Search

Previous studies suggested that differences between the behavioral effects of cocaine and analogs of benztropine were related to the relatively slow onset of action of the latter compounds. Several N-substituted benztropine analogs with a relatively fast onset of effects were studied to assess whether a fast onset of effects would render the effects more similar to those of cocaine. Only one of the compounds increased locomotor activity, and the increases were modest compared with those of 10 to 20 mg/kg cocaine. In rats trained to discriminate 10 mg/kg cocaine from saline none of the compounds produced more than 40% cocaine-like responds up to 2 h after injection. None of the compounds produced place-conditioning when examined up to 90 min after injection, indicating minimal abuse liability. The compounds had 5.6 to 30 nM affinities at the dopamine transporter (DAT), with uniformly lower affinities at norepinephrine and serotonin transporters (from 490-4600 and 1420-7350 nM, respectively). Affinities at muscarinic M(1) receptors were from 100- to 300-fold lower than DAT affinities, suggesting minimal contribution of those sites to the behavioral effects of the compounds. Affinities at histaminic H(1) sites were from 11- to 43-fold lower than those for the DAT. The compounds also had affinity for sigma, 5-hydroxytryptamine(1) (5-HT(1)), and 5-HT(2) receptors that may have contributed to their behavioral effects. Together, the results indicate that a slow onset of action is not a necessary condition for reduced cocaine-like effects of atypical DAT ligands and suggest several mechanisms that may contribute to the reduced cocaine-like efficacy of these compounds.

The syntheses of 2-methoxyestradiol analogs with modifications at the 3-position are described. The analogs were assessed for their antiproliferative, antiangiogenic, and estrogenic activities. Several lead substituents were identified with similar or improved antitumor activities and reduced metabolic liability compared to 2-methoxyestradiol.

The syntheses of 21 analogs of 2-methoxyestradiol are presented, including ENMD-1198 which was selected for advancement into Phase 1 clinical trials in oncology. These analogs were evaluated for antiproliferative activity using breast tumor MDA-MB-231 cells, for antiangiogenic activity in HUVEC proliferation assays, and for estrogenic activity in MCF-7 cell proliferation. The most active analogs were evaluated for iv and oral pharmacokinetic properties via cassette dosing in rat and in mice pharmacokinetic models.

A novel series of 17-modified and 2,17-modified analogs of 2-methoxyestradiol (2ME2) were synthesized and characterized. These analogs were designed to retain or potentiate the biological activities of 2ME2 and have diminished metabolic liability. The analogs were evaluated for antiproliferative activity against MDA-MB-231 breast tumor cells, antiangiogenic activity in HUVEC, and estrogenic activity on MCF-7 cell proliferation. Several analogs were evaluated for metabolic stability in human liver microsomes and in vivo in a rat cassette dosing model. This study lead to several 17-modified analogs of 2ME2 that have similar or improved antiproliferative and antiangiogenic activity, lack estrogenic properties and have improved metabolic stability compared to 2ME2.

A series of 3,3-diarylacrylonitriles were synthesized stereoselectively as tubulin polymerization inhibitors for potential use in cancer chemotherapy. This synthetic route features stannylcupration and palladium-catalyzed Stille cross-coupling chemistry, allowing both E and Z isomers of 3,3-diarylacrylonitriles to be prepared in a very short sequence of reactions.

