 H, m, CH2CH2CH2CH2CH2CH2 + But), 1.85 (1 H, br, NH), 3.11 (2 H, q, J ) 6 Hz, CH2N), 3.17 (2 H, m, CH2N), 4.85 (1 H, br, NH), 5.09 (2 H, s, PhCH2), 7.35 (5 H, s, Ar-H5); MS (EI) 350 (M). Anal. Calcd for C19H30N2O4: C, 65.12; H, 8.63; N, 7.99. Found: C, 65.41; H, 8.61; N, 8.17. Phenylmethyl N-(6-Aminohexyl)carbamate Hydrochloride (4). HCl was passed through 3 (960 mg, 2.7 mmol) in CH2Cl2 (100 mL) for 1 h. The solvent and excess reagent were evaporated to afford 4 as white crystals (680 mg, 99%): mp 177-178 °C [lit. (11) mp 177-178 °C]; IR 3470, 3340-3280, 1700, 1630 cm-1; NMR δ (CDCl3) 1.18 (4 H, m, 2 × CH2), 1.35 (4 H, m, 2 × CH2), 2.93 (2 H, t, J ) 6 Hz, CH2N+H3), 3.08 (2 H, q, J ) 6 Hz, CH2NH), 3.61 (3 H, br, N+H3), 5.07 (2 H, s, PhCH2), 7.39 (5 H, s, Ar-H5); MS (EI) 287 (M). 1,3-Dimethyl-6-[[[[6-(phenylmethoxy)carbonyl]amino]hexyl]amino]pyrimidine-2,4-dione (6a). 1,3-Dimethyl-6chloropyrimidine-2,4-dione (5) (490 mg, 2.8 mmol), 4 (840 mg, 3.7 mmol), and Na2CO3 (600 mg, 5.7 mmol) were boiled under reflux in dioxane (8 mL) for 4 h. The cooled mixture was diluted with CHCl3, and the solution was washed twice with water and once with saturated brine and was dried. The evaporation residue was recrystallized (EtOH) to give 6a as yellow crystals (600 mg, 54%): mp 128-130 °C; IR 3360, 3280, 1700, 1630 cm-1; NMR δ 0.90 (4 H, m, 2 × CH2), 1.23 (4 H, m, 2 × CH2), 3.15 (2 H, t, J ) 6 Hz, CH2N), 3.17 (2 H, t, J ) 6 Hz, CH2N), 3.06 (3 H, s, NMe), 3.23 (3 H, s, NMe), 4.21 (2 H, © 1997 American Chemical Society

612 Bioconjugate Chem., Vol. 8, No. 4, 1997

Yahioglu et al.

Scheme 1. Structures of Theophylline 1 and Fluorescein-5-isothiocyanate 12 and Synthetic Routes to the Fluorescent Theophylline Derivatives 13a-d and 14a

a Reagents: i, CbzCl, Et N, CH Cl ; ii, HCl, CH Cl ; iii, 5, 1,4-dioxane, Na CO , ∆; iv, C H ONO, HCl, EtOH; v, BuOH, ∆; vi, 3 2 2 2 2 2 3 5 11 HBr, HOAc; vii, 12, K2CO3, dioxane, water, pH 9.0 ( 0.2; viii, 5-(dimethylamino)naphthalene-1-sulfonyl chloride, Na2CO3, dioxane, water, pH 9.5-10.0.

m, pyrimidine 5-H + NH), 4.69 (2 H, s, PhCH2), 6.79 (5 H, m, Ph-H5), 7.70 (1 H, br, NH); m/z (EI) 388 (M), 91 (100%). Anal. Calcd for C20H28N4O4: C, 61.82; H, 7.27; N, 14.43. Found: C, 61.85; H, 7.36; N, 14.3. 1,3-Dimethyl-6-[[[[9-(phenylmethoxy)carbonyl]amino]nonyl]amino]pyrimidine-2,4-dione (6b). The chloropyrimidine 5 (1.00 g, 5.7 mmol) was boiled under reflux with nonane-1,9-diamine (1.81 g, 11 mmol) and Na2CO3 (1.21 g, 1.4 mmol) in EtOH (40 mL) for 3 h. The mixture was filtered and the solvent was evaporated. Recrystal-

lization (EtOH) afforded 1,3-dimethyl-6-[(9-aminononyl)amino]pyrimidine-2,4-dione (7b) as a white solid (1.09 g, 65%): mp 210-212 °C; IR 3500, 1660 cm-1; NMR δ 1.29 [14 H, m, (CH2)7], 2.54 (2 H, m, CH2NH2), 3.03 (2 H, m, NHCH2), 3.03 (3 H, s, NMe), 3.26 (3 H, s, NMe), 4.65 (1 H, br, NH), 6.72 (1 H , s, pyrimidine 5-H); m/z (EI) 297 (M), 159 (100%). This material (3.16 g, 11 mmol) was stirred with phenylmethyl chloroformate (1.91 g, 11 mmol) and Et3N (2.16 g, 21 mmol) in CH2Cl2 (150 mL) for 24 h. The suspension was filtered, and the filtrate

