2'-substituted analogs of cocaine: synthesis and dopamine transporter binding potencies.

A series of 2'-substituted cocaine analogs (4-8) was prepared and evaluated in an in vitro dopamine transporter (DAT) binding assay. Compounds 4-7 were prepared by esterifying the 3 beta-hydroxyl group of ecgonine methyl ester (3) using the appropriate acid chloride in the presence of Et3N and benzene. Compound 3 was obtained from cocaine (1) by hydrolysis using 1N HCl to afford ecgonine.HCl which was subjected to acid catalyzed esterification using methanol saturated with HCl gas. Compound 8 was obtained by hydrogenation of 7 using H2/Pd-C. The IC50 values were calculated from displacement experiment of the radioligand [3H]WIN-35,428 (2). 2'-Aminococaine (8) showed high binding affinity to the DAT (14- and 1.3-fold more active than cocaine and the radioligand 2, respectively). These results, along with previous results, emphasize the importance of a hydrogen-bond donor group at the 2'-position of cocaine to enhance binding affinity to the DAT.

Synthesis and biological activity of a photoaffinity etoposide probe.

The epipodophyllotoxin etoposide is a potent and widely used anticancer drug that targets DNA topoisomerase II. The synthesis, photochemical, and biological testing of a photoactivatable aromatic azido analogue of etoposide also containing an iodo group is described. This azido analogue should prove useful for identifying the etoposide interaction site on topoisomerase II. Irradiation of the azido analogue and an aldehyde-containing azido precursor with UV light produced changes in their UV--visible spectra that were consistent with photoactivation. The azido analogue strongly inhibited topoisomerase II and inhibited the growth of Chinese Hamster Ovary cells. Azido analogue-induced topoisomerase II--DNA covalent complexes were significantly increased subsequent to UV irradiation of drug-treated human leukemia K562 cells as compared to etoposide-treated cells. These results suggest that the photoactivated form of etoposide is a more effective topoisomerase II poison either by interacting directly with the enzyme or with DNA subsequent to topoisomerase II-mediated strand cleavage.

Design and synthesis of isoxazole containing bioisosteres of epibatidine as potent nicotinic acetylcholine receptor agonists.

An efficient synthesis of isoxazole containing isosteres of epibatidine is described. The synthesis proceeded from N-tert-butoxycarbonyl (Boc)-exo-2-(methoxycarbonyl)-7-azabicyclo[2.2.1]heptane (9). Compound 9 was reacted with the dilithium salt of an appropriately substituted oxime in tetrahydrofuran (THF). Cyclodehydration of the resultant beta-keto oxime and deprotection of the N-Boc group in 5 N aqueous HCl afforded the isoxazole containing isosteres of epibatidine (6-8). The binding affinities of these compounds were determined at the nicotinic acetylcholine receptor for the displacement of [3H]cystisine. The unsubstituted isoxazole containing isostere (6) showed the lower binding potency compared to the 3'-methylisoxazole isostere (7). Substitution with a phenyl group at the 3'-position of the isoxazole significantly reduced the binding potency. The in vivo toxicological studies of these analogs were also performed. The LD50 of the analogs ranged in the order: Me > H > Ph.

2beta-Substituted analogues of 4'-iodococaine: synthesis and dopamine transporter binding potencies.

A series of 2beta-substituted analogues of 4'-iodococaine (3) was synthesized and evaluated in an in vitro dopamine transporter (DAT) binding assay. Selective hydrolysis at the 2beta-position of 3 gave the carboxylic acid 15 that served as the intermediate for the synthesis of compounds 4, 5, and 6-11. The 2beta-alkyl derivatives were obtained from ecgonine methyl ester (17) through a series of reactions leading to the aldehyde 20. Wittig reaction of 20 with methyltriphenylphosphorane followed by hydrogenation and benzoylation gave the products 12 and 13. The binding affinity of 4'-iodococaine (3) was 10-fold less than that of cocaine. The hydroxymethane, acetate, amide, benzyl ester, oxidazole, and ethane derivatives of 3 exhibited decreased binding while the vinyl, phenyl, and ethyl esters showed a moderate increase in binding affinity. Only the isopropyl derivative 8 exhibited a 2-fold increase in binding affinity compared with 4'-iodococaine (3). Hydroxylation of 8 at the 2'-position gave 14 which enhanced not only the binding potency at the DAT by another 2-fold but also the selectivity at the DAT over the norepinephrine and serotonin transporters. Compound 14 failed to stimulate locomotor activity in C57BL/6J mice over a wide dose range and blocked cocaine-induced locomotor stimulant action.

Synthesis and ligand binding studies of 4'-iodobenzoyl esters of tropanes and piperidines at the dopamine transporter.