Clinical studies using the microtubule-targeting agent 2-methoxyestradiol (2ME2; Panzem) in cancer patients show that treatment is associated with clinical benefit, including prolonged stable disease, complete and partial responses, and an excellent safety profile. Studies have shown that 2ME2 is metabolized by conjugation at positions 3 and 17 and oxidation at position 17. To define structure-activity relationships for these positions of 2ME2 and to generate metabolically stable analogues with improved anti-tubulin properties, a series of analogues was generated and three lead analogues were selected, ENMD-1198, ENMD-1200, and ENMD-1237. These molecules showed improved metabolic stability with >65% remaining after 2-h incubation with hepatocytes. Pharmacokinetic studies showed that oral administration of the compounds resulted in increased plasma levels compared with 2ME2. All three analogues bind the colchicine binding site of tubulin, induce G(2)-M cell cycle arrest and apoptosis, and reduce hypoxia-inducible factor-1alpha levels. ENMD-1198 and ENMD-1200 showed improved in vitro antiproliferative activities. Significant reductions in tumor volumes compared with vehicle-treated mice were observed in an orthotopic breast carcinoma (MDA-MB-231) xenograft model following daily oral treatment with all compounds (ANOVA, P < 0.05). Significantly improved median survival time was observed with ENMD-1198 and ENMD-1237 (200 mg/kg/d) in a Lewis lung carcinoma metastatic model (P < 0.05). In both tumor models, the high-dose group of ENMD-1198 showed antitumor activity equivalent to that of cyclophosphamide. ENMD-1198 was selected as the lead molecule in this analogue series and is currently in a phase I clinical trial in patients with refractory solid tumors.

A prodrug strategy was investigated to address the problem of limited aqueous solubility and the resulting limited bioavailability of the antitumor agent 2-methoxyestradiol. The 3-phosphate, 17-phosphate, and 3,17-diphosphate of 2-methoxyestradiol were synthesized. 2-methoxyestradiol 3-phosphate was metabolized more efficiently to the parent compound in vivo than 2-methoxyestradiol 17-phosphate, and it was also more cytotoxic in cancer cell cultures than either the 17-phosphate or the 3,17-diphosphate. These results agree with the in vivo anticancer activity of 2-methoxyestradiol 3-phosphate in a mouse Lewis lung carcinoma experimental metastasis model as opposed to the 17-phosphate and 3,17-diphosphate, both of which were inactive. The in vivo antitumor activity of 2-methoxyestradiol 3-phosphate at a dose of 200 mg/kg per day was comparable to that of a maximally tolerated dose of cyclophosphamide.

A series of 16-modified 2-methoxyestradiol analogs were synthesized and evaluated for antiproliferative activity toward HUVEC and MDA-MB-231 cells, and for susceptibility to conjugation. In addition, the estrogenicity of these analogs was accessed by measuring cell proliferation of the estrogen-dependent cell line MCF7 in response to compound treatment. It was observed that antiproliferative activity dropped as the size of the 16 substituent increased. Selected analogs tested in glucuronidation assays had similar rates of clearance to 2-methoxyestradiol, but had enhanced clearance in sulfonate conjugation assays.

A series of new analogues of 2-methoxyestradiol (1) were synthesized to further elucidate the relationships between structure and activity. The compounds were designed to diminish the potential for metabolic deactivation at positions 2 and 17 and were analyzed as inhibitors of tubulin polymerization and for cytotoxicity. 17alpha-methyl-beta-estradiol (30), 2-propynyl-17alpha-methylestradiol (39), 2-ethoxy-17-(1'-methylene)estra-1,3,5(10)-triene-3-ol (50) and 2-ethoxy-17alpha-methylestradiol (51) showed similar or greater tubulin polymerization inhibition than 2-methoxyestradiol (1) and contained moieties that are expected to inhibit deactivating metabolic processes. All of the compounds tested were cytotoxic in the panel of 55 human cancer cell cultures, and generally, the derivatives that displayed the most activity against tubulin were also the most cytotoxic.