Technical Notes

was washed twice with water and once with hydrochloric acid (2 M) and was dried. Evaporation and chromatography (CHCl3/EtOAc 4:1 f EtOAc) gave 6b as white crystals (2.02 g, 43%): mp 105-106 °C; IR 3420, 1700, 1680 cm-1; NMR δ (CDCl3) 1.30 (4 H, br, 2 × CH2), 1.50 (2 H, quintet, J ) 7.3 Hz, CH2), 1.66 (8 H, m, 4 × CH2), 3.09 (2 H, q, J ) 7.3 Hz, NCH2), 3.18 (2 H, q, J ) 6.3 Hz, NCH2), 3.31 (3 H, s, NMe), 3.39 (3 H, s, NMe), 4.35 (1 H, br, NH), 4.79 (1 H, br, NH), 4.86 (1 H, s, 5-H), 5.09 (2 H, s, PhCH2), 7.35 (5 H, s, Ph-H5); MS (EI) 431 (M), 336 (M - PhCH2OH), 91 (100%). Anal. Calcd for C20H28N4O4: C, 61.84; H, 7.27; N, 14.42. Found: C, 61.9; H, 7.35; N, 14.39. 1,3-Dimethyl-6-[[[[10-(phenylmethoxy)carbonyl]amino]decyl]amino]pyrimidine-2,4-dione (6c). The chloropyrimidine 5 (1.35 g, 7.7 mmol) was treated with decane-1,10diamine (1.60 g, 9.3 mmol) as for the synthesis of 7b, except that recrystallization was omitted, to give crude 1,3-dimethyl-6-[(10-aminodecyl)amino]pyrimidine-2,4-dione (7c) (1.24 g, 52%) as a white solid: mp 72-74 °C; IR 3400, 1660 cm-1; NMR δ (CF3CO2D) 1.41 [16 H, m, (CH2)8], 1.82 (2 H, m, CH2N), 3.28 (2 H, m, CH2N), 3.60 (3 H, s, NMe), 3.69 (3 H, s, NMe); MS (EI) 310 (M), 173. The amine 7c (800 mg, 2.6 mmol) was treated with phenylmethyl chloroformate (440 mg, 2.6 mmol) as for the synthesis of 6b, except that the chromatographic eluant was CHCl3/MeOH 10:1, to afford 6c as white crystals (620 mg, 54%): mp 100-102 °C; IR 3360, 3280, 1700, 1640 cm-1; NMR δ (CDCl3) 1.29 [12 H, m, (CH2)6], 1.49 (2 H, m, CH2), 1.65 (2 H, quintet, J ) 6.8 Hz, CH2), 3.08 (2 H, dt, J ) 5.1, 7.1 Hz, CH2), 3.18 (2 H, q, J ) 6.6 Hz, NCH2), 3.31 (3 H, s, NMe), 3.39 (3 H, s, NMe), 4.40 (1 H, br, NH), 4.81 (1 H, s, pyrimidine 5-H), 5.09 (2 H, s, PhCH2), 7.35 (5 H, m, Ph-H5); MS (EI) 444 (M), 336 (M - PhCH2OH), 91 (100%). Anal. Calcd for C24H36N4O4: C, 64.82; H, 8.17; N, 12.61. Found: C, 65.1; H, 8.14; N, 12.65. 1,3-Dimethyl-6-[[[2-[2-(2-(phenylmethoxy)carbonyl]amino]ethoxyethoxyethyl]amino]pyrimidine-2,4-dione (6d). The chloropyrimidine 5 (2.82 g, 16 mmol) was treated with 3,6-dioxaoctane-1,8-diamine (6.0 g, 40 mmol) as for the synthesis of 7b, except that recrystallization was omitted, to give crude 1,3-dimethyl-6-[[2-[2-(2-aminoethoxy)ethoxy]ethyl]amino]pyrimidine-2,4-dione (7d) (3.03 g, 69%). This material was treated with phenylmethyl chloroformate (1.90 g, 11 mmol), as for the synthesis of 6b, except that the chromatographic eluant was EtOAc f EtOAc/MeOH 6:1, to afford 6d as a colorless gum (2.04 g, 44%): IR 3400-3280, 1740-1670 cm-1; NMR δ (CDCl3) 3.24 (2 H, q, J ) 6 Hz, NCH2), 3.31 (3 H, s, NMe), 3.36 (3 H, s, NMe), 3.40 (2 H, q, J ) 5.3 Hz, NCH2), 3.58 (2 H, t, J ) 6 Hz, NCH2CH2), 3.62 (4 H, s, OCH2CH2O), 3.68 (2 H, t, J ) 6 Hz, NCH2CH2), 4.83 (1 H, s, pyrimidine 5-H), 5.00 (1 H, br, NH), 5.09 (2 H, s, PhCH2), 5.29 (1 H, br, NH), 7.35 (5 H, s, Ph-H5); MS (EI) 421 (M), 91 (100%). Anal. Calcd for C20H28N4O6: C, 57.12; H, 6.72; N, 13.33. Found: C, 56.95; H, 6.78; N, 13.39. 1,3-Dimethyl-8-[[[5-(phenylmethoxy)carbonyl]amino]pentyl]purine-2,6-dione (10a). Pentyl nitrite (410 mg, 3.5 mmol) was added carefully to 6a (550 mg, 1.4 mmol) in EtOH (25 mL) at 40 °C, and the solution was stirred for 10 min. Hydrochloric acid (9 M, 0.3 mL) was added, and the mixture was stirred at ambient temperature for 16 h. The solvent was evaporated to afford crude 9a as a red gum (430 mg, 73%). This material was boiled under reflux in BuOH (3 mL) for 45 min until decolorization had occurred. The evaporation residue was washed with Et2O and recrystallized (BuOH) to give 10a as off-white crystals (400 mg, 71% from 6a): mp 182-184 °C; IR 3300, 1700, 1680, 1660 cm-1; NMR δ 1.55 (2 H, quintet,

