Synthesis of a Novel [125I] Neonicotinoid Photoaffinity Probe for the

Dec 1, 1996 - column for isolation of Drosophila and Musca nAChRs. (6). The design and development of the neonicotinoid photoaffinity probe involved t...
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Bioconjugate Chem. 1997, 8, 7−14

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ARTICLES Synthesis of a Novel [125I]Neonicotinoid Photoaffinity Probe for the Drosophila Nicotinic Acetylcholine Receptor Bachir Latli, Motohiro Tomizawa, and John E. Casida* Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720-3112. Received August 12, 1996X

The insect nicotinic acetylcholine receptor (nAChR) is the target for the major insecticide imidacloprid (IMI) and for the first candidate photoaffinity probe described here. Addition to 1-[(6-chloro-3pyridinyl)methyl]-4,5-dihydro-2-nitromethylene-1H-imidazolidine (the nitromethylene analog of the nitroimine IMI) of formaldehyde and any one of several primary amines is known to give hexahydro8-nitroimidazo[1,2-c]pyrimidine derivatives. These imidazopyrimidines with a wide range of Nsubstituents were found to inhibit [3H]IMI binding to the Drosophila or Musca nAChR by 50% (IC50) at 0.7-38 nM. Esterification of the N-(2-hydroxyethyl) derivative with 2-azido-5-(trimethylstannyl)benzoic acid and then iododestannylation using Na125I and chloramine-T provide the candidate photoaffinity probe 6-[2-(2-azido-5-[125I]iodobenzoyl)ethyl]-1-[(6-chloro-3-pyridinyl)methyl]-1,2,3,5,6,7hexahydro-8-nitroimidazo[1,2-c]pyrimidine. This compound (unlabeled) has an IC50 of 8 nM for [3H]IMI binding in Drosophila head membranes, and the 125I-labeled photoaffinity probe labels only a 66 kDa protein(s) at a specific site inhibited by (-)-nicotine, consistent with the insecticide-binding subunit of the nAChR.

INTRODUCTION 1

The nicotinic acetylcholine receptor (nAChR ) is the target in insects for 1-[(6-chloro-3-pyridinyl)methyl]-4,5dihydro-2-nitroimino-1H-imidazolidine (imidacloprid or IMI) and many analogs usually with (6-chloro-3-pyridinyl)methylamino and nitro substituents (1-3). This new class of insecticides is referred to as chloronicotinyls (4) or neonicotinoids (2). [3H]IMI is the principal radioligand for characterizing and quantitating the insecticide binding site of the nAChR of Drosophila melanogaster (fruit fly) and Musca domestica (house fly) (5, 6) (Figure 1). The nitromethylene analog of IMI (CH-IMI) (2, 7-9) and an acyclic nitromethylene compound (nitenpyram) (6, 10, 11) (Figure 1) are of interest not only as potent insecticides but also as probes for exploring the structure of Drosophila and Musca nAChRs. The insect nAChR is best understood for Drosophila, for which the structural features are deduced from molecular biology approaches (12) and the same three subunits of 69, 66, and 61 kDa have been isolated by chromatography on affinity columns based on R-bunga* Author to whom correspondence should be addressed at the Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, 114 Wellman Hall, University of California, Berkeley, CA 947203112 [telephone (510) 642-5424; fax (510) 642-6497; e-mail [email protected]]. X Abstract published in Advance ACS Abstracts, December 1, 1996. 1 Abbreviations: R-BGT, R-bungarotoxin; CH-IMI, nitromethylene analog of IMI; DCC, 1,3-dicyclohexylcarbodiimide; DMAP, 4-(dimethylamino)pyridine; EI, electron impact; EtOAc, ethyl acetate; FAB-HRMS, fast atom bombardment high-resolution MS; FAB-LRMS, fast atom bombardment low-resolution MS; Hex, hexane; IC50, concentration of test compound for 50% inhibition of specific radioligand binding; IMI, imidacloprid; nAChR, nicotinic acetylcholine receptor.

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rotoxin (R-BGT) (a toxin from the elapidae snake Bungarus multicinctus, which is the classical competitive antagonist of the nAChR) and demethylnitenpyram (6). R-BGT cross-links with a protein(s) in Drosophila head membranes calculated to have a molecular mass of 42 kDa (13) and, as an azidosalicylate derivative, it photoaffinity labels a 66 kDa polypeptide in isolated Drosophila and Musca nAChRs (6). Although the neonicotinoids and R-BGT share the same binding site(s) in insects, on the basis of competition studies (1, 14), the site of insecticide binding is only partially defined with R-BGT since the molecular size is different, i.e. masses of 256 for IMI versus 8000 for R-BGT (3, 15). The photoaffinity probes used in characterizing the vertebrate (Torpedo electric organ) nAChR (16, 17) are not suitable for insects because of relatively low affinity or binding to a site not directly relevant for insecticidal activity (1, 11). The alternative is to specifically design a photoaffinity probe based on an IMI analog as in the case of our recent development of a nitenpyram analog as an affinity column for isolation of Drosophila and Musca nAChRs (6). The design and development of the neonicotinoid photoaffinity probe involved the following steps: first, establish a site in the neonicotinoid for substitution allowing retention of moderate to high receptor potency in Drosophila brain membrane preparation; second, optimize the probe with candidate photoreactive substituents; and finally, prepare the radiolabeled photoaffinity ligand and test for light-dependent and protectable labeling of one or more nicotinic acetylcholine receptor subunits. The overall chemistry utilized is shown in Figure 1. Nitenpyram analogs were examined because of their successful use in preparing an affinity column (6). More extensive studies were made on nitromethylene imidazolidines such as CH-IMI since they are converted to insecticidal hexahydronitroimidazopyrimidines on ad© 1997 American Chemical Society