Previous studies demonstrated that analogs of benztropine (BZT) possess high affinity for the dopamine transporter, inhibit dopamine uptake, but generally have behavioral effects different from those of cocaine. One hypothesis is that muscarinic-M(1) receptor actions interfere with cocaine-like effects. Several tropane-nitrogen substitutions of 4',4"-diF-BZT have reduced M(1) affinity compared with the CH(3)-analog (AHN 1-055; 3alpha-[bis-(4-fluorophenyl)methoxy]tropane). All of the compounds displaced [(3)H]WIN 35,428 (2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane) binding with affinities ranging from 11 to 108 nM. Affinities at norepinephrine ([(3)H]nisoxetine) and serotonin ([(3)H]citalopram) transporters ranged from 457 to 4810 and 376 to 3260 nM, respectively, and at muscarinic M(1) receptors ([(3)H]pirenzepine) from 11.6 (AHN 1-055) to higher values, reaching 1030 nM for the other BZT-analogs. Cocaine and AHN 1-055 produced dose-related increases in locomotor activity in mice, with AHN 1-055 less effective than cocaine. The other compounds were ineffective in stimulating activity. In rats discriminating cocaine (29 micromol/kg i.p.) from saline, WIN 35,428 fully substituted for cocaine, whereas AHN 1-055 produced a maximal substitution of 79%. None of the other analogs fully substituted for cocaine. WIN 35,428 produced dose-related leftward shifts in the cocaine dose-effect curve, whereas selected BZT analogs produced minimal changes in the effects of cocaine. The results suggest that reducing M(1) affinity of 4',4"-diF-BZT with N-substitutions reduces effectiveness in potentiating the effects of cocaine. Furthermore, although the BZT-analogs bind with high affinity at the dopamine transporter, their behavioral effects differ from those of cocaine. These compounds have reduced efficacy compared with cocaine, a long duration of action, and may serve as leads for the development of medications to treat cocaine abuse.

Synthesis of a series of pure S-(+)-2beta-carboalkoxy-3alpha-[bis(4-fluorophenyl)methoxy]tropanes (>99% ee) was achieved by employing a chiral amine-induced asymmetric reaction of tropinone with methyl cyanoformate as the key step. In this series, all of the S-(+)-enantiomers were 2-fold more potent than their racemic mixtures and all displayed high-affinity binding for DAT (K(i)=13-40 nM). These data support previous findings of significant divergence in structural requirements for high-affinity DAT binding among tropane-based inhibitors. Furthermore, the 2-substituent in the 3alpha-[bis(4-fluorophenyl)methoxy]tropane series is well tolerated at the DAT but not at SERT (K(i)=690-2040 nM), or muscarinic M(1) receptors (K(i)=133-4380 nM) resulting in highly selective DAT ligands that may provide new leads toward a cocaine-abuse therapeutic.

Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.

Rationale: Several halogenated analogs of benztropine (BZT) have previously been characterized as potent DA uptake inhibitors with behavioral profiles that indicate diminished psychomotor stimulant effects relative to cocaine. In a previous study using a fixed-ratio 10 schedule, two chloro-analogs (3'-Cl-BZT and 4'-Cl-BZT) maintained i.v. self-administration in monkeys but appeared to be weak positive reinforcers., Objectives: The present experiments were designed to test the hypothesis that 3'-Cl-BZT and 4'-Cl-BZT are relatively weak reinforcers by evaluating reinforcing effects under increased response requirements. To examine further the effect of this halogen substitution on self-administration, 3',4"-diCl-BZT was also evaluated for reinforcing effects., Methods: Four rhesus monkeys self-administered cocaine (0.03 mg/kg per injection, i.v.) under a fixed-ratio 25 (FR25) schedule until stable responding was established. Saline, various doses of cocaine (0.003-0.2 mg/kg per injection), the BZT analogs (0.012-0.2 mg/kg per injection), GBR 12909 (0.012-0.2 mg/kg per injection), and compounds with known reinforcing effects (d-amphetamine, morphine, pentobarbital, ketamine) were then made available for self-administration. Various doses (0.01-0.3 mg/kg per injection) of the compounds that maintained self-administration under the FR schedule were then substituted for cocaine (0.1 mg/kg per injection) under progressive-ratio (PR) schedules., Results: Reinforcing effects were evident under the FR schedule for 3'-Cl-BZT, 4'-Cl-BZT, GBR 12909, and the control compounds, but not by 3',4"-diCl-BZT. Results with the PR suggested that the rank order of these compounds for their effectiveness as reinforcers was cocaine > GBR 12909 > 3'-Cl-BZT = 4'-Cl-BZT >> 3',4"-diCl-BZT., Conclusions: This study confirms and extends previous results suggesting that compounds with high DAT affinity can have strong, moderate, weak, or no effectiveness as reinforcers. The mechanisms that may underlie this variation in reinforcing effectiveness of these DAT ligands remain to be established.