Bioconjugate Chem., Vol. 8, No. 4, 1997 613

J ) 7.0 Hz, CH2), 1.67 (2 H, quintet, J ) 7 Hz, CH2), 1.90 (2 H, quintet, J ) 7.3 Hz, CH2), 1.92 (2 H, t, J ) 7.3 Hz, purine-CH2), 3.23 (2 H, q, J ) 6.4 Hz, CH2N), 3.45 (3 H, s, NMe), 3.59 (3 H, s, NMe), 5.23 (2 H, br, PhCH2), 7.54 (1 H, br, CbzNH), 7.58 (5 H, m, Ph-H5); MS (EI) 400 (M), 292 (M - PhCH2OH), 91 (100%). Anal. Calcd for C20H25N5O4: C, 60.14; H, 6.31; N, 17.53. Found: C, 60.09; H, 6.34; N, 17.50. 1,3-Dimethyl-8-[[[8-(phenylmethoxy)carbonyl]amino]octyl]purine-2,6-dione (10b). The pyrimidine 6b was treated with pentyl nitrite as for the synthesis of 9a, to give crude 9b as a purple gum. This material was heated, as for the synthesis of 10a, to give 10b as a cream solid (26%): mp 145-147 °C; IR 3320, 1740, 1680 cm-1; NMR δ 1.24 (8 H, m, 4 × CH2), 1.38 (2 H, quintet, J ) 6 Hz, CH2), 1.65 (2 H, quintet, J ) 6 Hz, CH2), 2.65 (2 H, t, J ) 6.2 Hz, purine-CH2), 2.95 (2 H, q, J ) 6.2 Hz, NCH2), 3.21 (3 H, s, NMe), 3.41 (3 H, s, NMe), 4.99 (2 H, s, PhCH2), 7.22 (1 H, br, NH), 7.33 (5 H, m, Ph-H5), 13.15 (1 H, s, NH); MS (EI) 442 (M), 334 (M - PhCH2OH), 91 (100%). Anal. Calcd for C23H31N5O4: C, 62.57; H, 7.08; N, 15.86. Found: C, 62.99; H, 7.35; N, 15.79. 1,3-Dimethyl-8-[[[9-(phenylmethoxy)carbonyl]amino]nonyl]purine-2,6-dione (10c). The 10-Cbz-aminodecylaminopyrimidine 6c was treated with pentyl nitrite, as for the synthesis of 9a, to afford crude 9c as a red-purple oil. This material was boiled under reflux for 40 min in BuOH, and the solvent was evaporated. Recrystallization (BuOH) gave 10c as pale buff crystals (300 mg, 45%): mp 95-96 °C; IR 3300, 1730, 1700, 1650 cm-1; NMR δ 1.24 [10 H, m, (CH2)5], 1.38 (2 H, t, J ) 6 Hz, CH2), 1.67 (4 H, m, 2 × CH2), 2.66 (2 H, q, J ) 7.6 Hz, NCH2), 2.97 (2 H, q, J ) 6.4 Hz, purine-CH2), 3.22 (3 H, s, NMe), 3.41 (3 H, s, NMe), 4.99 (2 H, s, PhCH2), 7.22 (1 H, t, J ) 6 Hz, NH), 7.34 (5 H, m, Ph-H5); MS (EI) 455 (M), 347 (M - PhCH2OH), 194 (100%). Anal. Calcd for C24H33N5O4: C, 63.26; H, 7.31; N, 15.38. Found: C, 63.6; H, 7.28; N, 15.4. 1,3-Dimethyl-8-[2-[2-(phenylmethoxy)carbonyl]ethoxyethoxymethyl]purine-2,6-dione (10d). The pyrimidine 6d was treated with pentyl nitrite, as for the synthesis of 9a, to give 9d as a purple gum (97%). This material was heated as for the synthesis 10a, to give 10d as a yellow gum (66%): IR 3420, 3250, 1740, 1680 cm-1; NMR δ 3.15 (2 H, q, J ) 5.9 Hz, NCH2), 3.23 (3 H, s, NMe), 3.40 (2 H, m, NCH2CH2), 3.42 (3 H, s, NMe), 3.55 (2 H, m) and 3.59 (2 H, m) (OCH2CH2O), 4.53 (2 H, s, purine-CH2), 5.00 (2 H, s, PhCH2), 7.33 (6 H, m, Ph-H5 + NH), 13.57 (1 H, br, purine-NH); m/z (EI) 431 (M), 91 (100%). Anal. Calcd for C20H25N5O6: C, 55.68; H, 5.84; N, 16.23. Found: C, 55.40; H, 5.72; N, 16.39. 8-(5-Aminopentyl)-2,4-dimethylpurine-1,3-dione Hydrobromide (11a). The Cbz-protected amine 10a (230 mg, 0.58 mmol) was stirred with HBr in AcOH (30%, 0.8 mL) for 15 min. Dry Et2O (10 mL) was added, the suspension was stirred vigorously for 5 min, and the Et2O was decanted. This washing procedure was repeated five times, and the solid was dried to afford 11a as an offwhite powder (200 mg, 100%); mp 250-252 °C; IR 35003400, 3240, 1740, 1680 cm-1; NMR δ 1.31 (2 H, quintet, J ) 6.7 Hz, CH2), 1.56 (2 H, quintet, J ) 7.3 Hz, CH2), 1.70 (2 H, quintet, J ) 7.3 Hz, CH2), 2.70 (2 H, t, J ) 7.7 Hz, purine-CH2), 2.75 (2 H, t, J ) 7.7 Hz, NCH2), 3.22 (3 H, s, NMe), 3.41 (3 H, s, NMe), 7.74 (3 H, br, N+H3); MS (FAB) 266 (M + H), 180 (100%). Anal. Calcd for C12H20BrN5O2: C, 41.63; H, 5.82; N, 20.23. Found: C, 41.57; H, 5.77; N, 20.22. 8-(8-Aminooctyl)-2,4-dimethylpurine-1,3-dione Hydrobromide (11b). The Cbz-protected amine 10b was treated with HBr, and the solid was washed with Et2O, as for