8 Bioconjugate Chem., Vol. 8, No. 1, 1997

Latli et al.

Figure 1. Structures of neonicotinoids including the radioligand ([3H]IMI), analogs (CH-IMI and nitenpyram), derivatives, and the photoaffinity probe ([125I]-14). Receptor potencies are indicated as concentrations for 50% inhibition (IC50) of the displacement of [3H]IMI binding from Drosophila head membranes (data from this paper and ref 6).

dition of formaldehyde and a primary amine (18, 19). We find that fortunately the imidazopyrimidines derived from CH-IMI retain much of the affinity at the receptor of the parent compound and therefore allow the introduction of a variety of substituents in optimizing a candidate photoaffinity probe. Optimization studies ultimately led to synthesis of 6-[2-(2-azido-5-[125I]-iodobenzoyl)ethyl]-1[(6-chloro-3-pyridinyl)methyl]-1,2,3,5,6,7-hexahydro-8-nitroimidazo[1,2-c]pyrimidine ([125I]-14) (Figure 1) as a candidate photoaffinity probe for the Drosophila nAChR. EXPERIMENTAL PROCEDURES

Materials. Silica gel TLC for analysis was performed with precoated plastic sheets (4 × 8 cm, 0.25 mm gel layer) with fluorescent indicator (Polygram R SILG/ UV254, Macherey-Nagel, Germany) and for preparative purposes with precoated silica gel GF plates (20 × 20 cm, Analtech). NMR spectra were recorded for CDCl3 solutions with a Bruker AM-300 or AM-400 spectrometer. Chemical shifts (δ in parts per million) are reported for 1 H at 300 Hz or 400 MHz and for 13C at 75 MHz relative to internal tetramethylsilane and CDCl3, respectively. Mass spectra were acquired by GC/MS with a HewlettPackard 5971A or 5985B instrument in the electron impact (EI) mode (70 eV, 200 °C). Fast atom bombardment (FAB)-MS (both low and high resolution) was conducted with the Fisons ZAB2-EQ spectrometer. UV absorbances were measured on a Hewlett-Packard 8452A diode array spectrophotometer. Photoreactivity of the candidate photoaffinity probe was determined for a solution in absolute ethanol in a 1 cm quartz cell positioned 3 cm from four 350 nm lamps in a Rayonet photochemical reactor (The Southern New England Ultraviolet Co., Hamden, CT). Melting points are uncorrected and were recorded on a Fisher-Johns melting point apparatus. All reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI) except EDTA, (-)nicotine, ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′tetraacetic acid, lithium dodecyl sulfate, and phenylmethanesulfonyl fluoride, which were obtained from Sigma Chemical Co. (St. Louis, MO). All solvents used were of reagent or HPLC grade. THF was distilled from sodium benzophenone under nitrogen in a recirculating still, with a deep blue color maintained in the distillation pot. Dioxane was dried by storage over molecular seives.