Rationale: Previous SAR studies demonstrated that small halogen substitutions on the diphenylether system of benztropine (BZT), such as a para-Cl group, retained high affinity at the cocaine binding site on the dopamine transporter. Despite this high affinity, the compounds generally had behavioral effects different from those of cocaine. However, compounds with meta-Cl substitutions had effects more similar to those of cocaine., Objectives: A series of phenyl-ring analogs of benztropine (BZT) substituted with 3'-, 4'-, 3',4"- and 4',4"-position Cl-groups were synthesized and their pharmacology was evaluated in order to assess more fully the contributions to pharmacological activity of substituents in these positions., Methods: Compounds were synthesized and their pharmacological activity was assessed by examining radioligand binding and behavioral techniques., Results: All of the compounds displaced [3H]WIN 35,428 binding with affinities ranging from 20 to 32.5 nM. Affinities at norepinephrine ([3H]nisoxetine) and serotonin ([3H]citalopram) transporters, respectively, ranged from 259 to 5120 and 451 to 2980 nM. Each of the compounds also inhibited [3H]pirenzepine binding to muscarinic M1 receptors, with affinities ranging from 0.98 to 47.9 nM. Cocaine and the BZT analogs produced dose-related increases in locomotor activity in mice. However, maximal effects of the BZT analogs were uniformly less than those produced by cocaine, and were obtained 2-3 h after injection compared to the relatively rapid onset (within 30 min) of cocaine effects. In rats trained to discriminate i.p. saline from 29 mumol/kg cocaine (10 mg/kg), cocaine produced a dose-related increase in responding on the cocaine lever, reaching 100% at the training dose; however, none of the BZT analogs fully substituted for cocaine, with maximum cocaine responding from 20 to 69%. Despite their reduced efficacy compared to cocaine in cocaine discrimination, none of the analogs antagonized the effects of cocaine. As has been reported previously for 4'-Cl-BZT, the cocaine discriminative-stimulus effects were shifted left-ward by co-administration of the present BZT analogs., Conclusions: The present results indicate that although the BZT analogs bind with relatively high affinity and selectivity at the dopamine transporter, their behavioral profile is distinct from that of cocaine. The present results suggest that analogs of BZT may be useful as treatments for cocaine abuse in situations in which an agonist treatment is indicated. These compounds possess features such as reduced efficacy compared to cocaine and a long duration of action that may render them particularly useful leads for the development of therapeutics for cocaine abusers.

In a continuing effort to further characterize the role of the dopamine transporter in the pharmacological effects of cocaine, a series of chiral and achiral N-substituted analogues of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (5) has been prepared as potential selective dopamine transporter ligands. These novel compounds displaced [(3)H]WIN 35,428 binding from the dopamine transporter in rat caudate putamen with K(i) values ranging from 13. 9 to 477 nM. Previously, it was reported that 5 demonstrated a significantly higher affinity for the dopamine transporter than the parent drug, 3alpha-(diphenylmethoxy)tropane (3; benztropine). However, 5 remained nonselective over muscarinic m(1) receptors (dopamine transporter, K(i) = 11.8 nM; m(1), K(i) = 11.6 nM) which could potentially confound the interpretation of behavioral data, for this compound and other members of this series. Thus, significant effort has been directed toward developing analogues that retain high affinity at the dopamine transporter but have decreased affinity at muscarinic sites. Recently, it was discovered that by replacing the N-methyl group of 5 with the phenyl-n-butyl substituent (6) retention of high binding affinity at the dopamine transporter (K(i) = 8.51 nM) while decreasing affinity at muscarinic receptors (K(i) = 576 nM) was achieved, resulting in 68-fold selectivity. In the present series, a further improvement in the selectivity for the dopamine transporter was accomplished, with the chiral analogue (S)-N-(2-amino-3-methyl-n-butyl)-3alpha-[bis(4'-fluorophenyl)metho xy] tropane (10b) showing a 136-fold selectivity for the dopamine transporter versus muscarinic m(1) receptors (K(i) = 29.5 nM versus K(i) = 4020 nM, respectively). In addition, a comparative molecular field analysis (CoMFA) model was derived to correlate the binding affinities of all the N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues that we have prepared with their 3D-structural features. The best model (q(2) = 0. 746) was used to accurately predict binding affinities of compounds in the training set and in a test set. The CoMFA coefficient contour plot for this model, which provides a visual representation of the chemical environment of the binding domain of the dopamine transporter, can now be used to design and/or predict the binding affinities of novel drugs within this class of dopamine uptake inhibitors.