614 Bioconjugate Chem., Vol. 8, No. 4, 1997

the synthesis of 11a, to give 11b as a white powder (100%): mp 239-241 °C; IR 3440, 3200, 1760, 1680 cm-1; NMR δ 1.27 [8 H, m, (CH2)4], 1.52 (2 H, m, CH2), 1.67 (2 H, quintet, J ) 6.7 Hz, CH2), 2.68 (2 H, t, J ) 7.6 Hz, purine-CH2), 2.76 (2 H, sextet, J ) 6.4 Hz, NCH2), 3.22 (3 H, s, NMe), 3.41 (3 H, s, NMe), 7.72 (3 H, br, N+H3); MS (FAB) 308 (M + H), 180 (100%). Anal. Calcd for C15H26BrN5O2: C, 46.42; H, 6.75; N, 18.04. Found: C, 46.41; H, 6.67; N, 18.10. 8-(9-Aminononyl)-2,4-dimethylpurine-1,3-dione Hydrobromide (11c). The Cbz-protected amine 10c was treated with HBr, and the solid was washed with Et2O, as for the synthesis of 11a, to give 11c as an off-white powder (87%): mp 218-219 °C; IR 3160, 1720, 1680 cm-1; NMR δ 1.25 (10 H, br, CH2CH2CH2CH2CH2CH2CH2CH2CH2), 1.51 (2 H, m, CH2), 1.67 (2 H, m, CH2), 2.68 (2 H, t, J ) 7.5 Hz, purine-CH2), 2.75 (2 H, m, NCH2), 3.22 (3 H, s, NMe), 3.41 (3 H, s, NMe), 7.71 (3 H, s, N+H3); MS (FAB) 322 (M + H), 180 (100%). Anal. Calcd for C16H28BrN5O2: C, 47.77; H, 7.01; N, 17.41. Found: C, 47.84; H, 7.11; N, 17.22. 8-[2-(2-Aminoethoxy)ethoxymethyl]-2,4-dimethylpurine1,3-dione Hydrobromide (11d). The Cbz-protected amine 10d was treated with HBr, and the solid was washed with Et2O, as for the synthesis of 11a, to give 11d as a pale yellow solid (100%): mp 220-221 °C; NMR δ 2.97 (2 H, sextet, J ) 6 Hz, NCH2), 3.23 (3 H, s, NMe), 3.43 (3 H, s, NMe), 3.62 (6 H, m, OCH2CH2OCH2), 4.55 (2 H, s, purine-CH2), 7.83 (3 H, br, N+H3); m/z (FAB) 298 (M + H), 180 (100%). Anal. Calcd for C12H20N5O4Br: C, 38.11; H, 5.33; N, 18.52. Found: C, 38.15; H, 5.27; N, 18.46. 5-[[N-[5-(2,4-Dimethyl-1,3-dioxopurin-8-yl)pentyl]thio]ureido]-3′,6′-dihydroxyspiro[isobenzofuran-1(3H),9′(9H)xanthen]-3-one (13a). 3′,6′-Dihydroxy-5-isothiocyanatospiro[isobenzofuran-1(3H),9′-(9H)xanthen]-3one (fluorescein-5-isothiocyanate; 12) (340 mg, 870 µmol) was suspended in water (12 mL) and brought to pH 9.0 by dropwise addition of aqueous K2CO3 (1 M). To this stirred solution was added 11b (300 mg, 870 µmol) in water (6.7 mL) and dioxane (3.3 mL) during 30 min. The mixture was stirred for 3 h. During this time, the pH was maintained at 9.0 ( 0.2 by dropwise addition of aqueous K2CO3 (1 M). The solution was acidified to pH 6.0 by dropwise addition of hydrochloric acid (2 M). Water (200 mL) was added, and the mixture was freezedried for 24 h to reveal an orange fluffy solid. Chromatography (EtOAc f EtOAc/MeOH 10:1) gave 13a as an orange-yellow solid (280 mg, 50%): mp 197-198 °C; IR 3480, 1710, 1660 cm-1; NMR δ 1.37 (2 H, m, CH2), 1.62 (2 H, m, NCH2CH2), 1.76 (2 H, m, purine-CH2CH2), 2.73 (2 H, t, J ) 7.7 Hz, purine-CH2), 3.24 (3 H, s, NMe), 3.40 (3 H, s, NMe), 3.53 (2 H, m, NCH2), 6.56 (2 H, dd, J ) 8.8, 2.2 Hz, xanthene 2′,7′-H2), 6.61 (2 H, d, J ) 8.8 Hz, xanthene 1′,8′-H2), 6.70 (2 H, d, J ) 2.2 Hz, xanthene 4′,5′-H2), 7.20 (1 H, d, J ) 8.2 Hz, Ar 3-H), 7.73 (1 H, m, Ar 4-H), 8.25 (1 H, br, NH), 8.27 (1 H, br, Ar 6-H), 9.93 (1 H, br, NH), 10.13 (2 H, s, 2 × OH), 13.21 (1 H, br, purine NH); MS (FAB) 655 (M + H). Anal. Calcd for C33H30N6O7S: C, 60.53; H, 4.62; N, 12.84. Found: C, 60.61; H, 4.57; N, 12.72. 5-[[N-[8-(2,4-Dimethyl-1,3-dioxopurin-8-yl)octyl]thio]ureido]-3′,6′-dihydroxyspiro[isobenzofuran-1(3H),9′(9H)xanthen]-3-one (13b). The 8-(8-aminooctyl)purine salt 11b was treated with 12, as for the synthesis of 13a, to give 13b as an orange-yellow solid (72%): mp 182183 °C; IR 3500-3100, 1710, 1660 cm-1; NMR δ 1.30 (8 H, m, 4 × CH2), 1.55 (2 H, m, NCH2CH2), 1.66 (2 H, m, purine-CH2CH2), 2.67 (2 H, t, J ) 7.3 Hz, purine-CH2), 3.22 (3 H, s, NMe), 3.41 (3 H, s, NMe), 3.49 (2 H, m,