Literature procedures were used to prepare 1-[(6-chloro3-pyridinyl)methyl]-1-(ethylamino)-1-(methylthio)-2-nitroethene (6) and CH-IMI (7, 8). [3H]IMI at 25 Ci/mmol was prepared in this laboratory (5). L-[N-methyl-3H]Nicotine ([3H]nicotine) at 78 Ci/mmol and Na125I at 584 MBq 125I/µg of iodine (∼2000 Ci/mmol) in dilute NaOH solution were purchased from DuPont NEN Research Products (Boston, MA) and Amersham Life Science Inc. (Arlington Heights, IL), respectively. Molecular mass markers were obtained from Bio-Rad (Richmond, CA). Liquid scintillation counting was performed with a Beckman LS 60001C, using High Flash Point Cocktail, SafetySolve, Research Products International Corp. (Mount Prospect, IL). Each intermediate and candidate probe was >98% pure on the basis of TLC and 1H and 13C NMR integrations. Compounds with photosensitive substituents were used in subdued light. Synthesis of Azidohydroxybenzoyl (1-4), Azidoiodobenzoyl (5-7), and Azidotrimethylstannylbenzoyl (8, 9) Intermediates (Scheme 1). 4-Azido2-hydroxybenzoic Acid (2). To a solution of 4-amino-2hydroxybenzoic acid (1) (4.6 g, 30 mmol) in concentrated HCl (70 mL, 12 N) was added dropwise and with care at 0 °C a solution of sodium nitrite (6.21 g, 90 mmol) in cold water (30 mL). The mixture was stirred for 30 min before a solution of sodium azide (10 g, 155 mmol) in cold water (50 mL) was added slowly and dropwise. The resulting suspension was stirred at 0 °C for 3 h and then filtered, washed with cold water, and dried under vacuum to give 4.6 g of 2 as a brownish solid in 86% yield: mp ) 165167 °C; 1H NMR (CDCl3) δ 7.88 (d, J ) 8.03 Hz, 1H), 6.62 (d, J ) 8.03 Hz, 1H), 6.60 (s, 1H); 13C NMR (CDCl3) δ 172.82, 164.47, 148.47, 133.26, 111.17, 110.83, 107.74. N-Hydroxysuccinimidyl-4-azido-2-hydroxybenzoate (3). To a stirred solution of 2 (2.33 g, 13 mmol) in dry THF (30 mL) was added N-hydroxysuccinimide (2.07 g, 18 mmol) at 0 °C followed by a solution of DCC (3.71 g, 18 mmol) in THF (2.0 mL), and the resulting mixture was stirred under nitrogen atmosphere for 6 h. It was then filtered and concentrated in vacuo to give 5.2 g of crude material. Purification by silica gel flash chromatography using CHCl3 as eluent gave 3.6 g of product as a white solid in 100% yield: mp ) 140-142 °C; Rf ) 0.73 in MeOH/CHCl3, 10:90; 1H NMR (CDCl3) δ 9.97 (d, J ) 8.64 Hz, 1H), 9.68 (s, 1H, OH), 6.68 (d, J ) 2.10 Hz, 1H), 6.62

Bioconjugate Chem., Vol. 8, No. 1, 1997 9

Photoaffinity Probe for Acetylcholine Receptor Scheme 1. Preparation Intermediatesa

of

Azidohydroxybenzoyl,

Azidoiodobenzoyl,

and

Azidotrimethylstannylbenzoyl

a (a) HCl, NaNO , NaN , 86-90%; (b) NHS, DCC, THF, 100%; (c) H N(CH ) NH , CH CN, THF, 91%; (d) MeOH, H SO , 100%; (e) 2 3 2 2 2 2 3 2 4 Pd(PPh3)4, Me3Sn-SnMe3 , dioxane, 67%; (f) LiOH, THF, 51%.

(dd, J ) 2.10, 8.64 Hz, 1H), 2.93 (brs, 4H); 13C NMR (CDCl3) δ 169.22, 163.83, 162.81, 149.32, 131.60, 111.11, 107.10, 104.62, 25.30. N-(4-Azido-2-hydroxybenzoyl)ethylenediamine (4). A solution of 3 (1.1 g, 4.0 mmol) in dry THF (10 mL) was stirred at 0 °C under nitrogen atmosphere. Ethylenediamine (0.4 mL, 6.0 mmol) was added in acetonitrile (5.0 mL), and the resulting mixture was stirred overnight in the dark. The mixture was concentrated in vacuo and purified by preparative TLC (2.0 mm thickness plate) to give 0.8 g of 4 as a yellowish oil in 91% yield: Rf ) 0.1 in MeOH/CHCl3, 10:90; 1H NMR (CDCl3) δ 7.73 (d, J ) 8.61 Hz, 1H), 6.54 (d, J ) 2.25 Hz, 1H), 6.48 (dd, J ) 2.25, 8.61 Hz, 1H), 3.49 (t, J ) 5.88 Hz, 2H), 2.91 (t, J ) 5.88 Hz, 2H), 2.74 (brs, NH2, NH); 13C NMR (CDCl3) δ 169.53, 165.1, 144.51, 129.61, 113.63, 108.51, 107.48, 41.26, 39.20; FAB-LRMS MH+(10%), M+(75%), 192(100%); FABHRMS C9H11N5O2H+, calcd 222.0991, found 222.0941. 2-Azido-5-iodobenzoic Acid (6). To a solution of 2-amino5-iodobenzoic acid (5) (5.26 g, 20 mmol) in concentrated HCl (60 mL, 12 N), stirred at 0 °C, was added a solution of NaNO2 (4.14 g, 60 mmol) in water (25 mL). The resulting mixture was stirred for 30 min before NaN3 (6.5 g, 100 mmol) in cold water (30 mL) was added dropwise, and the mixture was stirred overnight. It was then poured into ice and the solid was filtered and dried in vacuo to give 5.2 g of 6 as a cream colored solid in 90% yield: mp ) 118-120 °C; Rf ) 0.75 in MeOH/CHCl3, 10: 90; 1H NMR (CDCl3) δ 8.22 (d, J ) 2.21 Hz, 1H), 7.75 (dd, J ) 2.21, 8.61 Hz, 1H), 6.95 (d, J ) 8.61 Hz, 1H); 13C NMR (CDCl ) δ 169.31, 142.12, 141.12, 139.98, 3 123.72, 121.55, 87.50. Methyl 2-Azido-5-iodobenzoate (7). To a solution of 6 (1.0 g, 3.46 mmol) in MeOH (50 mL) was added a few drops of concentrated H2SO4 (95-98%), and the mixture was stirred under reflux overnight. After the mixture cooled to room temperature, the solvent was evaporated and the residue was diluted in EtOAc. Aqueous workup with NaHCO3 (5% solution), drying (MgSO4), filtration, and concentration in vacuo followed by purification by silica gel flash chromatography using CHCl3 as eluent gave 1.04 g (100% yield) of a solid as white needles: mp ) 68-69 °C; Rf ) 0.68 in EtOAc/Hex, 20:80; 1H NMR (CDCl3) δ 8.15 (d, J ) 2.12 Hz, 1H), 7.80 (dd, J ) 2.12, 8.62 Hz, 1H), 6.98 (d, J ) 8.62 Hz, 1H), 3.90 (s, 3H); 13C NMR (CDCl3) δ 163.83, 141.50, 140.08, 139.62, 123.86, 121.50, 87.15, 52.32. Methyl 2-Azido-5-(trimethylstannyl)benzoate (8). A mixture of 7 (130 mg, 0.43 mmol) and tetrakis(triphenylphosphine)palladium(0) (10 mg, 8.65 µmol) in anhydrous dioxane (5.0 mL) was made anaerobic by freezing, vacuum degassing, and introducing argon at-