Four analogs and two homologs of cocaine, designed as potent cocaine antagonists, were synthesized. The SN2 reaction between ecgonine methyl ester (13) or appropriately substituted piperidinol (19, 21) and appropriately substituted 4-iodobenzoyl chloride gave 4-iodobenzoyl esters of tropanes and piperidines (5-8). 2'-Hydroxycocaine (9) was obtained from 2'-acetoxycocaine (12) by selective transesterification with MeOH saturated with dry HCl gas. 2'-Acetoxycocaine (12) was synthesized from acetylsalicyloyl chloride (23) and ecgonine methyl ester (13). The binding affinities of these compounds were determined at the dopamine transporter for the displacement of [3H]WIN-35428. An iodo group substitution at the 4'-position of cocaine decreased dopamine transporter binding potency, while a hydroxy or acetoxy group at the 2'-position exhibited increased binding potency for the dopamine transporter compared to cocaine (10- and 3.58-fold, respectively). 2'-Hydroxylation also enhanced the bidning potency of 4'-iodococaine (5) by 10-fold. Replacement of the tropane ring with piperidine led to poor binding affinities.

Generation of polyclonal catalytic antibodies against cocaine using transition state analogs of cocaine conjugated to diphtheria toxoid.

Six novel transition state analogs (TSAs) of cocaine (10-14 and 17) and one non-cocaine, p-aminophenylphosphonyl ester of cyclohexanol (19), were synthesized and characterized by 1H- and 13C-NMR and FAB-MS. (1R)-ecgonine methyl ester or cyclohexanol were subjected to phenylphosphonylation in the presence of dicyclohexyl carbodiimde (DCC) and 4-N,N-dimethyl aminopyridine (4-DMAP). TSA-IV (10), however, was synthesized from norcocaine which was protected with dibromoethane to yield 4 before acid hydrolysis, esterification and phenylphosphonylation were carried out. TSA-III (11) TSA-I (12) and (19), using various length spacer arms, were coupled with the immunogenic protein, diphtheria toxoid (DT). The TSAs coupled with DT were used to immunize mice and after appropriate boosts their sera were tested for the presence and titer of anti-TSA polyclonal antibodies using ELISA. Preliminary results show that the mice immunized with these TSAs produced high titers of polyclonal catalytic antibodies, except for (19), with the ability to hydrolyze the substrate 125I-4'-iodococaine in an in vitro assay, even in the presence of noncatalytic anti-TSA antibodies.

Synthesis and biological evaluation of basic side chain derivatives of Analog II as pure antiestrogens and antitumor agents.

In an effort to prepare effective non-steroidal antiestrogens without intrinsic estrogenicity and with greater antagonism than those of the triarylethylenes (tamoxifen; TAM) four N-substituted (Z)-1,1-dichloro-2-[4-(2-aminoethoxy)phenyl]-3-phenylcyclopropa ne derivatives of the antiestrogen, Analog II, in which the basic side chains contain cyclic (piperidino and piperazino) and non-cyclic (dimethyl amino and diethyl amino) moieties, were synthesized. These compounds were prepared from an intermediate methanesulfonyloxyethoxy side chain ester of 1,1-dichloro-2,3-cis-diphenylcyclopropane using their respective side chain bases in triethylamine and acetonitrile. The gem-dichloro-cis-diarylcyclopropane derivatives were tested for their ability to inhibit the growth-stimulating effect of estradiol on immature mouse uteri and the growth of estrogen receptor (ER)-positive MCF-7E3, ER-negative MDA-MB-231 and the ER-positive MCF-7LY2 antiestrogen-resistant breast cancer cells in culture. The introduction of the various aminoethoxy side chains into Analog II did not improve its ER-binding affinity. Like Analog II, the derivatives did not exhibit any intrinsic estrogenicity, and compounds 9 and 10 antagonized estradiol action more completely than the parent compound. None of the compounds potentiated the uterine weight gain from the stimulating dose of estradiol (0.03 micrograms). Derivatives 9 (150 micrograms), 10 (150 micrograms) and 11 (150 micrograms) had uterine mean weights significantly below the estradiol-treated group, and were better antagonists than Analog II and MER25 at the same concentrations. All compounds exhibited a statistically significant (P < 0.01) reduction in control growth (antitumor activity) from 0.01 to 10 nM concentration in the MCF-7E3 cells. At 10 nM concentration, 8 (66%) and 9 (64%) had the greater antitumor activity over 10 (58%) and 11 (58%). No activity in this cell line was observed for Analog II, TAM and ICI 182,780. Antitumor action was also demonstrated in the MDA-MB-231 cells for all derivatives at 1.0 microM dose, with 9 having the greatest (27%) inhibition of control growth, followed by 8 (20%), 10 (18%) and 11 (12%). Analog II and ICI 182,780 had no antitumor activity in this cell line, while TAM exhibited only 8% inhibition. In the MCF-7E3 cell line at 1.0 microM, 9 exhibited 86% inhibition of the estradiol-stimulated growth (antiestrogenic activity), followed by 8 (64%), 10 (52%) and 11 (21%).(ABSTRACT TRUNCATED AT 400 WORDS)