Yahioglu et al.

NCH2), 6.53 (2 H, dd, J ) 8.8, 2.2 Hz, xanthene 2′,7′H2), 6.58 (2 H, d, J ) 8.8 Hz, xanthene 1′,8′-H2), 6.68 (2 H, d, J ) 2.2 Hz, xanthene 4′,5′-H2), 7.19 (1 H, d, J ) 8.4 Hz, Ar 3-H), 7.70 (1 H, m, Ar 4-H), 8.27 (1 H, br, Ar 6-H), 10.23 (2 H, s, 2 × OH); m/z (FAB) 697 (M + H). Anal. Calcd for C36H36N6O7S: C, 62.05; H, 5.21; N, 12.01. Found: C, 62.11; H, 5.27; N, 12.02. 5-[[N-[9-(2,4-Dimethyl-1,3-dioxopurin-8-yl)nonyl]thio]ureido]-3′,6′-dihydroxyspiro[isobenzofuran-1(3H),9′(9H)xanthen]-3-one (13c). The 8-(9-aminononyl)purine salt 11c was treated with 12, as for the synthesis of 13a, to give 13c as an orange-yellow solid (68%): mp 178179 °C; IR 3400-3000, 1740, 1700, 1650 cm-1; NMR δ 1.29 (10 H, br, 5 × CH2), 1.56 (2 H, m, NHCH2CH2), 1.68 (2 H, m, purine-CH2CH2), 2.67 (2 H, t, J ) 7.5 Hz, purineCH2), 3.22 (3 H, s, NMe), 3.41 (3 H, s, NMe), 3.48 (2 H, m, NCH2), 6.56 (2 H, dd, J ) 8.8, 2.2 Hz, xanthene 2′,7′H2), 6.59 (2 H, d, J ) 8.8 Hz, xanthene 1′,8′-H2), 6.67 (2 H, d, J ) 2.2 Hz, xanthene 4′,5′-H2), 7.17 (1 H, d, J ) 8.4 Hz, Ar 3-H), 7.70 (1 H, m, Ar 4-H), 8.06 (1 H, br, CH2NH), 8.22 (1 H, brs, Ar 6-H), 9.86 (1 H, br, Ar-NH), 10.18 (2 H, s, 2 × OH), 13.35 (1 H, purine NH); MS (FAB) 711 (M + H). Anal. Calcd for C37H38N6O7S: C, 62.52; H, 5.39; N, 11.82. Found: C, 62.45; H, 5.41; N, 11.75. 5-[N-[2-[2-(2,4-Dimethyl-1,3-dioxopurin-8-ylmethoxy)ethoxyethyl]thio]ureido]-3′,6′-dihydroxyspiro[isobenzofuran-1(3H),9′-(9H)xanthen]-3-one (13d). The purine derivative salt 11d was treated with 12, as for the synthesis of 13a, to give 13d as an orange-yellow solid (77%): mp 1971-1998 °C; IR 3400-3200, 1740, 1700, 1650 cm-1; δ 3.22 (3 H, s, NMe), 3.41 (3 H, s, NMe), 3.63 (8 H, m, OCH2CH2OCH2CH2), 4.54 (2 H, s, purine-CH2), 6.56 (2 H, dd, J ) 8.8 and 2.2 Hz, xanthene 2′,7′-H2), 6.59 (2 H, d, J ) 8.8 Hz, xanthene 1′,8′-H2), 6.66 (2 H, d, J ) 2.2 Hz, xanthene 4′,5′-H2), 7.18 (1 H, d, J ) 8.4 Hz, Ar 3-H), 7.73 (1 H, brd, J ) 8.5 Hz, Ar 4-H), 8.1 (1 H, br, NH), 8.26 (1 H, br, Ar 6-H), 10.05 (1 H, br, NH), 10.14 (2 H, s, 2 × OH), 13.6 (1 H, purine NH); MS (FAB) 687 (M + H). Anal. Calcd for C33H30N6O9S: C, 57.71; H, 4.41; N, 12.24. Found: C, 57.61; H, 4.45; N, 12.22. 2,4-Dimethyl-8-[5-[5-(dimethylamino)naphthalene-1sulfonylamino]pentyl]purine-1,3-dione Hydrobromide (14). The aminopentylpurine salt 11a (200 mg, 580 µmol) in water (8 mL) and dioxane (4 mL) was brought to pH 9.5 by addition of aqueous Na2CO3 (0.5 M). 5-(Dimethylamino)naphthalene-1-sulfonyl chloride (160 mg, 580 µmol) in dioxane (10 mL) was added. The mixture was stirred for 3 h. During this time, the pH was maintained at 9.5-10.0 by dropwise addition of aqueous Na2CO3 (0.5 M). The solution was neutralized to pH 7.0 by addition of hydrochloric acid (2 M). Water (25 mL) was added and the mixture was freeze-dried for 24 h to reveal an orange fluffy solid. The residue, in ethyl acetate, was washed with water and dried. The solvent was evaporated, and the residue was recrystallized (EtOH) to give 14 as a green-yellow solid (160 mg, 55%): mp 183-185 °C; NMR δ 1.14 (2 H, quintet, J ) 7 Hz, CH2CH2CH2CH2CH2), 1.29 (2 H, quintet, J ) 7 Hz, CH2CH2N), 1.46 (2 H, quintet, J ) 7 Hz, purine-CH2CH2), 2.76 (2 H, t, J ) 5.9 Hz, purineCH2), 2.81 (6 H, s, NMe2), 3.22 (3 H, s, NMe), 3.39 (5 H, m, NMe + CH2N), 7.24 (1 H, d, J ) 7.3 Hz, naphthalene 6-H), 7.58 (1 H, t, J ) 8.1 Hz, naphthalene 3-H or 6-H), 7.61 (1 H, d, J ) 7.7 Hz, naphthalene 8-H), 7.87 (1 H, t, J ) 7.9 Hz, naphthalene 6-H or 3-H), 8.08 (1 H, d, J ) 7.3 Hz, naphthalene 4-H), 8.29 (1 H, d, J ) 8.4 Hz, naphthalene 2-H), 13.1 (1 H, br, purine-NH); MS (FAB) 531.2020 (M + H) (C24H31N6O4S requires 531.2026). Immunoassay. The FCFD devices were fabricated according to the method of Badley et al. (7). The lower base plate was activated with (3-aminopropyl)trimeth-