mosphere three times at 0 °C. Then hexamethylditin (0.51 mL) was added, and the resulting mixture was stirred at 50 °C for 2 h. After the mixture cooled to room temperature, a saturated solution of NH4Cl was added and the organic layer was extracted with EtOAc, dried (MgSO4), filtered, and concentrated in vacuo. The pure product was isolated as a yellowish oil by silica gel flash chromatography using EtOAc in Hex (0-10%) as eluent to give 100 mg in 67% yield: Rf ) 0.78 in EtOAc/Hex, 20:80; 1H NMR (CDCl3) δ 7.87 (d, J ) 1.16 Hz, 1H), 7.58 (dd, J ) 1.16, 7.77 Hz, 1H), 7.17 (d, J ) 7.77 Hz, 1H), 3.87 (s, 3H), 0.27 (s, 9H); 13C NMR (CDCl3) δ 166.30, 140.35, 139.84, 138.61, 138.50, 122.17, 120.40, 52.30, -9.23; EI-MS max mass for C11H15SnN3O2 as 120Sn isotope, 341 (100%); EI-HRMS calcd 341.0276, found 341.0193 (M+ for tin and carbon isotopes pattern observed was similar to the one calculated). 2-Azido-5-(trimethylstannyl)benzoic Acid (9). To a solution of 8 (57 mg, 0.17 mmol) in THF (0.5 mL) was added a solution of LiOH (1 N solution, 0.34 mL). The mixture was stirred at 50 °C overnight and then cooled to room temperature and brought to pH ∼7.0 by adding dropwise a potassium biphthalate buffer (0.05 M, pH 4). The aqueous fraction was extracted with CHCl3, dried (MgSO4), filtered, and concentrated in vacuo. Purification by silica gel flash chromatography using 3:1 Hex/ EtOAc containing 2% acetic acid gave 28 mg of the desired acid as a yellowish solid: mp ) 126-128 °C; Rf ) 0.16 in EtOAc/Hex, 20:80; 1H NMR (CDCl3) δ 8.15 (d, J ) 2.1 Hz, 1H), 7.72 (dd, J ) 2.1, 7.73 Hz, 1H), 7.21 (d, J ) 7.73 Hz, 1H), 0.32 (s, 9H); 13C NMR (CDCl3) δ 169.21, 140.08, 139.95, 138.32, 132.98, 124.68, 119.03, -9.13. Synthesis of Azidohydroxybenzoyl and Azidoiodobenzoyl Candidate Hexahydronitroimidazopyrimidine Photoaffinity Probes (10-14) (Scheme 2). 6-[2(4-Azido-2-hydroxybenzamidyl)ethyl]-1-[(6-chloro-3pyridinyl)methyl]-1,2,3,5,6,7-hexahydro-8-nitroimidazo[1,2c]pyrimidine (10). To a solution of CH-IMI (127 mg, 0.50 mmol) and formaldehyde (85 µL, 37% solution in water) in THF (5.0 mL) was added the diamine derivative (4) (110 mg, 0.50 mmol) in THF (5.0 mL), and the resulting mixture was stirred at room temperature overnight. Concentration in vacuo followed by purification by preparative TLC (2.0 mm thickness plate) gave 73 mg of the desired product as a yellowish solid in 29% yield: mp ) 172-174 °C; Rf ) 0.63 in MeOH/CHCl3, 10:90; 1H NMR (CDCl3) δ 8.33 (d, J ) 2.4 Hz, 1H), 7.82 (dd, J ) 2.4, 8.2 Hz, 1H), 7.74 (d, J ) 8.2 Hz, 1H), 7.33 (d, J ) 8.61 Hz, 1H), 6.54 (d, J ) 2.25 Hz, 1H), 6.44 (dd, J ) 2.25, 8.61 Hz, 1H), 4.80 (s, 2H), 4.13 (s, 2H), 3.85 (s, 2H), 3.403.74 (m, 6H), 2.75 (t, J ) 5.88 Hz, 2H); 13C NMR (CDCl3) δ 169.94, 163.71, 158.41, 151.23, 149.37, 145.00, 139.93,