Benztropine and its analogs are tropane ring-containing dopamine uptake inhibitors that produce behavioral effects markedly different from cocaine and other dopamine transporter blockers. We investigated the benztropine binding site on dopamine transporters by covalently attaching a benztropine-based photoaffinity ligand, [125I]N-[n-butyl-4-(4"'-azido-3"'-iodophenyl)]-4', 4"-difluoro-3alpha-(diphenylmethoxy)tropane ([125I]GA II 34), to the protein, followed by proteolytic and immunological peptide mapping. The maps were compared with those obtained for dopamine transporters photoaffinity labeled with a GBR 12935 analog, [125I]1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)ethy l]p iperazine ([125I]DEEP), and a cocaine analog, [125I]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82), which have been shown previously to interact with different regions of the primary sequence of the protein. [125I]GA II 34 became incorporated in a membrane-bound, 14 kDa fragment predicted to contain transmembrane domains 1 and 2. This is the same region of the protein that binds [125I]DEEP, whereas the binding site for [125I]RTI 82 occurs closer to the C terminal in a domain containing transmembrane helices 4-7. Thus, although benztropine and cocaine both contain tropane rings, their binding sites are distinct, suggesting that dopamine transport inhibition may occur by different mechanisms. These results support previously derived structure-activity relationships suggesting that benztropine and cocaine analogs bind to different domains on the dopamine transporter. These differing molecular interactions may lead to the distinctive behavioral profiles of these compounds in animal models of drug abuse and indicate promise for the development of benztropine-based molecules for cocaine substitution therapies.

The design, synthesis and pharmacological evaluation of novel dopamine transporter ligands, based on Benztropine [3a-(diphenylmethoxy) tropane], has been a focus of our research efforts toward the development of novel cocaine-abuse pharmacotherapeutics. Structure-activity relationships at the dopamine transporter, for this series of compounds, have been derived and compared to those of cocaine and GBR 12909. These studies suggest that structurally diverse dopamine uptake inhibitors may access different binding domains on the dopamine transporter. The distinctive behavioral profile displayed in this series of compounds, as compared to cocaine and other dopamine uptake inhibitors, is of particular interest and is proposed to be relevant to the pharmacodynamic and pharmacokinetic properties of this class of tropane-based molecules.

A series of N-substituted 3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues has been prepared that function as dopamine uptake inhibitors. The N-methylated analogue of this series had a significantly higher affinity for the dopamine transporter than the parent compound, N-methyl-3 alpha- (diphenylmethoxy)tropane (benztropine, Cogentin). Yet like the parent compound, it retained high affinity for muscarinic receptors. A series of N-substituted compounds were prepared from nor-3 alpha-[bis(4'-fluorophenyl)methoxy]tropane via acylation followed by hydride reduction of the amide or by direct alkylation. All compounds containing a basic tropane nitrogen displaced [3H]WIN 35,428 at the dopamine transporter (Ki range = 8.5-634 nM) and blocked dopamine uptake (IC50 range = 10-371 nM) in rat caudate putamen, whereas ligands with a nonbasic nitrogen were virtually inactive. None of the compounds demonstrated high binding affinity at norepinephrine or serotonin transporters. Importantly, a separation of binding affinities for the dopamine transporter versus muscarinic m1 receptors was achieved by substitution of the N-methyl group with other N-alkyl or arylalkyl substituents (eg. n-butyl, allyl, benzyl, 3-phenylpropyl, etc.). Additionally, the most potent and selective analogue in this series at the dopamine transporter, N-(4"-phenyl-n-butyl)-3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogue failed to substitute for cocaine in rats trained to discriminate cocaine from saline. Potentially, new leads toward the development of a pharmacotherapeutic for cocaine abuse and other disorders affecting the dopamine transporter may be discovered.