Bioconjugate Chem., Vol. 8, No. 4, 1997 615

Technical Notes Table 1. Binding Affinities of the Theophylline-Fluorescein Conjugates 13a-d to the Antibody Immobilized in the FCFD Device conjugate

linker unit

binding affinity Ka (M-1)

theophylline (1) 13a 13b 13c 13d

(CH2)5 (CH2)8 (CH2)9 CH2O(CH2)2O(CH2)2

(0.06 ( 0.02) × 106 (3.1 ( 1.5) × 106 (1.4 ( 0.6) × 106 (3.2 ( 1.8) × 106 (1.0 ( 0.4) × 106

oxysilane. Anti-theophylline IgA mouse monoclonal antibody was dissolved in HEPES buffer (0.1 M) containing NaCl (0.2 M) to a final concentration of 200 µg mL-1. Aliquots of this solution (15 µL) were used for coupling of the antibody to the lower plate according to a carbonyldiimidazole method. The plates were then assembled in the usual way. Solutions of the theophyllinefluorescein conjugates 13a-d were prepared in Tris-HCl buffer at pH 8.8. All assays using the FCFDs were performed in duplicate following incubation at ambient temperature for 20 min to ensure that the system had come fully to equilibrium. Total binding of 13a-d to the antibody was measured by incubating the FCFDs with concentrations of conjugates from 0 to 5 µM. Nonspecific binding of 13a-d to the antibody-loaded devices was measured in the presence of theophylline (1 mM). The specific binding isotherms were constructed by plotting (total fluorescence signal observed minus signal in the presence of excess theophylline) vs concentration of 13ad. From these plots (not shown) were obtained the binding affinities of each conjugate for the immobilized antibody (Table 1). RESULTS AND DISCUSSION

Chemistry. Synthesis of 8-(2-aminoethyl)theophylline has been reported (12) involving acylation of 5,6-diamino1,3-dimethylpyrimidine-2,4-dione (a relatively unstable compound) with N-protected 3-aminopropanoic acid, followed by cyclization under drastic conditions. We sought to develop a method that started with more readily available 1,ω-diamines and which did not use vigorous conditions for cyclization. Fuchs et al. (12) have suggested that treatment of 6-chloro-1,3-dimethylpyrimidine (5) with 1,ω-diamines would give the products of 1,ωdisubstitution. Thus, our initial approach involved setting up a synthon for hexane-1,6-diamine, which is protected at one amine only. Exchange of protecting groups on the commercially available mono-Boc hexane1,6-diamine (2) was achieved by benzyloxycarbonylation at the free amine to give the orthogonally protected biscarbamate 3, followed by acidolytic removal of Boc in excellent yield. The Cbz protection in 4 was designed to survive the reaction conditions in later steps, which would cleave Boc. Substitution of the chlorine of the 6-chloropyrimidinedione 5 with the amine 4 was effected in boiling 1,4-dioxane in the presence of inorganic base to give 6a in satisfactory yield, as shown in Scheme 1. An attempt to raise the yield by use of refluxing ethanol as the reaction medium gave only a small amount of 6a but encouraged substitution of chlorine by the solvent to give largely 1,3-dimethyl-6-ethoxypyrimidine-2,4-dione. Following the general method of Goldner et al. (13), nitrosation at C-5 of the pyrimidine was carried out with pentyl nitrite, in a reaction catalyzed by a small quantity of acid. Thermal cyclization/condensation of the brightly colored 5-nitrosopyrimidine 9a to the Cbz-protected 8-(5aminopentyl)theophylline 10a was rapid and high yielding in boiling butanol. Deprotection using hydrogen bromide in acetic acid was virtually quantitative in furnishing the 8-(5-aminopentyl)theophylline salt 11a,