10 Bioconjugate Chem., Vol. 8, No. 1, 1997 Scheme 2. Preparation of Azidohydroxybenzoyl imidazopyrimidine Photoaffinity Probesa

Latli et al. and

Azidoiodobenzoyl

Candidate

Hexahydronitro-

a Structures of intermediates are given in Scheme 1. (a) HCHO, THF, 29%; (b) HCHO, H N(CH ) OH, THF, 96%; (c) DCC, DMAP, 2 2 2 CH2Cl2, 60-94%; (d) Na125I, chloramine-T, MeOH, 20%.

131.54, 130.68, 125.02, 109.68, 107.44, 107.22, 103.66, 65.89, 50.88, 47.62, 37.86, 52.54, 52.18, 51.90; FAB-LRMS MH+ (60%), 154 (100%); FAB-HRMS C21H22ClN9O4H+, calcd 500.1561, found 500.1551; UV λ270  ) 20 290, λ348  ) 19 380. 1-[(6-Chloro-3-pyridinyl)methyl]-1,2,3,5,6,7-hexahydro6-(2-hydroxyethyl)-8-nitroimidazo[1,2-c]pyrimidine (11). To a solution of CH-IMI (254 mg, 1.0 mmol) in THF (20 mL) was added a solution of formaldehyde (0.153 mL, 37% solution in water) and ethanolamine (0.06 mL, 1.0 mmol). The resulting mixture was stirred overnight, concentrated in vacuo, and purified by preparative TLC (2.0 mm thickness plate) to give 312 mg of the desired product as a white solid in 96% yield: mp ) 146-148 °C; Rf ) 0.26 in MeOH/CHCl3, 10:90; 1H NMR (CDCl3) δ 8.34 (d, J ) 2.45 Hz, 1H), 7.86 (dd, J ) 2.45, 8.26 Hz, 1H), 7.34 (d, J ) 8.26 Hz, 1H), 4.84 (s, 2H), 4.11 (s, 2H), 3.88 (s, 2H), 3.73 (t, J ) 5.16 Hz, 2H), 3.62 (m, 4H), 2.73 (t, J ) 5.16 Hz, 2H); 13C NMR (CDCl3) δ 157.45, 151.01, 149.07, 139.18, 130.76, 124.41, 102.97, 65.92, 59.63, 55.04, 52.17, 51.03, 49.29, 46.69; FAB-LRMS MH+ (26%), MLi+(346, 12%); FAB-HRMS C14H18ClN5O3H+, calcd 340.1176, found 340.1169. 6-[2-(4-Azido-2-hydroxybenzoyl)ethyl]-1-[(6-chloro-3pyridinyl)methyl]-1,2,3,5,6,7-hexahydro-8-nitroimidazo[1,2-c]pyrimidine (12). To a solution of 11 (68 mg, 0.20 mmol) in dry CH2Cl2 (12 mL) was added acid 2 (44 mg, 0.25 mmol), DMAP (30 mg, 0.25 mmol), and then DCC (66 mg, 0.32 mmol) in CH2Cl2 (2.0 mL). The resulting mixture was stirred overnight, concentrated in vacuo, and purified using preparative TLC (2.0 mm thickness plate) to give 94 mg of the product in 94% yield as a yellowish solid: mp ) 142-144 °C; Rf ) 0.7 in MeOH/ CHCl3, 10:90; 1H NMR (CDCl3) δ 8.34 (d, J ) 2.40 Hz, 1H), 7.83 (m, 2H), 7.32 (d, J ) 8.2 Hz, 1H), 6.62 (d, J ) 2.10 Hz, 1H), 6.56 (dd, J ) 2.10, 8.59 Hz, 1H), 4.83 (s, 2H), 4.49 (t, J ) 5.55 Hz, 2H), 4.16 (s, 2H), 3.95 (s, 2H), 3.61 (m, 4H), 2.96 (t, J ) 5.55 Hz, 2H); 13C NMR (CDCl3) δ 169.21, 163.35, 157.28, 150.01, 149.06, 147.66, 139.24, 131.55, 130.58, 124.53, 110.66, 107.54, 107.24, 102.97, 65.97, 57.02, 52.21, 51.93, 50.83, 49.26, 46.51; FAB-LRMS MH+(66%), 154 (100%); FAB-HRMS C21H21ClN8O5H+, calcd 501.1402, found 501.1393; UV λ272  ) 24 020, λ352  ) 13 790. 6-[2-(2-Azido-5-trimethylstannylbenzoyl)ethyl]-1-[(6chloro-3-pyridinyl)methyl]-1,2,3,5,6,7-hexahydro-8-nitroim-