which carries the required link and nucleophilic amine for attachment of electrophilically substituted fluorophores. Selective monoprotection of longer chain alkane-1,ωdiamines, particularly 3,6-dioxaoctane-1,8-diamine, proved to be troublesome. However, by careful selection of reacting quantities and conditions, it proved possible to obtain good yields of the unprotected 6-[ω-amino(oxa)alkylamino]pyrimidines 7b-d, which could be separated with difficulty from traces of the products of 1,ω-disubstitution. Nevertheless, direct reaction of the crude product mixture with benzyl chloroformate gave the easily purified Cbz-protected 6-[ω-amino(oxa)alkylamino]pyrimidines 6b-d. As before, the sequence of nitrosation, thermal cyclization, and deprotection furnished the required 8-[ω-amino(oxa)alkylamino]theophylline salts 11b-d. The aminoalkyltheophyllines 11a-c enabled study of the effect of the length of the link between hapten and fluorophore on the immunoreactivity of the final derivatives, whereas comparison of these with the diether linked compounds derived from 11d indicated the importance of hydrophilicity of the link. Fluorescein has absorption and fluorescence emission maxima at wavelengths that are appropriate for the FCFD device, with a large Stokes shift and a high quantum yield. The 5-isothiocyanate or mixtures of the 4- and 5-isothiocyanates have frequently been used for fluorescent labeling of amino groups of biological molecules in aqueous media (15, 16). Fluorescein also confers aqueous solubility on its derivatives, a factor important for the target fluorescent theophylline derivatives. To have a chemically defined system, the 5-isothiocyanate was used to thiocarbamoylate the amines 11ad. It was found necessary to control closely the pH of the aqueous reaction mixture in the range 9.0 ( 0.2 to ensure that the amine was not protonated and that the isothiocyanate was not subject to base-catalyzed hydrolysis. To provide an alternative fluorescent derivative of theophylline for our studies with different wavelength maxima for excitation and fluorescence, 8-(5-aminopentyl)theophylline (11a) was also coupled with dansyl chloride [5-(dimethylamino)naphthalene-1-sulfonyl chloride] under aqueous basic conditions, although this reaction tolerated a higher pH (9.5-10.0), owing to the lower lability of dansyl chloride to hydrolysis. The dansylaminopentyltheophylline 14 was formed in good yield. The 1H NMR spectra of the fluorescein-theophylline derivatives 13a-d were examined in solution in deuteriodimethyl sulfoxide. The signals for the xanthene moiety show considerable symmetry, in which H-1′ and H-8′ are magnetically equivalent, as are H-2′ and H-7′ and H-4′ and H-5′, respectively. Most interestingly, the phenolic OH signal appears as a 2 H singlet at δ 10.2, confirming that the fluorescein is present in this solvent as the tautomer shown, rather than the lactone-opened carboxylate tautomer. Straightforward syntheses of a range of 8-[ω-amino(oxa)alkyl]theophyllines have been achieved, providing convenient access to fluorescent derivatives with applications in automated immunoassays for theophylline. Immunoassay. The initial requirement of the study was to determine the antibody-binding affinities of the fluorescent theophylline conjugates 13a-d, and it was sensible to carry out these experiments on the immobilized antibody within the FCFD by introducing the conjugates in solution to the FCFD in the presence and absence of excess theophylline. The FCFD reader was able to determine the relative intensity of surface fluo-