idazo[1,2-c]pyrimidine (13). Acid 9 (17 mg, 0.052 mmol), alcohol 11 (17 mg, 0.050 mmol), and DMAP (8 mg, 0.065 mmol) in dry CH2Cl2 (3.0 mL) were stirred at room temperature under nitrogen atmosphere. Then DCC (16 mg, 0.077 mmol) was added in CH2Cl2 (0.50 mL), the mixture was stirred overnight and concentrated in vacuo, and the residue was purified by preparative TLC (1.0 mm thickness plate) using MeOH/CHCl3, 10:90, as eluent to give 10 mg of pure product as a yellowish solid in 31% yield: mp ) decomposition at 130 °C; Rf ) 0.73 in MeOH/ CHCl3, 10:90; 1H NMR (CDCl3) δ 8.34 (d, J ) 2.35 Hz, 1H), 7.91 (d, J ) 1.25 Hz, 1H), 7.86 (dd, J ) 2.46, 8.24 Hz, 1H), 7.64 (dd, J ) 1.25, 7.86 Hz, 1H), 7.31 (d, J ) 8.24 Hz, 1H), 7.23 (d, J ) 7.86 Hz, 1H), 4.83 (s, 2H), 4.47 (t, J ) 5.75 Hz, 2H), 4.12 (s, 2H), 3.96 (s, 2H), 3.62 (m, 4H), 2.97 (t, J ) 5.75 Hz, 2H), 0.32 (s, 9H); 13C NMR (CDCl3) δ 165.84, 159.28, 151.03, 149.13, 140.71, 139.84, 139.34, 138.85, 138.71, 130.67, 124.59, 121.47, 119.27, 103.18, 65.53, 63.10, 52.24, 52.05, 51.93, 49.22, 46.54, -9.34; FAB-LRMS C24H29ClN8O4SnH+ (649, 70%), 267 (100%); FAB-HRMS max MH+ peak for 120Sn isotope calcd 649.1101, found 649.1104. 6-[2-(2-Azido-5-iodobenzoyl)ethyl]-1-[(6-chloro-3-pyridinyl)methyl]-1,2,3,5,6,7-hexahydro-8-nitroimidazo[1,2-c]pyrimidine (14) from 11. A solution of alcohol 11 (68 mg, 0.20 mmol) in dry CH2Cl2 (12 mL) was stirred at room temperature under nitrogen atmosphere. Then acid 6 (58 mg, 0.20 mmol) and DMAP (30 mg, 0.25 mmol) were added followed by DCC (66 mg, 0.32 mmol) in dry CH2Cl2 (2.0 mL). The resulting mixture was stirred overnight and then concentrated in vacuo. The solid residue was purified by preparative TLC to give 110 mg of a yellowish solid in 91% yield (decomposed at 160 °C): Rf ) 0.57 in MeOH/CHCl3, 10:90; 1H NMR (CDCl3) δ 8.20 (d, J ) 2.17 Hz, 1H), 8.14 (d, J ) 2.11 Hz, 1H), 7.88 (dd, J ) 2.11, 8.23 Hz, 1H), 7.82 (dd, J ) 2.17, 8.49 Hz, 1H), 7.33 (d, J ) 8.23Hz, 1H), 7.00 (d, J ) 8.49 Hz, 1H), 4.84 (s, 2H), 4.46 (t, J ) 5.77 Hz, 2H), 4.09 (s, 2H), 3.96 (s, 2H), 3.61 (m, 4H), 2.94 (t, J ) 5.77 Hz, 2H); 13C NMR (CDCl3) δ 169.78, 163.68, 157.43, 151.22, 149.10, 142.03, 140.42, 140.32, 139.25, 130.65, 124.51, 121.76, 103.00, 87.40, 65.57, 63.24, 52.20, 51.74, 50.94, 49.21, 46.51; FAB-LRMS MH+ (58%), 225 (100%), 154 (30%); FABHRMS C21H20ClIN8O4H+, calcd 611.0419, found 611.0414; UV λ268  ) 18 600; λ352  ) 14 300.

Photoaffinity Probe for Acetylcholine Receptor

Bioconjugate Chem., Vol. 8, No. 1, 1997 11

Scheme 3. Preparation of Nitenpyram Analogs Including an Agarose Derivative as an Affinity Columna

a

(a) EtOH, reflux, 29%; (b) H2N(CH2)5OH, EtOH, reflux, 30%; (c) 2-hydroxybenzoic acid, DCC, DMAP, CH2Cl2, 44%.