616 Bioconjugate Chem., Vol. 8, No. 4, 1997

rescence, and the antibody-bound fluorescence was determined by subtracting the nonspecific fluorescence, in the presence of excess theophylline, from the total fluorescence. Initial experiments were conducted to establish the equilibration time of the system, by recording the total fluorescence at 30 s intervals over a 30 min period. Binding equilibrium was achieved within approximately 10 min, and a standard incubation time of 20 min was adopted for further studies. Specifically bound fluorescence was plotted against concentration of each conjugate, according to the Langmuir equation, to allow determination of the binding affinities of 13a-d (Table 1). The binding affinity of theophylline could then be determined by competition with a fluorescent derivative, in this case with 13a (Table 1). The binding affinities of the conjugates compared well with that of theophylline, indicating that this antibody could tolerate substitution of theophylline at position 8. The affinities were almost independent of the length [(CH2)5 vs (CH2)9] or the hydrophobic/hydrophilic nature of the link [(CH2)9 vs CH2O(CH2)2O(CH2)2] between the theophylline hapten and the fluorophore. Compound 13a was identified by a small margin as being the conjugate of choice and was selected for further testing and development of assay procedures. Briefly, it was established that the fluorescent conjugates had the appropriate antibody-binding properties for development of an FCFD-based assay for theophylline, within a concentration range which would be useful for clinical use (0-300 µM theophylline). Compound 13a was used at a concentration of 10-6 M within the FCFD device, with low levels of interference from nonspecific surface-bound fluorescence. Theophylline competed with 13a such that the signal recorded by the FCFD reader decreased from maximum to minimum levels between theophylline concentrations of 5 and 500 µM. Precision was determined at theophylline concentrations of 25, 50, and 125 µM, the coefficients of variation being 6.5%, 6.0%, and 5.2% (within-assay) and 8.0%, 7.5%, and 5.0% (between-assay), respectively. This concentration range corresponded to a useful clinical range, although the precision of the assay in estimating the concentration of theophylline was compromised by insufficient total fluorescence. This total fluorescence could be increased by immobilizing a higher mass of antibody to the bottom plate of the FCFD. Nevertheless, the FCFD devices studied here would be capable of distinguishing between low (5 µM) and high (250 µM) clinical levels of theophylline, which could be useful in acute conditions to establish whether a patient is presenting with overdose or underdose of theophylline. Preliminary experiments were carried out to test the cross-reactivity of the system with other xanthines, including caffeine, theobromine, and metabolites of theophylline. Initial results suggested that the concentration of each xanthine required to displace 13a was at least 100 times greater than that for theophylline. Thus, little interference can be expected from other blood-borne xanthines. It was also established that the assay worked as well in the presence of 5 mg mL-1 human serum albumin, with a slight loss of sensitivity presumably due to interactions between xanthines and albumin, which reduced the effective concentration of free xanthines in the FCFD.

Yahioglu et al.

The results of further studies on the development and validation of the immunoassays will be reported in full elsewhere. ACKNOWLEDGMENT

We thank Dr. G. Robinson and Dr. J. Deacon (Serono Diagnostics) for helpful discussions, Mr. R. R. Hartell and Mr. D. Wood (University of Bath) for the NMR spectra, Mr. C. Cryer (University of Bath) for the mass spectra, and Serono Diagnostics for financial support. F.Y. held an EPRSC CASE studentship. LITERATURE CITED (1) Hendeles, L., and Weinberger, M. (1983) Theophylline: a “state of the art review”. Pharmacotherapy 3, 2. (2) Bierman, C. W., and Williams, P. V. (1989) Therapeutic monitoring of theophyllinesrationale and current status. Clin. Pharmacokinetics 17, 377. (3) Self T., and Abou-Shala, N. (1994) Theophylline. Lancet 343, 1226. (4) Danilova, N. D., and Vasilov, R. G. (1991) Production and characterisation of anti-theophylline monoclonal antibodies suitable for immunoassay. Immunol. Lett. 28, 79. (5) Neese, A. L., and Soyka, L. F. (1977) Development of a radioimmunoassay for theophylline. Application to studies in premature infants. Clin. Pharmacol. Ther. 21, 633. (6) Kester, M. B., Saccar, C. L., Rocci, M. L., and Mansmann, H. C. (1986) New simplified microassay for the quantitation of theophylline and its major metabolites in serum by high performance liquid chromatography. J. Chromatogr. 380, 99. (7) Badley, R. A., Drake, R. A. L., Shanks, I. A., and Smith, A. M. (1987) Optical biosensors for immunoassays: the fluorescence capillary-fill device. Philos. Trans. R. Soc. London 312, 143. (8) Parry, R. P., Love, C., and Robinson, G. A. (1990) Detection of rubella antibody using an optical immunosensor. J. Virol. Methods 27, 39. (9) Robinson, G. A. (1991) Optical immunosensing systemss meeting the market needs. Biosensors Bioelectronics 6, 183. (10) Deacon, J. K., Thomas, A. M., Page, A. L., Stops, J. E., Roberts, P. R., Whiteley, S. C., Attridge, J. W., Love, C. A., and Robinson, G. A. (1991) An assay for human chorionic gonadotrophin using the capillary fill immunosensor. Biosensors Bioelectronics 6, 193. (11) Jeno, K. (1978) Synthesis of 8-aminomethyl- and 8-aminoethyl-theophylline. Juhasz Gyula Tanarkepzo Foiskola Tud. Kozl. 133; Chem. Abstr. 93, 239365. (12) Fuchs, H., Gottlieb, M., and Pfleiderer, W. (1978) Purine, XIIsU ¨ ber die Cyclisierung von 4-Alkylamino-5-nitrosouracilen und die Synthese von 8-substitutierten Xanthinen und Bis(theophylline-8-yl)-alkan-Derivaten. Chem. Ber. 111, 982. (13) Goldner, H., Dietz, G., and Carstens, E. (1966) Eine neue Xanthin-Synthese. Liebigs Ann. Chem. 692, 134. (14) Mann, K. G., and Fish, W. W. (1972) Protein polypeptide chain molecular weights by gel chromatography in guanidinium chloride. Methods Enzymol. 26, 28. (15) Miller, L., Phillips, M., and Riesler, E. (1988) Polymerisation of actin modified with fluorescein isothiocyanate. Eur. J. Biochem. 174, 23. (16) Atwell, G. J., and Denny, W. A. (1984) Monoprotection of R,ω-alkanediamines with the benzyloxycarbonyl group. Synthesis, 1032.

BC9700648