Synthesis of 14 from 13. The method was validated by preparing unlabeled 14. Thus, to a solution of 13 (1.3 mg, 2 µmol of 1.0 mg/mL solution in MeOH) were added NaI (0.3 mg, 2 µmol, 3 mg/mL solution in MeOH) and then chloramine-T (0.5 mg, 2 µmol, 0.1 mL of a solution of 5 mg/mL in 200 mM phosphate buffer, pH 7.5). The resulting mixture was stirred for 10 min, the solvent evaporated under nitrogen, and the product taken up in CHCl3. 1H NMR data were identical to those of 14, prepared via intermediates 11 and 6, and comigrated with it on TLC. Radiosynthesis of [125I]-14 from 13. The above procedure was then used in the preparation of [125I]-14. Thus, to a solution of Na125I (5.6 mCi, 11 µL) in dilute NaOH solution (pH 9.0) was injected the organotin precursor (13) (15 µL, 0.1 mg/mL MeOH solution), followed by chloramine-T (6.0 µL, 0.1 mg/ mL 200 mM phosphate buffer solution, pH 7.5). The resulting mixture was shaken occasionally for 10 min before transfer to a vial for evaporation of the solvents under nitrogen. The residue was then dissolved in CHCl3 and transferred to another vial, leaving insoluble materials behind. The 125I-labeled product (1.0 mCi) cochromatographed with unlabeled 14 on TLC with the MeOH/CHCl3 system above as observed by UV light and PhosphorImager (Molecular Dynamics, Sunnyvale, CA) detection. Synthesis of Nitenpyram Analogs (15-19) (Scheme 3). 1-[(6-Chloro-3-pyridinyl)methyl]-1-(ethylamino)-1-[2(2-hydroxybenzamidyl)ethyl]amino-2-nitroethene (17) via 1-[(6-Chloro-3-pyridinyl)methyl]-1-(ethylamino)-1-(methylthio)-2-nitroethene (15) and N-(2-Hydroxybenzoyl)ethylenediamine (16). A solution of 15 (100 mg, 0.35 mmol) and 16 (60 mg, 0.33 mmol, prepared similarly to intermediate 4 in Scheme 1) in EtOH (5.0 mL) was refluxed for 2 h and then concentrated in vacuo and purified by preparative TLC (1.0 mm layer thickness) to give 20 mg of product as a white solid (decomposed at 160 °C) in 29% yield: Rf ) 0.5 in MeOH/CHCl3, 10:90; 1H NMR (CDCl3) δ 8.23 (d, J ) 2.52 Hz, 1H), 7.72 (dd, J ) 1.75, 7.92 Hz, 1H), 7.52 (dd, J ) 2.52, 8.25 Hz, 1H), 7.35 (s, OH), 7.30 (dt, J ) 1.75, 8.29 Hz, 1H), 7.23 (d, J ) 8.25 Hz, 1H), 6.83 (d, J ) 8.29 Hz, 1H), 6.72 (t, J ) 7.92 Hz, 1H), 6.49 (s, 1H), 4.48 (s, 2H), 3.76 (brs, 4H), 3.61 (m, 4H), 3.27 (q, J ) 7.13 Hz, 2H), 1.16 (t, J ) 7.13 Hz, 3H); 13C NMR (CDCl3) δ 170.75, 163.59, 160.39, 150.66, 148.02, 137.98, 133.37, 130.11, 128.34, 128.13, 124.51, 119.16, 116.34,

101.01, 49.87, 46.47, 44.86, 38.84, 12.0; FAB-LRMS MH+ (420, 20%), 164 (100%); FAB-HRMS C19H22ClN5O4H+, calcd 420.1439, found 420.1448. 1-[(6-Chloro-3-pyridinyl)methyl]-1-(ethylamino)-1-(5hydroxypentyl)amino-2-nitroethene (18). The above methylthio derivative (15) (144 mg, 0.50 mmol) was refluxed in EtOH (5.0 mL) with 5-amino-1-pentanol (53 mg, 0.51 mmol) for 2 h and then concentrated in vacuo. Purification by preparative TLC (1.0 mm thickness plate) gave 51 mg of a white solid in 30% yield: Rf ) 0.67 in MeOH/CHCl3, 20:80; 1H NMR (CDCl3) δ 9.69 (s, 1H, NH), 8.30 (d, J ) 2.40 Hz, 1H), 7.53 (dd, J ) 2.40, 8.22 Hz, 1H), 7.36 (d, J ) 8.22 Hz, 1H), 6.51 (s, 1H), 4.33 (s, 2H), 3.64 (t, J ) 7.73 Hz, 2H), 3.37 (m, 2H), 3.13 (q, J ) 7.04 Hz, 2H), 1.45-1.82 (m, 8H), 1.19 (t, J ) 7.04 Hz, 3H); 13 C NMR (CDCl3) δ 150.23, 148.77, 143.32, 137.97, 130.25, 124.65, 104.28, 62.18, 50.00, 45.90, 44.88, 31.98, 29.91, 23.00, 12.18. 1-[(6-Chloro-3-pyridinyl)methyl]-1-(ethylamino)-1-[5-(2hydroxybenzoyl)pentyl]amino-2-nitroethene (19). To the above alcohol (18) (17 mg, 0.050 mmol) in dry DMF (0.50 mL) was added 2-hydroxybenzoic acid (7 mg, 0.053 mmol), DMAP (6.1 mg, 0.050 mmol), and DCC (10.3 mg, 0.050 mmol) in DMF (0.50 mL). The mixture was stirred overnight and then concentrated in vacuo and purified by preparative TLC to give 13 mg of 19 as a white solid in 44% yield: mp ) 190-192 °C; Rf ) 0.67 in MeOH/ CHCl3, 10:90; 1H NMR (CDCl3) δ 9.68 (s, 1H), 8.28 (d, J ) 2.32 Hz, 1H), 7.83 (d, J ) 7.97 Hz, 1H), 7.46 (m, 2H), 7.34 (d, J ) 8.15 Hz, 1H), 6.97 (d, J ) 7.97 Hz, 1H), 6.90 (t, J ) 8.15 Hz, 1H), 6.51 (s, 1H), 4.34 (t, J ) 6.12 Hz, 2H), 4.32 (s, 2H), 3.34 (t, J ) 6.12 Hz, 2H), 3.10 (q, J ) 7.03 Hz, 2H), 1.42-1.82 (m, 8H), 1.17 (t, J ) 7.03 Hz, 3H); 13C NMR (CDCl3) δ 170.35, 162.32, 160.81, 156.38, 151.72, 148.52, 137.98, 135.61, 130.25, 129.73, 124.60, 119.10, 117.38, 103.82, 64.69, 49.11, 45.81, 44.71, 29.64, 28.05, 23.14, 12.13; FAB-LRMS MH+ (80%), 225 (100%); FAB-HRMS C22H27ClN4O5H+, calcd 463.1748, found 463.1752. Receptor Assays. Binding studies with Drosophila and Musca head membranes (6) involved ∼300 µg of protein incubated with 2.5 nM [3H]IMI (25 Ci/mmol) for 60 min at 25 °C in a total volume of 250 µL of 10 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCl, 5 mM EDTA, 3 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 0.1 mM phenyl-

12 Bioconjugate Chem., Vol. 8, No. 1, 1997

methanesulfonyl fluoride, and 0.02% NaN3. Alternatively, rat whole brain membranes (20) (∼800 µg of protein) were incubated for 10 min at 25 °C with 5 nM [3H]nicotine (78 Ci/mmol) in 250 µL of 20 mM Tris buffer, pH 7.4, containing 118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, and 1 mM EDTA. The incubated mixtures were rapidly filtered on Whatman GF/B filters presoaked in 0.1% polyethylenimine. The filter was rinsed twice (for rat) or three times (for insects) with 2.5 mL of ice-cold 0.9% NaCl. Radioactivity remaining on the filter was determined by liquid scintillation counting. Specific binding was defined as the difference in radioactivity in the absence or presence of 25 µM unlabeled IMI (for [3H]IMI binding) or 10 µM unlabeled (-)-nicotine (for [3H]nicotine binding). IC50 values were calculated by iterative nonlinear least-squares regression. Photoaffinity Labeling. The procedure was similar to that we used before with the azidosalicylate derivative of [125I]-R-BGT and irradiation at 350 nm (6). Drosophila head membranes photoaffinity labeled with ∼4 nM [125I]14 (∼2000 Ci/mmol) in the absence or presence of 10 mM (-)-nicotine were separated by lithium dodecyl sulfatepolyacrylamide gel electrophoresis. The radioactive band(s) was detected by PhosphorImager analysis and compared with molecular mass markers on the same gel. RESULTS

Synthesis of Hexahydronitroimidazopyrimidines (Figure 1; Schemes 1 and 2). Reaction of an enamine, possessing an NH substituent, with suitable biselectrophiles leads to heterocyclization by bond formations at both nucleophilic sites (21). The desired product in the present study is obtained by stirring CH-IMI with a primary amine and formaldehyde in THF. The benzoyl derivatives 10 and 12 were initially examined because of extensive background on their use in the preparation of 125I-labeled ligands (22, 23). Synthesis included the preparation, on a multigram scale, of N-hydroxysuccinimidyl-4-azidosalicylic acid (3) (available commercially from Pierce) (22). Compound 3 on treatment with ethylenediamine gave amino analog 4, which upon coupling with CH-IMI in the presence of formaldehyde gave the penultimate intermediate (10). This step, however, was very sluggish; the yields were improved greatly by using the benzoyl rather than the benzamidyl series. Ethanolamine is preferred over ethylenediamine in coupling with CH-IMI because the diamine can react with formaldehyde and give many cyclic products (24). Thus, ethanolamine and formaldehyde were reacted directly with CH-IMI to give the alcohol (11) in 96% yield (19) and then coupled to 4-azidosalicylic acid (2) using DCC in 94% yield. Iodination of 12 failed to give a clean reaction, and HPLC would be needed to isolate the product from the monoiodo and diiodo compounds and the starting material to determine the specific activity (25). Synthesis of [125I]Azidobenzoyl Photoaffinity Probe (Schemes 1 and 2). Iodine-125 can be easily introduced by destannylation of compound 13 in the presence of chloramine-T in methanol (26). Thus , 2-azido-5-iodobenzoic acid (6) was prepared in 90% yield from the amino precursor (5) and then coupled to the alcohol derivative (11) with DCC in 91% yield to give 14. This compound has suitable photoreactivity with a halflife of